INVESTIGATION OF SELECTED
POTENTIAL ENVIRONMENTAL CONTAMINANTS
NITROAROMATICS
June 1976
FINAL REPORT
Office of Toxic Substances
U.S. Environmental Protection Agency
Washington, D.C. 20460
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EPA-560/2-76-010 TR 76-573
INVESTIGATION OF SELECTED POTENTIAL
ENVIRONMENTAL CONTAMINANTS:
NITROAROMATICS
Philip H. Howard
Joseph Santodonato
Jitendra Saxena
Judith Mailing
Dorothy Greninger
June 1976
Contract No. 68-01-2999
SRC No. L1257-05
Project Officer - Frank Kover
Prepared for
Office of Toxic Substances
U.S. Environmental Protection Agency
Washington, D.C. 20460
Document is available to the public through the National Technical Information
Service, Springfield, Virginia 22151
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NOTICE
This report has been reviewed by the Office of Toxic Substances, EPA,
and approved for publication. Approval does not signify that the contents
necessarily reflect the views and policies of the Environmental Protection
Agency, nor does mention of trade names or commercial products constitute
endorsement or recommendation for use.
ii
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TABLE OF CONTENTS
Page
Executive Summary xxv
Introduction 1
I. Physical and Chemical Data 2
A. Structure and Properties 2
I. Chemical Structure 2
2. Physical Properties 3
3. Principal Contaminants and Specifications of 16
Commercial Products
B. Chemistry 21
1. Reactions Involved in Uses 21
2. Hydrolysis 29"
3. Oxidation 31
4. Photochemistry 31
II. Environmental Exposure Factors 47
A. Production, Consumption 47
1. Quantity Produced and Imported 47
2. Producers and Production Sites 57
3. Production Methods and Processes 57
a. General .57
b. Nitrobenzene 65
c. Dinitrotoluene 66
d. Chloronitrobenzene Process 68
e. Trinitrotoluene Processes 70
f. Nitrophenol Processes 73
4. Market Prices 73
5. Market Trends 75
B. Uses 79
1. Major Uses 79
2. Minor Uses 86
3. Possible Alternatives to Use 98
iii
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Table of Contents
(continued)
Page
C. Environmental Contamination Potential 101
1. General 101
2. From Production and Uses 101
3. From Transport and Storage 102
4. From Disposal 102
5. Potential Inadvertent Production of Nitroaromatics 104
in Other Industrial Processes as a By-Product
- 6. Potential Inadvertent Production in the Environment 104
D. Current Handling Practices and Control Technology 104
1. Special Handling in Use 104
2. Methods for Transport and Storage 105
3. Disposal Methods 106
4. Accident Procedures 107
5. Current Control Technology 108
E. Monitoring and Analysis 111
1. Analytical Methods 111
a. Explosives 111
b. Pesticides 112
c. Miscellaneous Nitroaromatic Methods and 122
Monitoring Studies
2. Monitoring Studies 127
III. Health and Environmental Effects 133
A. Environmental Effects 133
1. Persistence 133
a. Biological Degradation, Organisms, and Products 133
(i) Nitrobenzenes and Chloronitrobenzenes 133
(ii) Nitrobenzoic Acids 140
(a) Mono Nitrobenzoic Acids 141
(b) Di- and Trinitro-substituted Benzoic 150
Acids
(c) Halogen Analogues of Nitrobenzoic Acids 151
iv
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Table of Contents
(continued)
Page
(iii) Nitrophenols and Related Compounds 152
(a) _pj-, m-, and p-Nitrophenols 154
(b) M- and Trinitrophenols 162
(c) Nitrocresols 164
(d) Nitroresorcinol 165
(iv) Nitrotoluenes 167
(a) Degradation by Pure Cultures of 168
Microorganisms
(b) Degradation by Natural Communities of 171
Microorganisms
(c) Degradation Under Sewage Treatment Plant 172
Conditions
(d) Routes of Degradation of Nitrotoluenes 177
(v) Nitroanilines 178
(vi) Summary of the Biodegradation Studies with 181
Nitroaromatics
2. Environmental Transport 184
a. Volatility 184
(i) Volatilization From Water 184
(ii) Volatilization From Soil and Other Surfaces 186
b. Leaching and Downward Movement of Nitroaromatics 187
c. Mobility in Water 188
3. Bioaccumulation 189
4. Biomagnification 192
B. Biology 194
1. Absorption and Elimination 194
a. Nitrophenol Derivatives 198
b. Nitrobenzene 214
2. Transport and Distribution 217
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Table of Contents
(continued)
Page
3. Metabolism and Excretion 221
a. Nitrobenzene Derivatives 221
b. Nitrotoluene Derivatives 231
c. Nitroaniline Derivatives 236
d. Nitrophenol Derivatives 240
e. Metabolic Reduction of Nitroaromatic Compounds 244
f. Metabolism by Gastro-Intestinal Microorganisms 247
4. Metabolic and Pharmacologic Effects 252
a. Hematologic Effects 252
b. Skin Sensitization 259
c. Uncoupling of Oxidative Phosphorylation 261
(i) Metabolic Disruption 266
(ii) Neurologic Effects 274
d. Organoleptic Properties 276
C. Toxicity - Humans 277
1. Occupational Studies 279
a. Trinitrotoluene 286
b. Tetryl 291
c. Dinitrobenzene 292
d. Nitrochlorobenzene 294
e. l-Chloro-2,4-dinitrobenzene (DNCB) 296
f. 2,4-Dinitrophenol 298
g. 4,6-Dinitro-ortho-cresol (DNOC) 299
h. Other Nitrophenol Derivatives 301
i. Nitroanilines 305
2. Non-Occupational Exposures 305
a. 2,4-Dinitrophenol and Derivatives 306
b. Nitrobenzene 309
c. Nitroaniline 311
3. Epidemiological and Controlled Human Studies 314
a. Tetryl 314
b. Trinitrotoluene 315
vi
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Table of Contents
(continued)
D.
Page
c. Dinitrochlorobenzene (DNCB)
d. Nitrobenzene
e. Dinitro-ortho-cresol (DNOC)
Toxicity - Birds and Mammals
1. Acute Animal Toxicity
a. Dinitrophenol and Derivatives
(i) Acute Cataract Development
b. Nitrobenzene and Derivatives
c. Trinitrotoluene (TNT)
d. Structure-Activity Relationships
2. Subacute and Chronic Toxicity
a. Dinitrophenol Derivatives
(i) Chronic Cataract Development
b. Nitroaniline
c. Chloronitrobenzenes
d. Nitrobenzene
e. Nitrotoluenes
f. Trinitrotoluene (TNT)
3. Sensitization
4. Mutagenicity
5. Teratogenicity
a. 2,4-Dinitrophenol
b. 2-sec-Butyl-4,6-dinitrophenol
c. Pentachloronitrobenzene
6. Carcinogenicity
a. 4-Nitroquinoline-N-oxide (4NQO)
b. Nitrobenzene Derivatives
c. Heterocyclic Nitro Compounds
d. Nitro Derivatives of Aromatic Amine Carcinogens
e. Miscellaneous Nitroaromatic Carcinogens
317
318
321
322
322
323
331
339
344
344
375
385
386
389
389
390
393
393
395
399
403
403
406
410
411
414
419
427
427
433
7. Possible Synergisms 435
vii
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Table of Contents
(continued)
E. Toxicity to Lower Animals 437
1. Nitrophenols 437
2. Halogen-substituted Nitrophenols 439
3. Nitrosalicylanilide 446
4. Agricultural Chemicals 449
5. Trinitrotoluene 453
F. Toxicity - Plants 457
1. Nitrophenols 457
2. Nitrotoluenes 461
3. Nitrobenzenes 466
G. Toxicity - Microorganisms 467
1. Effects on Bacteria 467
a. Growth Inhibition 470
b. Effect on Cell Permeability 472
c. Effect on Protein Synthesis 473
d. Influence on Oxidative Enzyme Systems 475
e. Mutagenic Effects 476
f. Miscellaneous Effects 477
2. Effect on Yeast and Fungi 477
3. Effect on Protozoa 481
4. Effect on Unicellular Algae 482
5. Influence of Nitroaromatics on the Microbiological 485
Systems Concerned with Waste Treatment
6. Effect on Natural Microbial Populations 491
IV. Regulations and Standards 493
A. Current Regulations 493
B. Concensus and Similar Standards 495
C. Foreign Authority 497
V. Summary and Conclusions 499
A. Summary 499
B. Conclusions 517
Chemical Index 525
References
viii
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List of Tables
Number
? 1 Physical Properties of Significant Nitroaromatic Chemicals 4
2 Water Solubility of Chlorine Substituted Nitrobenzenes 3
3 Analysis of Several Nitroaromatic Chemical Intermediates Before 18
and After Recrystallization
4 Crude Sample Analyses 19
5 Percentage Concentration of Impurities in Typical Samples of 19
Production TNT (2,4,6-Trinitrotoluene)
6 Sales Specifications and Contaminants in Technical Grades of 20
Nitroaromatic Compounds
7 Alkaline and Neutral Hydrolysis Rates of Nitroaromatic Compounds 30
in Water
8 Ultraviolet Spectra of Representative Nitroaromatic Compounds 33
9 Solvent Effects on Nitrobenzene Ultraviolet Spectra 34
10 Ultraviolet Absorption Spectrum Changes Caused by Adsorption on 35
Silicic Acid
11 Quantum Yield and Products from the Disappearance of Substituted 38
Nitrobenzenes in 2-Propanol Under Nitrogen Atmosphere
12 Solvent Effect for Photoreduction of £-Nitrobenzonitrile 39
13 Solvent Dependence of the Products of the Photoreduction of 40
Nitrobenzene with Light X >290 nm
14 Photolysis Studies of Nitroaromatic Pesticides 42
15 Photolysis Products from an Aqueous Solution of TNT 44
16 U.S. Production and Sales of Nitroaromatic Compounds 48
17 Imports of Nitroaromatic Chemicals 54
18 Production Volumes of Major Commercial Nitroaromatic Compounds 58
19 Major Nitroaromatic Compound Producers, Capacities, and Plant 61
Locations
20 .Comparative Prices and Production Volumes of Some Nitroaromatic 74
Chemicals
21 Recent Market Prices of Nitroaromatic Chemicals 76
22 Market Trends of Major Nitroaromatic Chemicals 78
23 Uses of Major Nitroaromatic Chemicals 80
24 Large Volume Aromatic Amines Produced by Reduction of 87
Nitroaromatic Compounds
25 Uses of Minor Nitroaromatic Chemicals 88
ix
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List of Tables (Cont'd)
Number Page
26 Estimates of Trinitrotoluene and Dinitrotoluene from Army 103
Munitions Plants
27 Analytical Methods for Nitroaromatic Explosives 113
28 Methods Used for Analysis of Nitroaromatic Pesticides and 119
Related Compounds
29 Relative Electron Capture Sensitivities of Nitroaromatic Compounds 122
30 Miscellaneous Nitroaromatic Analytical and Monitoring Techniques 123
31 Ambient or Effluent Monitoring of Nitroaromatic Compounds 128
32 Monitoring Studies Reporting No Detectable Quantity of 130
Nitroaromatic Compounds
33 Summary of the Degradation Studies with Unsubstituted and Halogen 134
Substituted Nitrobenzenes
34 Oxidation of Nitro-Substituted Benzenes by Phenol-Adapted Culture 138
35 Summary of the Studies Dealing with Biodegradation of Substituted 142
and Unsubstituted Nitrobenzoic Acids
36 Time to Reach 100 mg/fc Level of Soluble C.O.D. Based on Projected 145
Curves
37 Metabolic Activities of 34 Strains of Soil Bacteria Belonging 145
to Pseudomonas Group Towards Derivatives of Benzoic Acid
38
39
40
41
42
43
44
45
46
47
48
49
Experimental Conditions Used by Various Investigators in Studying
the Fate of Nitrophenols and Related Compounds
Degradation of Mononitrophenols by DNOC-Grown Bacteria
Salient Features of the Biodegradation Studies with Nitrotoluenes
Results of the Biodegradability Test on TNT and TNT Waste in
Combination with Ammunition Plant Domestic Waste and Glucose
Effect of TNT Concentration on the Biodegradability of TNT-Waste
Summary of the Degradation Studies with Nitroanilines
Biodegradability of Nitroaromatic Compounds Under Varying Test
Conditions
Rate of Evaporation of Nitroaromatics from Bodies of Water
Bioconcentration Factor of Nitroaromatics in Trout Muscle
Ecological Magnification in the Model Aquatic Ecosystem for
Nitroaromatics
Intestinal Absorption in Rats From Solutions of Various pH Values
Absorption of Organic Acids From the Rat Small Intestine
155
158
169
175
175
179
182
185
191
193
195
196
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List of Tables (Cont'd)
Number Page
50 Absorption of Organic Bases From the Rat Small Intestine 197
51 Absorption of DNOC Following Single Dose of 30 mg/kg Given by 201
Stomach Tube to Albino Rats
52 Blood DNOC Levels in Animals Following Single Doses 202
53 Decaying Blood DNOC Values in Man and in Animals 206
54 Distribution of DNOC in Man Following Single Dose of 75 mg DNOC 207
55 Absolute Rates of Elimination of Four Nitre-Compounds 208
56 Comparison of Rates of Elimination 210
57 Blood Levels in Guinea Pigs After Oral Doses of Binapacryl and 211
Dinoseb
58 Blood Levels in Rabbits After Dermal Absorption on Binapacryl 212
and Dinoseb
59 Comparison of DNOC Concentration in Serum, Kidney, and Liver of 220
Rats After Single and 40 Successive Daily Injections Each of
20 mg DNOC per kg
14
60 Metabolic Fate of a Single Oral Dose of C-Nitrobenzene in the 221
Rabbit During 4-5 Days After Dosing
14
61 Metabolites of m-Dinitro [ C]benzene Excreted in Urine by 224
Rabbits
62 Excretion of Metabolites of jj-, m-, and £-Chloronitrobenzenes 226
by the Rabbit
63 Excretion of Metabolites of 2,3,5,6- and 2,3,4,5-Tetrachloro- 228
nitrobenzenes and of 2,3,5,6-Tetrachloroaniline by the Rabbit
64 PCNB Studies on Twenty-Four Month Male Beagle Dog Tissues 232
65 PCNB Studies on Rat Fat 233
66 Acid Hydrolysis vs. Direct Solvent Extraction 233
67 Excretion of 14C by Rats Dosed with [14C]2,6-Dichloro-4- 237
nitroaniline
68 Excretion of 14C by Rats Dosed with [14C]4-Nitroaniline 238
69 Summary of the Metabolism of Mononitrophenols 240
14
70 Excretion of [ C]Dinoseb by Female Mice Following Oral or IP 243
Administration
71 Reduction of Various Nitro Compounds by Liver Homogenates 245
72 The Reduction of PNBA by Conventional and Cecectomized Rats 251
73 Per Cent Circulating Methemoglobin at Various Times After the 253
Injection of Aromatic Hydroxylamines in Female Mice
xi
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List of Tables (Cont'd)
Number Page
- 74 Per Cent Circulating Methemoglobin at Various Times After 254
the Injection of Aminophenols in Female Mice
75 The Occurrence of Heinz Bodies in the Peripheral Blood of 256
Rabbits Following the Administration of a Single Oral Dose
of 3-Nitronaphthalene (BNN)
76 Content of Phosphorylase a and Total and of Phosphorylase b 269
Kinase in Hearts of Rats Poisoned With DNP 2.5 mg/100 g
77 Effective Concentration of Nitrocompounds Tested on the Small 275
Intestine of Guinea Pig
78 Effect of 2,4-Dinitrophenol Upon Total Acetylcholine Content of 276
Guinea Pig Hemi-Diaphragms
79 The Chemical Cyanosis Anemia Syndrome-Hazards of the Nitroaromatic 280
Compounds
80 Blood Examinations of Workmen in Aromatic Chemical Production 282
Plants
81 Mean Values of UIBC in Workers Exposed to Nitro- and Amino- 285
Aromatic Derivatives and in Control
82 Last Reticulocyte Count at Factory Compared with Value on Return 288
to Oxford Approximately 48 Hours Later
83 Dermal Exposure of Spraymen to DINOSEB and Na-DNOC 303
84 Incidence of Disturbances of Occupational Origin Among 203 314
Tetryl Workers
85 Incidence of Different Symptoms in Exposed Workers Compared to 316
the Control Group
86 Results of Determination of Olfactory Sensation Threshold of 319
Nitrobenzene
87 Effect of Logarithmically Increasing Intraperitoneal Doses of 327
2,4-DNP on Rectal Temperature and Lethality in Rats
88 Comparison of LD,._ Values of Dinitrophenols 329
89 Comparison of LD Values in Rats for Dinitro- and Trinitro- 329
cresol
90 Nitroaromatic Compounds with Cataractogenic Activity in Chickens 333
91 Comparison of Cataract Producing Activities of Various Nitro 334
Compounds in Chickens
92 Incidence of Cataracts in Ducks Following the Administration of 335
a Single Dose of 2,4-Dinitrophenol
93 Incidence of Cataracts in Ducklings Following a Single Injection 336
of 2,4-Dinitrophenol Into the Posterior Chamber of the Eye
xii
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List of Tables (Cont'd)
Number Page
94 Incidence of Cataracts in 90-Day-Old Rabbits Following a 337
Single Injection of 2,4-Dinitrophenol Into the Posterior
Chamber of the Eye
95 Cataract Production in Rabbit Lenses Incubated for 24 Hours at 337
37° in KEI-4 Media Containing Varying Concentrations of 2,4-
Dinitrophenol
96 The Dose of 2,4-Dinitrophenol Causing a 50% Incidence of Cataracts 338
in Various Age Groups of Rabbits Following Intraperitoneal
Administration
97 Comparative Toxicity of Some Nitroaromatic Compounds in Cats 340
98 Acute Effects of Several Nitroaromatic Compounds Given by 343
Intraperitoneal Injection to Rats
99 Comparative Toxicity of Various Nitroaromatic Structures in the 346
Rat
100 Potency of Various 2,4-Diiiitrophenols as Uncouplers of Oxidative 349
Phosphorylation In Vitro and Their Toxicity to Mice, Houseflies,
and Honey Bees
101 Uncoupling and Inhibition of Brain and Liver Mitochondria After 350
Injection of Mice with Various Dinitrophenols
102 Acute Animal Toxicity of Various Nitroaniline Derivatives 351
103 Acute Animal Toxicity of Various Nitrobenzene Derivatives 354
104 Acute Animal Toxicity of Various Nitrophenol Derivatives 358
105 Acute Animal Toxicity of Various Nitrotoluene Derivatives 368
106 Acute Animal Toxicity of Miscellaneous Nitroaromatic Compounds 371
107 Subacute and Chronic Animal Toxicity 376
108 Chronic Effects of Subcutaneous Nitrobenzene Injections on 392
Tissues of the Rabbit
109 Tetryl Exposure at Picatinny Arsenal; Incidence of Dermatitis 398
Treated
110 Frequency of Chromosomal Aberrations Induced by Saturated Solution 400
of DNP After 24 Hours of Treatment
111 Mutagenicity of 2-Nitro-p_-Phenylenediamine and 4-Nitro-o- 402
Phenylenediamine in S_. typhimurium TA1538 With or Without
Liver Microsomal Activation
112 Effect of 2-Nitro-£-Phenylenediamine on Cultured Human Lymphocytes 403
113 Results of Experiments in Which Either Insulin or 2,4-Dinitro- 405
phenol or Both Compounds Were Injected into the Yolk Sac of Eggs
of White Leghorn Fowl After 96 Hours of Incubation
xiii
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List of Tables (Cont'd)
Number Page
114 Effect on Resorption Rate and Fetal Size of 2,4-Dinitro- 407
phenol (DNP) Administered to Mice During Early Organogenesis
115 Gross, Soft-Tissue, and Skeletal Anomalies in Offspring of 408
Pregnant Mice Given Dinoseb by I.P. Injection During Early
Organogenesis
116 Nitroaromatic Compounds Tested for Carcinogenicity 415
117 Incidence of Skin Tumors on Mice During and After Treatment 420
with Croton Oil, Following Applications of Some Chloromono-
nitrobenzenes
118 Tumor Formation in Male and Femal Mice Receiving PCNB 423
119 Tumor Promotion in Female Mice by l-Fluoro-2,4-dinitrobenzene 425
120 Tumor Promotion by l-Fluoro-2,4-dinitrobenzene (DNFB) in Various 426
Stocks of Female Mice
121 Carcinogenesis by l,2-Dichloro-3-nitronaphthalene and 3-Nitro- 431
2-naphthylamine
122 Incidence of Mammary Tumors in Female Rats Treated with Hexa- 434
nitrodiphenylamine
123 Unadjusted Ratios of Predicted to Observed LD Q Values of Nitro- 436
benzene Mixed by Volume with Various Chemicals
124 Differential Toxic Effects Among Larval Lampreys and Fishes of 440
Certain Mononitrophenols Containing Halogens
125 Toxic Effects of 31 Mononitrophenols Containing Halogens or a 442
Trifluoromethyl Group on Larval Lampreys and Fingerling Rainbow
Trout
126 Biological Activity of Phenol and Some Substituted Phenolic 444
Compounds Other Than Mononitrophenols Containing Other Halogens
127 Comparison of Molecular Requirements for Substituted Mono- 446
halo-nitrosalicylanilides Exhibiting Selective Toxicity to
Larval Sea Lamprey and Fingerling Rainbow Trout
128 Comparative Toxicity of Halonitrosalicylanilides to Larval Sea 447
Lamprey and Fingerling Rainbow Trout as a Function of Substituent
Loci
129 Selective Toxicity of Polysubstituted 3-Nitrosalicylanilides to 448
Larval Sea Lamprey and Fingerling Rainbow Trout as a Function of
Atomic Loci
130 Estimated 48 Hour TL_Q Values of Trifluralin to Six Species of 450
Freshwater Crustaceans and One Species of Fish
131 Estimated TL n Values and Confidence Limits for Several Herbicides 450
to Scud
xiv
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List of Tables (Cont'd)
Number Page
132 Toxicity of Dinitramine to Nine Species of Freshwater Fish 451
in Soft Water at 12°
133 Toxicity of Trifluralin, Dinoseb, and Dinocap to Halequin Fish 452
(Rasbora Heteromorpha)
134 Mortality in Fish and Wildlife by DCNA, DDT, and Diphenamid 453
135 Acute Toxicity of Several Nitroaromatic Compounds to Fish 454
136 Relative Toxicity of Mono- and Dinitro-substituted Phenols on 458
Plants
137 Effect of 2,4-Dinitrophenol (pH 5.8) on Exogenous Respiration 460
by Segments of Sunflower Hypocotyl
138 Summary of Lemna perpusilla Colony Growth When Exposed to Various 462
Nitrotoluenes
139 Plant Genera and Species Affected by the Introduction of TNT Plant 465
Effluent in New River (Virginia) (Radford Army Ammunition Plant)
June, 1971
140 Summary of the Studies Dealing with Toxicity of Nitroaromatics to 468
Bacteria
141 Bacteriostatic Activity of Certain Nitrophenols 471
142 Effect of 2,4-Dinitrophenol on the Regeneration of Flagella, and 474
on Protein and Nucleic Acid Synthesis by Salmonella typhimurium
143 Effect of 2,4-Dinitrophenol on the Oxidation of Saturated Aliphatic 476
Fatty Acids by Pseudomonas aeruginosa
144 Effect of 2,4-Dinitrophenol on Nitrogen Assimilation and 478
Respiration in the Fungus Scopulariopsis brevicaulis
145 Influence of Nitrophenols on the Metabolic Quotient, Oxygen 479
Consumption and Carbon Dioxide Evolution by Conidia of Ifl.
Sitophila
146 Effect of Nitrophenolic Compounds on the Growth and Respiration 482
of Chlorella vulgaris
147 Effect of 2,4-Dinitrophenol on the Uptake of 2,4-Dichlprophenoxy- 483
acetic Acid by Chlorella pyrenoidosa
148 Adopted Threshold Limit Values for Nitroaromatic Compounds 496
149 Summary of Information on Nitroaromatic Chemicals 500
150 Nitroaromatic Compounds Detected in River, Drinking, or Waste 513
Waters That Are Not Commercial Products
151 Tumor Production in Animals by Nitroaromatic Chemicals 516
152 Nitroaromatic Compounds Which Have a High Potential for Being 518
Environmental Pollutants
xv
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List of Figures
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Conjugative Interaction Between Aromatic Systems and Nitro
Functional Groups
Reduction Products of Nitroaromatic Compounds
Reduction of Nitrobenzene Using Zinc
Synthesis of Benzidine and Benzidine Derivatives from Nitro-
aromatics
Resonance Structures of Nitroaromatics
Commercial Chemistry of Nitroaromatic Compounds
Suggested Mechanism to Explain Photochromism of o-Alkylnitro-
benzenes
Proposed Mechanism for the Primary Photochemical Step for the
Photodecomposition of TNT in Water
U.S. Production Trends of Nitroaromatic Compounds
Nitrobenzene Process
2,4-Dinitrotoluene Process
Monochloronitrobenzene Process
Continuous Countercurrent Trinitrotoluene Process
Continuous Fluid-Bed Vapor Phase Reduction of Nitrobenzene
Mechanism of Bucherer Reaction
Metabolic Pathways of Degradation of Nitrobenzoate by Species
of the Genus Nocardia
Metabolism of p-Nitrobenzoate by Pseudomonas sp.
Metabolism of o-Nitrobenzoate by Pseudomonas sp.
Oxidation of Halogenonitrobenzoates by j>-Nitrobenzoate-Grown
N. erythropolis
Sequence of the Reduction of Nitro Group in Fusarium sp.
Metabolism of 4,6-Dinitro-^-Cresol by Soil Microorganisms
Proposed Pathway for TNT Metabolism
Intermediate Products of £-Nitroaniline Decomposition
Changes in Serum Level of DNOC After a Single Subcutaneous
Injection of 10 ing DNOC/kg
Effect of Repeated Subcutaneous Injections (10 mg DNOC/kg) on
Serum Level of DNOC in Rabbit
Blood Levels of DNOC in the Dog After Repeated Subcutaneous
Page
2
23
24
24
25
26
36
45
53
67
69
71
72
86
100
147
148
149
153
163
166
177
178
198
199
200
Injections
xv i
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List of Figures (Cont'd)
Number Page
27 Effect of DNOC Aerosol on Rats; Blood DNOC Values After 203
Exposure at 25° to Concentration of 0.1 mg/cu m
28 Skin Absorption of DNOC by Twelve Chinchilla Rabbits 204
29 Comparison of 24-Hour Blood Levels 205
30 Decay Curves of Blood DNOC of Man, Rat, and Rabbit 206
31 Elimination of Nitro Compounds By the Monkey 209
32 Concentration of DNBP in Blood of Individual Rabbits at Various 213
Times After DNBP (50 mg/kg) Had Been Applied to the Skin
33 The Percentage Retention of Nitrobenzene Vapor in the Lungs in 215
the Course of a Six-Hour Exposure (Mean Values)
34 The Concentration of £-Nitrophenol in Urine Collected Two to Three 215
Hours After the End of Exposure, as a Function of the Absorbed
Dose of Nitrobenzene
35 The Excretion Rate of £-Nitrophenol in the Urine Collected During 216
the First Two to Three Hours After the End of Exposure, as a
Function of the Absorbed Dose of Nitrobenzene
36 Binding of £-Nitroaniline to Suspensions of Broken Red Cells and 218
to Hemoglobin
37 Suggested Pathways in the Metabolism of Nitrobenzene 222
38 Metabolism of m-Dinitrobenzene 225
39 Phenolic Metabolites Excreted (Free or Conjugated) in the Urine 227
by the Rabbit After Dosage with £-, m-, and o-Chloronitrobenzene
and £-, m-, and £-Chloroaniline
40 Metabolism of 2,3,5,6-Tetrachloronitrobenzene 229
41 Metabolism of 2,3,4,5-Tetrachloronitrobenzene 230
42 Proposed Mechanism for the Displacement of Nitro by Hydroxyl in 239
the Metabolism of 2,6-Dihalo-4-Nitroanilines
14
43 Rate of C-Trifluralin Degradation and Formation of Degradation 248
Products in Artificial Rumen Fluid
44 Typical Time Course of Reduction of PNBA by Rat Cecal Contents 250
45 Methemoglobin Formation by Nitrobenzene In Vitro . 255
46 Plots of the Amount of Methemoglobin and Oxygen Affinity of 257
Hemoglobin (p02 -,_) versus Time After Injection
47 The Platelet-Count of Rats After the Administration of Phenol 259
Derivatives
48 The Electron Transport Sequence and Probable Sites of Coupled 262
Oxidative Phosphorylation
xvii
-------
List of Figures (Cont'd)
Number
49 Structural Similarities Among Various Uncouplers of Oxidative 265
Phosphorylation
50 Effect of Logarithmically Increasing Intraperitoneal Doses of 267
2,4-DNP on Rectal Temperature in Rats
51 Epinephrine and Glucagon Control of Glycogen Metabolism 268
52 Response to Intravenous Dinitrophenol Injection in Dogs 271
53 Oxygen Consumption of Guinea Pigs — Average Weight of Group 272
1500 g, in Response to Varied Doses of DNOC Given Intraperitoneally
54 Effect of Temperature Upon Cyanosis Occurrence 281
55 Total Reticulocyte Count During and After TNT Exposure 288
56 Hematologic Effects From Nitrochlorobenzene Poisoning 295
57 The Level of Methemoglobin and Hemoglobin in Blood During the 312
First 12 Hours of Treatment
58 The Level of Methemoglobin and Hemoglobin During the Entire 313
Treatment Period
59 The Excretion Rate (mg/day) of Nitrobenzene Metabolites jp_- 313
Nitrophenol (PNP) and prAminophenol (PAP) During the Entire
Period of Observation
60 Change in the Light Sensitivity of the Eye During Inhalation of 319
Nitrobenzene in Subject S
61 Changes in the Amplitude of Reinforced Intrinsic Potentials of 320
the Brain in Subject L
62 Infusion of Dinitrophenol in Conscious Dog - Rate: 0.4 mg/kg/ 324
minute, Right External Jugular Vein
63 Effect of Logarithmically Increasing Intraperitoneal Doses of 2,4- 326
DNP on Rectal Temperature in Rats
64 Effect of Environmental Temperature on Mortality in Rats Caused 328
by a Single Dose of Dinitro-o-cresol
65 Mortality From a Single Dose of DNP by Oral and Dermal Administra- 330
tion
66 Mortality From a Single Dose of DNOC by Oral and Dermal Administra- 330
tiori
67 Mortality From a Single Dose of Dinoseb by Oral and Dermal 330
Administration
68 Mortality From a Single Oral Dose of 2-Cyclohexyl-4,6-dinitrophenol 330
in the Rat
69 Acute Oral Toxicity of Dinoseb to Various Animal Species 332
xviii
-------
List of Figures (Cont'd)
Number Page
70 Metabolism of Dinitrophenol and Production of a 387
Cataractogenic Substance
71 Polycyclic Aromatic Carcinogen Skeletons 413
72 Metabolic Conversions of 4NQO 414
73 Average Number of Skin Tumors/Surviving Mouse During and 421
After Treatment with Croton Oil
74 Mortality Curves for Various ^-Nitrophenol Concentrations 438
75 Enhancement of the Acute Toxicity of TFM to Rainbow Trout 445
by Salicylamide
76 Relative Susceptibility of Bluegills to TNT at Different Water 455
Hardness* and Temperature
77 Mortality Curves at Four Days 456
78 The Effect of Waste Discharges from Radford Army Ammunition 463
Plant on the Biota of the New River, Virginia - Location of
Sampling Points
79 Histogram Representing the Number of Species of Aquatic Plants 464
Present Below the High-Water Mark in Ten Samples Along the
New River, June 1971
80 Respiration Rate of TNT Acclimated and Nonacclimated Micro- 487
organisms at Various Concentrations of TNT
81 Effect of Trinitrophenol on the Biochemical Characteristics of 488
Activated Sludge
82 Concentration-Time Profiles of Carbon Dioxide in Exit Stream at 490
Different Bulk DNP Concentrations
83 Effect of Bulk DNP Concentration on Cell Growth 490
84 Inhibition of Casamino Acid Oxidation by 2,4-Dinitrophenol 492
xix
-------
XX
-------
EXECUTIVE SUMMARY
This report considers the large number of chemicals which contain at
least one nitro substituent on an" aromatic ring. Approximately 250-300 chemi-
cals are listed as commercial nitroaromatic compounds. However, only about
40 compounds are produced or consumed annually in quantities over 500,000 pounds
and perhaps another 50-100 compounds exceed 100,000 pounds. Nitroaromatic
compounds are used as pesticides, perfumes, explosives, and chemical intermedi-
ates. This report focuses upon the non-pesticidal nitroaromatics.
Because of the large number of compounds considered in this report, com-
prehensive information on individual compounds could not be developed. However,
adequate information is available to provide priorities for further study and
research. Production volume, uses, environmental fate, monitoring, and bio-
logical effects were considered. In general, nitroaromatic compounds appear
to be fairly persistent and exhibit either hematologic or metabolic effects
at high levels of exposure. Most of the large-volume nitroaromatics have not
been screened for carcinogenic, mutagenic, or teratogenic effects. Nevertheless,
the following compounds appear to have high contamination potential: nitrobenzene
(655 million Ibs/year; detected in drinking water); 2,4- (and 2,6-) dinitrotoluene
(471 million Ibs/year; detected in drinking water); 2,4,6-trinitrotoluene (TNT)
(432 million Ibs/year; demonstrated to have considerable pollution problems;
not detected in drinking water; may be biodegradable); o-, m-, p_-chloronitro-
benzene (60, 110, and 8 million Ibs/year, respectively; persistent; meta-
isomer detected in drinking water); and 1,3-dinitrobenzene (12 million Ibs/year;
persistent; detected in drinking water). For a more detailed list of chemicals,
see the Summary and Conclusions in Section V, p. 499.
xxi
-------
xxii
-------
Investigation of Selected Potential Environmental Contaminants:
Nitroaromatic Compounds
Introduction
This report reviews the environmental hazard involved in the commercial use
of an important and large group of chemicals, nitroaromatics. Information on
chemical and physical properties, production, uses, environmental contamination
potential, and biological effects of the compounds is reviewed. The group in-
cludes any compound that has an aromatic system with a nitro group directly attached
to the aromatic ring. Most of the compounds are substituted benzenes, but naph-
thalene and other aromatic systems are frequently encountered. For the most part,
nitroaromatics have direct uses or are used as chemical intermediates in the ex-
plosive, dye, pigment, pharmaceutical, rubber, pesticide, and perfume industries.
Because of the large number of compounds considered in this report, only
limited information on any individual chemical can be presented. For this reason,
pesticide nitroaromatic compounds have not been as thoroughly treated as they could
be from the available information. The degree of detail for the other chemicals
was frequently dependent upon the commercial importance of the compounds, although
the availability of information was often a major factor.
The nitroaromatic compounds that are produced in commercial quantities
($1,000 or a 1,000 Ibs per year) are listed with the name of the producer(s) in
the Chemical Index. Other nitroaromatics that do not appear to be commercial
products but for which information is available are also listed in the Appendix,
and that appendix can be used as an index to information on individual compounds.
-------
I. Physical and Chemical Data
A. Structure and Properties
1. Chemical Structure
Nitroaromatic compounds have at least one nitro substituent
attached to an aromatic ring. The nitrogen and two oxygens of the nitro group
are kept a large percentage of the time in the same plane as the aromatic ring
2
by conjugation between the sp orbitals of the ring and the nitro group (see
Figure 1).
Figure 1. Conjugative Interaction Between Aromatic Systems and Nitro
Functional Groups
This conjugative interaction between the nitro substituent and the aromatic ring
has considerable impact on the chemical reactions that the compound may undergo,
and affects the electron (UV) spectra of the chemicals (See Section I-B, p. 21
+ ^ 0~
for examples). The nitro functional group is polar and electrophilic (-N^ ),
and this results in inductive withdrawal of electrons from the ring through the
a bond (deactivates the ring to electrophilic substitution) and a high dipole
moment.
-------
2. Physical Properties
The physical properties of 96 important nitroaromatic
compounds are presented in Table 1. The compounds in the table were selected
because (1) production data were available or (2) environmental fate or biological
effects data were available and the compound was listed as a commercial product.
The data presented in Table 1 illustrate that the physical properties are very
dependent upon the type, number, and position of the substituents.
In general, nitroaromatic compounds are not very soluble in
water. Water solubility usually decreases with increasing nitro substitution.
Other substituents, like chlorine groups, can have considerable effects upon the
water solubility. For example, Eckert (1962) has shown that the water solubility
of chloronitrobenzenes decreases with an increase in the degree of chlorination
(See Table 2). In contrast, groups such as amino, hydroxyl, carboxyl, etc. in-
crease the water solubility of nitroaromatic compounds.
Table 2. Water Solubility of Chlorine Substituted Nitrobenzenes
(Eckert, 1962)
Water Solubility
. Compound # CSL yM/liter. 20°C
Nitrobenzene 0 15,100
4-Chloronitrobenzene 1 • 2,877
2-Chloronitrobenzene 1 2,800
3-Chloronitrobenzene 1 1 732
l-Chloro-2,4-dinitrobenzene 1
l,2-Dichloro-4-nitrobenzene 2 ^•/f,>
l,4-Dichloro-2-nitrobenzene 2 48Q
l,2,4,5-Tetrachloro-3-nitrobenzene 4 g
Pentachloronitrobenzene 5
-------
Table 1. Physical Properties of Significant Nitroaromatic Chemicals
Compound
Synonym
Chemical
Registry
Formula
Structure
2-Amino-4-nitro- 4-Amino-4' -nitro-
phenol 2,2' -st ilbene-
disulfonic acid
2-hydroxy-5-
nitroaniline
4-nitro-2-amino-
1-hydroxy benzene
Abstract
No. 99-57-0
W2°3 C14H12N2°8S2
— u HOT.
2-Bromo-4,6-
dlnitroanillne
6-bromo-2 ,4-
dinitroaniline
1817-73-8
C6H43rN3°4
W«4
AIC^^ _•*
2-sec-Buty 1-4,6-
dlnitrophenol
4 , 6-dlnitro-o-sec-
butylphenol
2,4-dinltro-6-sec-
butylphenol
DNOSBP
DNSBP
DtlBP
88-85-7
C10H12N2°5
aH CM<«
on *-«5
. .. -A. lufu rU-
6-tert-Butyl-3-
methy 1-2,4-
dinitroanisole
4-tert-butyl-3-
• methoxy-2 , 6-
dinitro toluene
Musk ambrette
83-66-9
C12H16N2°5
?'"'
l-Chloro-2,4-
dlnltrobenzene
2,4-dinitro-l-
chlorobenzene
DNCB
97-00-7
C6H3C1N204
<*
4-Chloro-3-
nitroanillne
3-nitro-
chloraniline
635-22-3
C6H5C1N202
WM,
2-Chloro-4-
nitroaniline
l-amlno-2-chloro-
4-nltrobenzene
o-chloro-£-
nitroanillne
OCPNA
121-S7-9
C6H5C1N202
N«a.
Ho.
Molecular
Weight
Melting
Point (°C)
Boiling
Point (°C)
Specific Gravity
or Density
Water Solubility
(g/100E)
Vapor Pressure
(mmHg)
, Octanol-Water
Partition
Coefficient
(log of)
154.12
80-90
(1 mol. H.O)
143
anhydrous
Ultra-
^lolet ,
SPectral
Xmax 373, 308
cmax 4250, 4780
400.24
153-4
241.11
0.073
(250)-25°
336, 268, 229
12000, 8700, 10100
268
265
202.56
172.58
53.4 (alpha, stable)
43 (beta) 102-3
27 (gamma)
315
Insoluble
206, 238
172.58
104-5
-------
Table 1. Physical Properties of Significant Nitroaromatic Chemicals (Cont'd.)
Compound 4-Chloro-2-
nicroaniline
Synonym l-amino-4-chloro-
2-nltrobenzene
£-chloro-o-nitro-
aniline
Azoic dlazo
component 9
CI #37040
Chemical Abstract
Registry No. 89-63-4
Formula C6H5C1N202
Structure
w#t
$T
a
Molecular 172.58
Weight
Melting 116-7
Point (°C)
Boiling
Point (°C)
Specific Gravity
or Density
Water Solubility
(g/lOOg)
Vapor Pressure
(mmHg)
Oc tanol-Water
Partition
Coefficient
(log of)
Ultra-
violel; Ana* 234.5
Spectral ena*
Data
l-Chloro-2-
nitrobenzene
chloro-£-
nitrobenzene
o-nitrochloro-
benzene
ONCB
o-nitrophenyl
chloride
88-73-3
C5H4C1N02
'fr
157.6
35.5
32.2 (HCP)
245
1.368
10 ran Hg
(230°F)
2.24
l-Chloro-3- l-Chloro-4- 4-Chloro-3-nitro-
nitrobenzene nitrobenzene benzenesulfonamlde
m-nitrochloro- ^-chloronitro- 4-chloro-3-nitro-
benzene benzene sulfamylbenzene
l-nitro-4- l-chloro-2-nitro-4-
chlorobenzene aulfonamidobenzene
PNCB c— nitrochlorobenzene
£-nltrophenyl sulfooamide
chloride Yellow sulfone
121-73-3 100-00-5 97-09-6
C6H4C1N02 C6H4C1N02 CgHjClN^S
CJL fJL fO,WM»
p.... p ^U. .
X
157.6 157.6 236.4
24 (unstable) 83 172-3
44
235-6 242
1.534 1.520
2.41 2.41
257, 206 270 292, 222.5
814, 20300
2-Chloro-5-nitro-
benzenesulf onic
acid
p_-nitrochlorobenzi
o-sulfonic acid
2-chloro-5-nitro-
sulfobenzene
C.H.C1NO.S
6 4 - 5
soj*
JP"
N^^<*^
L
237.43
2-Chloro-4-
nltrobenzenesulfonlc
acid
4-Chloro-3-nitro-
be nzenesulfonic
acid
237.43
237.43
72
-------
Table 1. Physical Properties of Significant Nitroaromatic Chemicals (Cont'd.)
Compound
Synonym
2-Chloro-5-nit.ro-
benzenesulfonic
acid, sodium salt
4-Chloro~3-nitro-
benzenesulfonv I
«^hloride
2-Chloro-4-
nitrobenzoic
• acid
£-(4-Chloro-3-
ni' obenzoyl)
benzoic acid
.'-carboxy-4'-
chloro-3"nitro-
ta en zophen one
2-Chloro-4-nitro- 2-Chloro-6- 4-Chloro-2- A-Chloro-3- 2,6-Dichloro-i-
toluene nitrotoluene nitrotoluene nitrotoluene nitroaniline
c>-chloru-j>-
nitrotoluene
l-amino-2,6-
dichloro-4-
nitrobenzene
Chemical Abstract
Registry No. 946-30-5
Formula C.
Structure
97-08-5
99-60-5
85-54-1
121-86-8
83-42-1
89-59-8 89-60-1
C.H..C. ..,
/ o Z 762
99-30-9
Molecular
Weight
Melting
Point (°C)
Boiling
Point (°C).
Specific Gravity
or Density
Water Solubility
(g/lOOg)
Vapor Pressure
(mmHg)
Octanol-Water
Partition
Coefficient
(log of)
259.6
Dltra-
violet
Xmax
345, 222
201.57
140
139-141
262, 209
271.23
171.58
65
171,58
37
236-8
171. 58
37
239.5-240
(215.5)
171.58
7
304, 254
248
1,2559
300, 251
298, 250
207.03
245, 350
-------
Table 1. Physical Properties of Significant Nitroaromatic Chemicals (Cont'd.)
Compound
1 , 2-Dichloro-4- 1 , 4-Dichloro-2- 2 , 5-Dichloro-3- 2 , A-Dichlorophenyl-
nitrobenzene
Synonym
Chemical Abstract
Registry No. 99-54-7
Formula C6H3C12N°2
Structure
($"'
NO,.
Molecular 192.0
Weight
Melting 43
Point (°C)
Boiling 255-6
Point (°C)
Specific Gravity 1.4558
or Density
Water Solubility insoluble
(g/lOOg)
Vapor Pressure
(nmHg)
Octanol-Water
Partition
Coefficient
(log of)
Ultra-
*10let , Ana* 266, 212.5
Sl>ectral Enax
Data EnaX
nitrobenzene nitrobenzoic - 4-nitrophenyl
acid . ether
2,5-dichloro- Dichloronitro- Nitrofen
nitrobenzene benzoic acid, NIP
isomerlc
mixture
89-61-2 88-86-8 1836-75-5
C6H3C12N02 C7H3C12N04 ^2H7C12"°3
C*- CO>H ci
$r jte^^
ci
192.0 236.02 284.1
56 71-2
266
1.669
299, 219.7 293
293
0,O-Diethyl-j>T
nitrophenyl-
phosphorothloate
0,0-Dlmethyl-£- 2,4-Dlnitro-
nitrophenyl- aniline
phosphoro thloate
£-(2,4-Dlnltro-
anilino) phenol
0,0,diethyl-0-(p- 0,0-dlmethyl-£- l-amino-2,4-
nitrophenyl) thiono- (j-nitrophenyl) dinitrobenzene
phosphate thionophosphate
Ethyl parathion Methyl parathion
Paridol
MPT
56-38-2
298-00-0
263.2
97-02-9
119-15-3
C12H9N3°5
336, 257, 225 224
16600
-------
Table 1. Physical Properties of Significant Nitroaromatic Chemicals (Cont'd.)
Compound 2,4-Dinicro- 3',4-Dinitro- 1,.3-Dinitro- 4,4'-Dinitro- Dlnitrobutyl- Dinitrocaprylphenyl 4,6-Dinitro-o- 2,4-Dlnitro-=- 2,4-Dinitro-
anisole benzanilide benzene biphenyl phenol, crotonate cresol naphthol phenol
ammonium salt
Synonym l-methoxy-2,4- 3'-nitro-N-(4- m-dinitro-
dlnltrobenzene nitrophenyl) benzene
benzamide
2-(l-methylhepCyl)- DNC, DNOC 2,4-dinitro- «-dinitro-
4,6-dlnitrophenyl 2-methyl-3,5- 1-naphthol phenol
crotonate dlnicrophenol Martlus yellow l-hydroxy-2,4-
Karathane, Dlnocap 4,6-dinitro-2- dlnitrobenzene
hyd roxytoluene
00
Chemical Abstract
Registry No. 119-27-7
Formula
Structure
Molecular
Weight
Melting
Point (°C)
95.5-6.0
287.2
223-4
99-65-0
1528-74-1
39300-45-3
534-52-1
C7H5N205
168.1
89.75
244.2
234-5
364,4
138-40
6.05 mm.
198.1
85, 85.8
605-69-6
234.2
51-28-5
184.11
114
Boiling
Point (°C)
Specific Gravity
or Density
Water Solubility
(g/100g)
Vapor Pressure
(mmHg)
Octanol-Water
Partition
Coefficient
(log of)
Ultra-
violet .
Spectral im""
Data «
29°- 251-5, 213. ^66. .'35
10900, 7430, 14000
302
1.571
0.05 (15°C)
0.32 (100°C)
1.49
305
25900
0.013 (15°)
105 x 10"
(25*C)
1.683
0.6 (25°)
0.02 (12.5°)
1.32 (100°)
1.51
263
-------
Table 1. Physical Properties of Significant Nitroaromatic Chemicals (Cont'd.)
Compound 4,4' -Dinitro- 3 , 5-Dini tro- 2 , 4-Dinitro- 2 , 4- (and 2,6-) l-Fluoro-2 , 4- £-Fluoronitro-
st ilbene-2 , 2 ' - toluamide toluene Dinit rotoluene dinitrobenzene benzene
disulfonic
acid
Synonym 2 , 2' - (1 ,2- 2-methyl-3 , 5- 2 ,4-dinitrotoluol 2 ,4-dinitro-l- 1-f luoro-4-
ethanediyl) dinitro- l-methyl-2,4- f luorobenzene nitrobenzene
bis [5=nitro] benzamide dinitrobenzene
benzenesul- Zoalene
fonic acid
Chemical Abstract
Registry No. 128-42-7 148-01-6 121-14-2 121-14-2 70-34-8 350-46-9
606-20-2
Formula WuPfu?! C^O, . W^ CJifft ^^2\ V«"°2
Structure c^ 0 CHj CJ», F p
**,»»*?. °^(^^ rAr"0> ^(r^** ifV'0* ^ &
V^O-^-O^M' 'yj ]©j v o^*c
^ — ' ^ — ' w» wo,. °j I NO^ ]£^
Molecular 430.4 225.13 182.1 182.1 186.1 141.0
Weight
Melting 69.5-70.5
Point (°C) 71-2
Boiling decomposes at 300
Point (°C)
Specific Gravity 1.521
or Density
Water Solubility 0.027g/100ml (22°)
(g/lOOg)
Vapor Pressure
(mmHg)
Octanol-Water
Partition
Coefficient
(log of)
2,2' ,4,4' ,6,6'- 4-(Hethylsul£onyl)-
Hexanitrostilbene 2,6-dinitro-N,N-
dipropylaniline
Nitralin
20062-22-0 4726-14-1
C14H6N6°12 C13H19N3°6S
vfcwti
_>»»» V} — . oaw^xSyWok
^C/»=«/T-O[[^II^ LL^J
"Vfot ojt 5oAc«,
450.2 345.4
0.6
, -6
1.5 x 10
(25°)
-------
Table 1. Physical Properties of Significant Nitroaromatic Chemicals (Cont'd.)
Compound
3'-Nitroacetanilide 3*-NItro- m-Nitroaniline o-Nltroan 1line
acetophenone
p-Nitroaniline 4-Nitro-o-
anisidine
5-Nitro-o-
anisldine
Synonym mracetylamino-
nitrobenzene
N-acetyl-ja-
nitroaniline
N-(ja-nitrophenyl)
acetamide
ffi-Nitroacetanillde
Chemical Abstract
Registry No. 122-28-1
Formula
121-89-1
3-nitroaniline l-amlno-2-
nttrobenzene
2-nltroaniline
Azoic diazo
c omponent 6
2-nicrobenzenamine
99-09-2
88-7A-4
l-smino-4- 2-amino-5-
nitrobenzene nitroanlsole
4-nitroanillne 2-methoxy-4-
nitroanillne
Azoic diazo
100-01-6
"2W3
2-amino-4-
nitroanisole
2-methoxy-5-
nltroanillne
2-anisid±ne nitrate
component 5 (salt) Azoic diazo component
CI #37125 (salt) 13 (salt)
CI #37130 (salt)
97-52-9 99-59-2
C7B8N2°3 C7H8N2°3
Structure
NO.
Molecular 180.0
Weight
Melting.
Point (°C)
Boiling
Point (°C)
Specific Gravity
or Density
Water Solubility -
(g/lOOg)
Vapor Pressure
(mmHg)
Octanol-Water
Partition
Coefficient
(log of)
Ultra-
vlolec . Imax
165 138.12 138.12 138.12 168.2
76-8 114 71-2 146-8 139-140
286 270 with 200"C
decomposition (10 mmHg)
1.437
0.11 0.1 0.08
1.46 1.79 1.19
225.5 371, 233 277, 232 388,
168.2
Spectral
Data
1091, 12000
14700, 5580
374, 303, 257, 227
3900, 5300, 14200, 11300
-------
Table 1. Physical Properties of Significant Nitroaromatic Chemicals (Cont'd.)
Compound
-Nitroanisole £-Nitroanlsole £-Nitroben- Nitrobenzene m-Nitrobenzoic o-Mitrobenzoic j>-Nltrobenzoic
zaldehyde acid acid acid
0- and £-Nitro- nr-Nitrobenzoic
benzole acids acid, sodium
salt
Synonym 2-nitroanisole 4-nltroanisole
2-methoxynitro- 4-methoxynitro-
benzene benzene
3-nitrobenzoic 2-nitrobenzoic 4-nltrobenzoic
ac id acid 4-ni trod racy lie
acid
Chemical Abstract
Registry No. 91-23-6
100-17-*
555-16-8
98-95-3
552-16-9
62-23-7
C7
54
827-95-2
C7H5H04
co^H
CO^Ne.
Molecular 153. 1
Weight
Melting 9.4
Point (°C)
Boiling 272.3
Point CO
Specific Gravity
or Density
Water Solubility 0.17
(g/lOOg) (30^
Vapor Pressure
(mmHg)
Octanol-Water
Partition
Coefficient
(log of)
Ultra-
violet ,2 •
, A max jA-i. . --'j. -
SP««al emax
Data
153.1 151.12 123.1 167.1 167.1 167.1 167.1
54 42-44 5.1 141.4 147.5 242.4
(144-5)
274 153 210.9
(23 mmHg)
1.203
0.06 . 0.23 0.1
(30°)
1 (44°C)
10 (85.4°C)
1.85 J.31 1.85
i . J04 *ib- 23y .>!>. 4 ^59
10600 7180
189
>300
9fc A
ZDU,
7870
-------
Table 1. Physical Properties of Significant Nitroaromatic Chemicals (Cont'd.)
Compound
Synonym
Chemical Abstract
Registry No. 86-00-0
Formula
Structure
Molecular 199.21
Weight
Melting
Point (°C)
Boiling 325
Point (°C)
Specific Gravity
or Density
Water Solubility
(g/lOOg)
Vapor Pressure
(nmHg)
Octanol-Water
Partition
Coefficient
(log of)
Ultra-
violet Xmax 231
Spectral emax
Data
2-Nitro-£- 1-Nitro- 3-Nitro-l,5- 7- (and 8-)
cresol naphthalene ' naphthalene- Nltronaph (1,
disulfonic 2-d)(l,2,3)
acid oxadiazole-5-
sulfonic acid
nitro casella 7-nitrodiazo
acid acid
119-33-5 86-57-7 117-86-2
OH jflu SOjH
153.1 173.2 333.3 295.2
57.8
304
276, 214 J31 . 212.5
o-Nitrophenol £-Hitrophenol 4'-(p-Nitro- 2-Nitro-£-
phenyl) aceto- phenylene-
phenone dlamine
2-hydroxynitro- 4-amlno-2-
benzene nitroaniline
l,4-diamlno-2-
nitro benzene
88-75-5 100-02-7 135-69-3 5307-14-2
C6H5H03 C6H5N03 C14B11N°3 Wft
., aU MM
J-39-1 139.1 241 153.1
44-45 n4 220
214-216 279,
decomposes
1.2942 L47,
0.2 0.804 (15°)
1.6 (25°)
29.1 (90°)
1 mm. Hg
(49.3°)
1-79 1.91
345, 272 3K 530, 340, 287, 275, 216 441 231
3330, 6300
-------
Table 1. Physical Properties of Significant Nitroaromatic Chemicals (Cont'd.)
Compound 4-Nitro-o- 4-Nitrostilbene 3-Nitrotoluene 2-Nitrotoluene 4-Nitrotoluene i-Nitro-p_- 4-Nitro-o-toluene- 3-Nltro-p_-
phenylene- toluenesulfonlc sulfonic acid toluidlne
diamine acld
4-Nltro-o-
toluldtne
Synonym
u>
2-amino-4-
nitroaniline
1,2-diamlno-4-
nitrobenzene
Chemical Abstract
Registry No. 99-56-9
Formula
Structure
4003-94-5
m-nitrotoluene
3-methylnitro-
benzene
3-nitrotoluol
l-methyl-3-
nltrobenzene
2-methylnitro- 4-raethylnItro-
l-methyl-4-nitro- 4-amino-3-
benzene
l-methyl-2-
nitrobenzene
o-nitrotoluene
88-72-2
benzene
l-methyl-4-
nitrobenzene
4-nitrotoluol
£-nitrotoluene
99-99-0
97-06-3
2-sulfobenzene
4-nltro-2-sulfo-
toluene
p—nitro-toluene-
o-sulfonic acid
121-03-9
nitrotoluene
4-methyl-2-
nitroaniline
3-nitro-4-
toluidine
2-amino-4-
nitrotoluene
l-amino-2-methyl-
5-n1trobenzene
2-methyl-5-nitro-
aniline
Azoic dlazo compo- 4-nitro-2-
nent 8 (salt)
CI $37110 (salt)
119-32-4
toluidine
99-52-5
Molecular 153.1
Weight
Melting 201-2
Point (°C)
Boiling
Point (°C)
Specific Gravity
or Density
Water Solubility
(g/lOOg)
Vapor Pressure
(nnnHg)
Octanol-Water
Partition
Coefficient
(log of)
226.3
137.1
Ultra-
violet
• Spectral
Data
,,..
-63
16.1
232.6
1.1618
0.0498
(30°)
-10. 5-,
-4. IB
(stable)
221.7
1.1643
0.0652
(30°C)
51.9
238.3
1.1226
0.004g/100ml
(15°)
1 (50"C)
265
7700
1 mm Hg 50°C 1 (53.7°C)
13 100.2
30 119.2
2.37
2.30
264
217.2
133.5 anydrous,
152.15
77-8
152.15
129-132
279.5, 228
225
-------
Table 1. Physical Properties of Significant Nitroaromatic Chemicals (Cont'd.)
Compound 5-Nitro-o-
toluidine
Synonym 2-amino-5-
nitrotoluene
4-amino-3-methyl'
nitrobenzene
2-methyl-A-
nitroaniline
5-nitro-2-
toluidine
Chemical Abstract
Registry No. 99-55-8
Formula C^gN^
Structure
V^
Molecular 152.15
Weight
Helting
Point (°C)
Boiling
Point (°C)
Specific Gravity
or Density
Water Solubility
(g/lOOg)
Vapor Pressure
(mrnHg)
Octanol-Water
Partition
Coefficient
(log of)
Ultra-
violet . --.,
, Xmax 227
Spectral
!. emax
Data
Nltroxylenes Pentachloro- Picric acid
nitrobenzene
Terrachlor carbazotic acid
2-hyuroxy-l,3,5-
trinl robenzene
nitroxa thic acid
phenol rinitrate
plcroni ric acid
2,4,6-t initrophenol
82-68-8 88-89-1
C8HgN02 C6C15N02 C(,H3N307
/CH\ HOi. • on
Ci. W0t
151.2 295.3 229.1
146.5 122.5
decomposes
above 300
1.763
1.4 (20°C)
6.8 (100°C)
2mm. Hg 195°C
50mm. Hg 255T
301, 212.5 352
635, 72900
1,2,4,5-Tetrachloro- 2,3,4,6-Tetra- Tetryl
Tecnazene l-amino-2,3,4,6- N-methyl-N,2,4-
2,3,5,6-Tetrachloro- tetrauitrobenzene tetranitroaniline
1-nit robenzene N-methyl-N-nitro-2,4,
6-trinitroaniline
oethyl 2,4,6-trinitro-
phenylnitramine
117-18-0 3698-54-2 479-45-8
C6HC14N02 C6H3N5°8 C7H5H5°8
C(i yo
NOj NJf» if ""
DgC ^:: "i^r
260.9 273.1 287.15
99 (2,3,5,6) 129.45 vith slight
65-7 (2,3,4,5-) decomposition
128.75 (military)
0.005 (0°C)
0.0195 (50°C)
0.184 I'jOO'C)
(2,3,5,6-) (2,3,4,5-)
296, 208.5 205 224
1100, 53400 44000 2280
-------
Table 1. Physical Properties of Significant Nitroaromatic Chemicals (Cont'd.)
Compound 1.2, 4-Tr ichloro- .
5-nitrobenzene
Synonym
Chemical Abstract
Registry No. 89-69-0
Formula ^6H2^"3^2
Structure
MOL
&" v
« >^
CL
Molecular 226. 4
Weight
Melting 57
Point ,(°C)
Boiling
Point (°C)
Specific Gravity
or Density
Water Solubility
(g/100g)
Vapor Pressure
(mmHg)
Octanol-Water
Partition
Coefficient
(log of)
Ultra-.
violet Xmax 305, 260, 22.'.
Spectral
Data Ema*
-,-,«-Trlfluoro- 2,4,6-Trinitro- 2,4,6-Trinitro-
2,6-dinitro-N, benzenesulfonic resorcinol
N-dlpropyl-p- acid.
toluidine
Trifluralin picryl sulfonic styphnic acid
Treflan acid 2,4-dihydroxy-l,
3,5-trinitro-
benzene
1582-09-8 2508-19-2 82-71-3
C13H16F3N3°4 C6H3N3°9S C6H3N3°8
CFj NO, NO,
^Y^ ojt^^s^NOi airX-x^wo^
NfcH^CH^CHj)^ OH
335.3 293.2 245.1
48.5-49 175.5 (180°)
96-7 sublimes
(0.18 mm Hg)
1 ppm ' 0.45 (15°C)
0.57 (20'C)
0.68 (25°C)
1.99 x 10"4 1-136 (62°C)
(24.5")
192, 336. 265, 208
9780. 8820. 12500
2,4,6-Trinltro-
toluene
«-trinitro-
toluene
TNT
118-96-7
C7H5N306
e*i
y0rNOi
wo^
227.1
82 (80.2)
explodes 240
(varies)
0.013
0.053 (85'C)
-------
The melting and boiling points generally increase with the number
of nitro groups, as does the thermal instability of the compounds. The polynitro-
aromatic compounds frequently decompose before they melt.
The reported vapor pressures of nitroaromatic compounds are listed
in Table 1. Lenchitz and Velicky (1970) used the measured vapor pressure of
three nitrotoluenes to calculate the heats of sublimation. They found that
the change in the heat of sublimation was not equal for each successive nitro
group substitution. They attributed this result to the high polarity of the i
nitro group, which influences inter- and intramolecular forces.
3. Principal Contaminants and Specifications of Commercial Products
Nitroaromatic compounds are produced by such processes as nitration,
sulfonation, chlorination, reduction, and oxidation. Whenever a new substituent is
added to the ring, a number of isomers are usually formed. For example, mononitration
of toluene will yield approximately 62-63% £-nitrotoluene, 3-4% m-nitrotoluene, and
33-34% p-nitrotoluene, depending upon the -nitration conditions (Matsuguma, 1967a).
The intended use of the compound determines the degree of purification. For example,
dinitrotoluene is available from Du Pont in five different grades: (1) Dinitrotoluene
Mixture Technical (ratio between 2,4- and 2,6-isomers is about 80:20), (2) Dinitro-
toluene Mixture Blend G Technical (isotner ratio 3 to 2), (3) Dinitrotoluene Oil
Technical (isomer ratio 1 to 1), (4) Dinitrotoluene Oil 26° Technical (isomer ratio
1 to 1), and (5) 2,4-Dinitrotoluene Technical (high percentage of 2,4-isomer) (See
Table 6, p. 20). In contrast, 2,4,6-trinitrotoluene (TNT) must be substantially free
from other unsymmetrical isomers because the unsymmetrical isomers explode randomly
(Nay, 1972). Frequently, the final product requires more than one synthesis step,
and, as a result, the contaminant in the final product may have been formed several
16
-------
steps before the final synthesis step. The similarity between the physical prop-
erties of the isomers frequently makes complete purification economically infeasible.
Other contaminants besides related isomers may be found in the
final product due to by-product formation, retention of the reagents, or unreacted
starting material. For example, nitrobenzoic acids and tetranitromethane may be
formed as by-products from the nitration of toluene (Matsuguma, 1967a); some of the
mixed acid (sulfuric and nitric) used in nitration steps may be retained, and some
iron or sulfur might remain when reduction is the final synthesis step. However,
these contaminants are usually found in very low concentration because their physical
properties allow easy removal.
Several researchers have analyzed commercial grades of nitroaromatics
to determine their purity. Tucker and Schwartz (1971) found that certain defects in
oxidation hair colors could be traced to impurities in the chemical intermediate.
They analyzed a number of chemical intermediates before and after purification (by
recrystallization) (See Table 3), but did not attempt to identify the organic con-
taminants (However, infrared spectra of the sludges from recrystallization were
given).
Venturella e_t al. (1973) developed a gas chromatographic method
which they used to determine the purity of crude samples of a variety of nitroaro-
matic compounds. Their results are listed in Table 4. A similar GC study of raw
and crude TNT by Gehring and Shirk (1967) identified the concentrations of impurities
(See Table 5).
17
-------
Table 3. Analysis of Several Nitroaromatic Chemical Intermediates Before and After Recrystallization
(Tucker and Schwartz, 1971)
00
Comp ound
Ni tro- o-pheny lenediamine
Raw
Purified
Ni t r o-p-pheny lene di amine
Raw
Purified
4-Amino-2-nitrophenol
Raw
Purified
2-Amino-5-nitrophenol
Raw
Purified
2-Amino-A-nitrophenol
Raw (11.6% water)
Purified
Purity
(%)
95.4
99.5
90.1
98.1
95.1
99.4
95.0
98.8
75.0
97.9
Ash
(%)
0.20
0.025
0.20
Trace
0.82
Trace
0.39
0.02
0.06
0.07
Iron
(ppm)
178
36
363
Trace
382
Trace
28
34
230
69
Melting
Point
199.4
202.5
134.0
143.6
130
136
186
201
93.0 (hydrate
110.0 (hydrate
-------
Table 4. Crude Sample Analyses
(Venturella e± al., 1973)
Compound
2,6-Dichloro-4-nitroaniline
2-Chloro-4,6-dinitroaniline
2-Bromo-4,6-dinitroaniline
3-Nitro-4-chloroaniline
2-Chloro-5-nitroaniline
2-Nitro-4-chloroaniline
2,5-Dichloro-4-nitroaniline
2-Amino-5-nitroanisole
2-Amino-4-nitroanisole
3-Nitro-4-aminoanisole
2-Nitro-5-chloroanisole
% Purity
86.0
90.47
98.33
93.56
92.75
100.3
87.25
94.65
97.52
90.93
98.51
Table 5. Percentage Concentration of Impurities in Typical Samples
of Production TNT (2,4,6-Trinitrotoluene)
(Gehring and Shirk, 1967)
Crude TNT
Refined TNT
2,5-DNT
Nitrotoluene Impurities (wt %)
2.4-DNT 3.5-DNT 2.3.5-TNT 2.4,5-TNT 2.3.4-TNT
0.02-0.05 0.40-0.81 0.02-0.03
0.02-0.04 0.25-0.40
- 0.02 1.35-2.22 0.80-1.30
0.34-0.45 0.30-0.42
Table 6 provides information on a number of commercial products
offered by the Du Pont Company. Both the sales specifications and contaminants are
noted; these are typical of the information available on the purity of the commercial
nitroaromatics.
19
-------
Table 6. Sales Specifications and Contaminants in Technical Grades of
Nitroaromatic Compounds
Compound
Nitrobenzene
o-Nitrotoluene ,
technical
p_-Nitrotoluene ,
technical
2-Chloro-5-ni trobenzene-
sulfontc acid , sodium
salt , technical
2 ,4-Olnit rotoluene ,
technical
Specifications
wt Z mln. 99.8
distill, range °C max - 0.8
(first drop to 95%, incl. 210. 8°C)
freezing point, "C min. 5.1
color - yellow
distill, range *C max - 0.5
(5 - 95%, Incl. 222. 0°C)
freezing point, *C mln. 51.2
rain, purity 40. 0%
max impurities insoluble
in water - 0.15 wt%
solid state - light yellow to buff
crystals, 90% pass 2 mm screen
molten state - crystalline solid below
68.5°C
Contaminants
dinitrobenzene, wt% max 0.1Z
nl trothiophene - none
water, wt% max - 0.1%
acidity (cacld. as HN03), wt% max - 0.001
pj- and m-nltrotoluene - 0.5%
water, wtZ max - 0.2%
ro-nitrotoluene - 0.3%
o-nitrotoluene - 0.3%
water, wt% - 0.3%
acid free
water, wt% - 50%
sulfuric acid and sodium sulfate - 5%
p_-chloronltrobenzene - traces
water, wtZ solid - 0.25%
molten - none
max acid content (as H2S(V " 0<005 wt
Reference
Du Pont (1974)
Du Pont (1965L)
Du Pont (19G6c)
Du Pont (1965b)
Du Pont U965a)
Dinit rotoluene mixture ,
technical
!>ini trotolucne oil.
technical
t) lend 0, technical
Dini t rotoluene
oil 26°, technical
2,4 - isoroer -
2,6 - isomer -
freezing point.
2,4 - isomer -
2,6 - isomer
freezing point
nitrogen content
2,6 - Isomer
freezing point,
2,4 - la one r
2,6 - isonjer, -
freezing point
nitrogen content
80 + 1%
20 + 1%
mln. - 56.0°C
50%
50%
30.0 + 5.0°C
, min. - 15.15%
40%
max 44.0°C
50%
50%
26.0 + 3.0°C
, mln - 15.15%
o- or £-dinitro isomers - 5%
water, wt% -
0.
acid or alkali
acidity, max
t t%
'
idi
y,
d
51
- none
is H SO^) - 0.052
53E
2 4
Uu Pont (196f.ii)
Uu Pont (1970b)
no odor of o-nitrotoluene
acidity, max
water, wtZ -
(/
0
as H SO.) - 0.05Z
.SI
Du Pont (1970o)
VNitro-4-arninoanisole,
technical
o-Nitroanisole, technical
£-Ni tro;jnisole, technical
freezing point, rain. 122.8°C
purity, mln. - 99.0% by wt
light transmittance, mln. 50%
solution in alcohol at 525 nm - 40Z
freezing point, min. 10.0"C
jo-chloronitrobenrene - trace
nitrophenole, max - 0.01% by wt
water, max - 0.5% by wt
freezing point, min. 51.0°C
P_-nitrophenol - trace
chloronitrobenzene - trace
water, max - 0.5% by wt
i'u Pnnt (1966b)
Uu Pont (1966f)
I)u Pont (1966f)
p-Nitrobrnzoic acid,
technical
purlty, min. - 99.5%
min. light transmission of 6.3% solution
in aqueous alcohol - NaOH -, 75Z
£-nitrotoluene - trace
mineral acids - trace
water, max - 0.25%
o-NItrochlorobenzene,
technical
p~N(trochlorobenzene,
technical
freezing point, min. 31.8°C
matter insoluble in alcohol,
max -.0.2%
m~ or £-chlorbnltrobenzene - 1%
m- or £-chloronitrobenzene - trace
water, max - 0.1%
Du Pont (19711.)
Du Pont QJ,971c)
purity - 99.5%
freezing point, min. 82.5°C
£-chloronltrobenzene - trace
water, max - 0.15%
Du PonL (1966g)
p-NitrophenoJ, technical
purity, min. - 99.5% by wt
melting point 111 - 114CC
water - 1.0%
ash, max - 0.10%
iron, max - 25 ppm
p_-Nitro Sodium Phenolate,
technical
purity, min. - 76.0% by wt
optical density of chloroform
extract at 400 nm - 0.10%
material insoluble in hot water - 0.05%
sodium chloride
£~chloronltrobenzene - trace
Du Pont (1971ji)
Du Pont (1966d)
20
-------
B. Chemistry
1. Reactions Involved in Uses
Historically, the first commercial uses of nitroaromatic
compounds were as chemical intermediates for the dye and pigment industry. Much
of the chemistry was developed in the late 1800's (e.g., Aniline was first
commercially manufactured from nitrobenzene in 1874; Kouris and Northcott, 1963).
Presently, the chemical intermediate uses of nitroaromatics for rubber chemicals,
photographic chemicals, and drugs are similar in commercial importance to the
applications in dyes and pigments. The thermal instability imparted by multiple
nitro group substitution is also important to the use of polynitroaromatic com-
pounds as explosives.
Explosives are materials "that can undergo very rapid self-
propagating decomposition or reaction of ingredients with the consequent formation
of more stable materials, the liberation of heat, and the development of a
sudden pressure through the action of its heat on produced or adjacent gases"
(Rinkenbach, 1965). The chemistry of the decomposition process is not well
understood, but it appears to be closely related to the high oxygen content of
compounds containing several nitro substituehts. Rinkenbach (1965) indicates
that the oxygen balance (oxygen content relative to the total oxygen required for
complete oxidation) is a very important property of most explosives. Maksimov
(1972) concluded that the autocatalytic decomposition of gaseous nitroaromatic
compounds at 270-350°C was quite dependent upon the substituents on the ring.
He found that the tendency for decomposition increases with the number of nitro
groups and is accelerated by the presence of hydroxyl, methyl, bromo, fluoro,
chloro, and amino substituents. The amino and chloro derivatives were the most
21
-------
stable, while the stability of nitrobenzenes decreases in the order: m-dinitro-
benzene > jD-dinitrobenzene > o-dinitrobenzene > 1,2,3-trinitrobenzene > 1,3,5-
trinitrobenzene.
Substitution of a nitro group on an aromatic ring may have
several synthetic functions. Perhaps the most important use of nitro substitution
is as a starting point for the preparation of amines (Bannister and
Olin, 1965). There are two major processes that can be used to prepare amines:
(1) amination by ammonolysis and (2) amination by reduction of nitro groups
(Shreve, 1963). The commercial approach selected depends upon several factors,
such as the other ring substituents and process economics. The production of
aniline by vapor phase reduction of nitrobenzene (about 97% of the nitrobenzene
produced is consumed in this way) is typical of reductive amination.
Actually, reduction of nitro functional groups that are
attached to aromatic rings can result in a variety of products, depending upon
the reaction conditions used (see Figure 2). Reduction can be accomplished
by employing any of the following reagents (Shreve, 1963).
(1) Hydrogen or carbon monoxide in the presence of a catalyst,
in liquid or vapor phase.
(2) Iron in acid or neutral solutions (tin or zinc occasionally
used).
(3) Zinc or iron in alkaline solution.
(4) Sulfides in alkaline solution.
22
-------
(5) Miscellaneous reducing agents
(a) Sodium hydrosulfite, Na2S204, in alkaline solution.
(b) Sodium sulfite, Na2S03, in solution.
(c) Electrolytic action.
(d) Metal hydrides.
The old Bechamp process, which used iron turnings and acid, is gradually being
replaced by more efficient catalytic hydrogenation in large-scale manufacturing
process (e.g.,production of aniline, toluidines, xylidines, phenylenediamines,
and toluenediamines).
alkaline j! electro-
H202 reduction
H202 and glac
CH3COOH
distillation
with Fe filings
{
jhenylhydroxylamine
Zn dust
and
CH3COOH
Zn dust
and
alkali
hydrazobenzene
electro-
reduction
o
Figure 2. Reduction Products of Nitroaromatic. Compounds (Shreve, 1963)
23
-------
As can be seen in Figure 3, reduction under alkaline
conditions usually results in some form of dimerization. The reduction of
tiitrobenzene with zinc dust under acid, neutral, and basic conditions
illustrates the variety of products that can be formed.
NH2
O /-NHOH
O >—N02
Zn + water
2n + base
NHNH ( o
Figure 3. Reduction of Nitrobenzene Using Zinc
The reductive coupling of nitro groups under basic conditions happens to have
considerable commercial importance. The hydrazobenzenes that are formed can be
rearranged by treatment with acid (benzidine rearrangement) to form benzidine
and benzidine derivatives (see Figure 4). Benzidine and benzidine derivatives
are important intermediates in the production of azo dyes.
( O .' NO
V.
CH3
( C )— N02
• Cl
' c ,-
\_y
•NO:
Zn
2 NaOH
H2N—< c .)—< o > (3-15%)
H2N—'
Figure 4. Synthesis of Benzidine and Benzidine Derivatives from Nitroaromatics
(Lurie, 1964).
24
-------
Nitro groups are usually introduced into aromatic systems
by direct nitration with mixed sulfuric and nitric acids. The process is
considered to be an electrophilic attack by the nitronium ion, +N02. Because
nitro groups inductively withdraw electrons out of the aromatic ring, they
deactivate the ring to further electrophilic substitution; therefore, subsequent
nitration, or other electrophilic substitution (e.g., chlorine, Friedel-Crafts
alkylation, etc.), is much more difficult. The nitro group is also a meta
director for electrophilic substitution, due to the resonance structures
depicted in Figure 5 (the relatively negative meta-position is more susceptible
to electrophilic attack).
>CH2-H < >
Figure 5. Resonance Structures of Nitroaromatics
The resonance structures in Figure 5 also explain the susceptibility of ortho-
or para-, but not met a-, substituted chloronitrobenzenes to nucleophilic substitution
(e.g. ,£-chloronitrobenzene hydrolysis to p-nitrophenol). The increased acidity
of methyl hydrogens or _o- or p-nitrotoluenes is explained by the resonance
structures in the lower portion of Figure 5.
The commercial use of nitration and the use of the resulting
nitroaromatics as chemical intermediates are illustrated in Figure 6. In order
to understand the logic behind the synthetic approaches, the resonance structures
discussed above, as well as the directing affects of various substituents, must be
considered (e.g.^H3,-OH,-Cl,-Br,-OR,-NHCR are ortho-para directors, while
25
-------
NaUH
3% -
KOh.MeOH
high temp.
4 press.
(KO)2PCl
OH 87% » parathlons
References
Chem. Marketing Reponvr
1974a) , Matminiiro:i (IMh/a. l>
Koucis and Northcott (1963)
Shreve (196'J)
reduction,, H2N-
NH3,H-0
- i - »
NH2
C V-N02
NaOH,H20
Cl
Figure 6. Commercial Chemistry of Nitroaromatic Compounds
26
-------
Reactions uf Nitrutolueues
o)-CHi
HN03
'S03M
Format Ion (»C Aromatic :Amlnes
CU.CUO
NH2
H2N-/O)-CH3
MutsiiKuma (19fi7.i), Snruve
(1963), SonvandiT and
Domlnguez (19f>9)
NCO
r-<
OCN /p \ .CH.
polyurot hnm.'S
Cherair.Hj Markt-t my.
Reporter (1974b), Shrev,
(19M-. rhlrtlfi (1968)
N02
•^"X'^-v
("") 1 O i
3f+&
N02
S03H
NH2
N02
Na^S, alkaline
HNOj
HU3S
S03H
,O>-NH2
Fe.HCt
N02
H03S
S03H M02
-^^~.
o]o
Fe
NH2
-* (O)-NH2
NH2
„ HCt ^ .
3H H03:
S03H NH2
Wo"
Figure 6. Commercial Chemistry of Nitroaromatic Compounds (Cont'd)
27
-------
Miscellaneous
N02
Q y~ CH = CHC02H
KMnO,,
NO,
Matsuguma (l%7u)
CH
HNO,
NO,
~ C02H
Uuncker (19f>«)
4 /
CH3- / r )- NHj
N02
HNO i
Oti
N02 OH
....^
Toluidine Red
(Pigment Red 3)
no reaction
Typical diazotization
reaction
Ehrlch (19M)
02N
Na2S03
N02
02N.-O-CH3
Sellite process for removing
unsynmetrical Isomers of
trinitrotoluenes from TNT
Figure 6. Commercial Chemistry of Nitroaromatic Compounds (Cont'd)
28
-------
0
-CH.-COH,-NO-,-NH.,-S00H are meta directors). Also important in determining
2 2. j
the appropriate synthetic approach is the fact that nitric acid used as
a nitrating reagent is also capable of oxidizing some of the starting material.
As a result, poor yields preclude the commercial use of direct nitration of
chemicals such as phenol and benzoic acid. For this reason, chloronitrobenzenes
are used to synthesize nitrophenols and aminophenols.
2. Hydrolysis
Nitroaromatics are generally very stable in water under
neutral conditioning. Hoffsommer and Rosen (1973) examined the hydrolysis of
2,4,6-trinitrotoluene (TNT) and N-methyl-N-nitro-2,4,6-trinitroaniline (tetryl)
in sea water. For several months, they stored approximately saturated solutions
(TNT = 95 ppm; tetryl = 26 ppm) of the compounds in sea water (pH = 8.1). After
101 days, only 12% of the tetryl remained, as measured by gas chromatography with
an electron capture detector (GC-EC). The ultraviolet spectrum of the aqueous
solution suggests the formation of picric acid (2,4,6-trinitrophenol), an
expected hydrolysis product. With TNT, no change in concentration was observed
by GC-EC measurement after 108 days at room temperature. The nucleophilic
reactivity of positions ortho or para to nitro substituents has been discussed
in Section I-B-1 (see Figure 5, p. 25). In fact, this hydrolytic reactivity
is utilized in the synthesis of nitrophenols and nitroethers from chloronitro-
benzene. The difference in the reactivities of tetryl and TNT in sea water can
be attributed to the difference in leaving groups; the methyl anion ( CH_) in
trinitrotoluene makes a poor leaving group compared to the substituted amino
group in tetryl.
Aromatic nucleophilic substitution at positions ortho or
para to nitro groups is well known (Murto and Murto, 1966; Murto, 1966). The
neutral hydrolysis rate constants for some 2,4,6-trinitrobenzene derivatives
29
-------
are fast enough to be measured. In contrast, only the alkaline hydrolysis
rate constants of 2,4-dinitrobenzene derivatives can be measured. The kinetic
data developed by Murto (1966) and Murto and Murto (1966) are presented in
Table 7. Both the neutral and basic hydrolysis reactions probably proceed by
an S 2 mechanism. The kinetic data demonstrated that the reaction is dependent
N
upon the hydroxyl ion concentration (i.e.,it is pH dependent) and the concentration
of the nitroaromatic compound. The effect of pH can be demonstrated with 2,4-
dinitrochlorobenzene: at pH 13.3, the measured half-life at 25°C is 3800 seconds;
at neutral conditions (pH 7.0), the calculated half-life is 1 x 107 hours.
Elevated temperatures may affect the reaction rate significantly. For example,
Urbanksi (1964) notes that when 2,4-dinitrochlorobenzene and 2,4,6-trinitrochloro-
benzene are boiled in water for 5 hours, the percent that hydrolyzes is «.!% and
26.6%, respectively.
Table 7. Alkaline and Neutral Hydrolysis Rates of Nitroaromatic Compounds in
Water (Murto, 1966; Murto and Murto, 1966)
Compound
2,4,6-(N02)3C6H2OCH3
2,4,6-(N02)3C6H2OC6H5
2,4,6-(N02)3C6H2F
2,4,6-(N02)3C6H2«
2,4,6-(N02) 3C6H2OCH3
2,4,6-(N02)3C6H2OC6H5
2,4,6-(N02)3C6H2N02
2,4,6-(N02)3C6H2F
2,4,6-(N02)3C6H2a
2,4-(N02)2C6H3OCH3
2,4-(N02)2C6H3OC6H5
2,4-(N02)2C6H3N02
2,4-(N02)2C6H3F
2,4-(N02)2C6H3CJ>
Initial NaOH
Concentration
(N)
0.005
0.005
0.005
0.005
-
-
-
-
-
0.05
0.05
0.05
0.05
0.05
Initial
PH
12.3
12.3
12.3
12.3
7.0
7.0
7.0
7.0
7.0
13.3
13.3
13.3
13.3
13.3
k
(i mole'V1)
25°C
1.28
1.56
700
0.506
0.199 x 10"5
0.144 x 10~5
75.7 x 10" 5
144 x 10" 5
0.00644 x 10" 5
34 x 10~5
20 x 10~5
8520 x 10"5
12900 x 10~5
185 x 10"5
\
(sec)
0.5
0.4
0.001
1.4
3.5 x 105
4.8 x 105
9.2 x 102
4.8 x 102
1.1 x 107
2.0 x 103
3.5 x 10-1
8.2
5.4
3.8 x 103
30
-------
3. Oxidation
Nitro groups are already in a high state of oxidation
and, therefore, have little susceptibility to oxidation conditions. In fact,
nitrobenzene has been used as a mild oxidizing agent (Matsuguma, 1967a). The
pxidative stability of the nitro group, as well as the instability of other
functional groups, is illustrated by the following equations:
_ xCH3 C02H
©- »o
_ >N02 N02
cH=CHC02H
x_. — '
4. Photochemistry
In order for a compound to react photochemically, it has to
be able to derive energy from the available light and then convert the energy
into some type of chemical transformation. Photochemical processes are extremely
complex and are difficult to predict; they are dependent upon such parameters
as the ultraviolet absorption spectrum and quantum yields (efficiency of reaction
based on the quantity of incident light absorbed) of the chemicals or available
sensitizers, and the reaction medium in which the chemical is irradiated (this
affects the previous parameters as well as the reaction that the excited state
may undergo). As a result, environmental photochemistry is extremely difficult
to simulate because the conditions can vary so much. However, there is fairly
good agreement that the incident light (sunlight) contains only wavelengths
longer than 290 nm because the ozone in the stratosphere effectively filters out
the shorter wavelengths (higher energy light) (Roller, 1965).
31
-------
Substitution of a nitro functional group on an aromatic
»Y\
system has a distinctive bathochrorfic effect upon its ultraviolet spectrum.
In fact, nitrobenzene (Amax = 268.5, emax = 7800) shows the greatest red shift
(toward longer wavelengths) of all the common monosubstituted benzenes (Jaffe
and Orchin, 1962) (for comparison benzene Amax = 203.5, emax = 7400). This has
been attributed to the particularly strong resonance interaction between the
nitro group and the aromatic ring. This resonance interaction and, consequently,
the ultraviolet absorption spectrum, is affected by other substitutents and by
their position on the aromatic ring. This is illustrated for a number of nitro-
aromatic compounds in Table 8. The Amax for the ultraviolet absorption spectrum
of the majority of the compounds considered for this review are listed in Table 1.
The reported Amax in Tables 1 and 8 are only for the primary absorption band
(*L band, IT >• ir* transition). Frequently, there is a much less intensive
3,
band located at longer wavelength (1L, band, n >- TT* transition) which is
capable of absorbing sunlight wavelengths (see Table 10 for examples). With
nitrobenzene, the ^L, band is considered to be under the *L band.
b a .
The ultraviolet absorption spectrum of any given nitroaromatic
compound can be considerably affected by the medium or solvent in which the spectrum
is measured. This is generally true for aromatic systems with electron-accepting
substituents. This shift of the UV absorption spectra is illustrated for nitro-
benzene in Table 9 and for a number of compounds in Table 8.
32
-------
Table 8. Ultraviolet Spectra of Representative Nitroaromatic Compounds (Sandus
and Slagg, 1972; Hashimoto and Kano, 1972; Jaffe and Orchin, 1962)
Compound
Nitrobenzene
Nitrobenzene
-------
Table 9. Solvent Effects on Nitrobenzene Ultraviolet Spectra
Solvent Water Ethanol Heptane Vapor
A , nm 265.5 259.5 251.8 239.1
max
A similar effect may occur for nitroaromatic compounds in the absorbed state,
a condition that frequently takes place in the environment and can alter the
energy required to bring about photpdecomposition (Plimmer, 1972). The
absorption spectrum shifts result from perturbations of the electronic states
of the molecules upon adsorption. Robin and Trueblood (1957) observed such
absorption spectra changes for several nitroaromatic compounds after adsorb-
ing the compounds on silicic acid in cyclohexane solution. Their results are
presented in Table 10. In a similar study of pesticides adsorbed on silica
gel, Plimmer (1972) noted that trifluralin (a, a, a-trifluoro-2,6-dinitro-
N,N-dipropyl-p_-toluidine) exhibited the largest red shift (60 nm) of all
the compounds tested. Keeping in mind the many factors mentioned above that
may affect the propensity of nitroaromatic compounds to photochemically re-
act, the available studies on photochemistry will be reviewed in the following
paragraphs.
A number of researchers have studied the fundamental and
mechanistic aspects of nitroaromatic photochemistry. The relevance of these
studies to environmental conditions is unknown, but they do provide some in-
sight into the process. Frequently, these studies use light sources that con-
tain wavelengths of higher energy than those found in sunlight (254 nm light
is used frequently).
34
-------
Table 10. Ultraviolet Absorption Spectrum Changes Caused by Adsorption on
Silicic Acid (Robin and Trueblood, 1957)
Compound
2-Nitroaniline
4-Nitroaniline
4-Nitroanisole
Nitrobenzene
4-Nitro-N-ethylaniline
4-Nitro-N ,N-diethylaniline
2-Nitrophenol
X (nm)
Cyclohexane
(C6H12)
375-378
270
246
229
323
294
253
344
^277
360-366
234
345-349
272
X (nm)
Silicic Acid
(SA)
and ec R /£ .
Cyclohexane °6 12 b
413
285
240
374-382 3.6
316 1.2
271 1.3
420
420
352-356
284-286
4-Nitrophenol
286
315
1.3
35
-------
Several investigators have used high energy light to study
the photochromism (formation of colored species upon exposure to light) of
£-alkylnitrobenzenes which contain at least one benzylic hydrogen. Using a
flash photolysis technique with light containing wavelengths greater than
250 nm, Wettennark (1962a,b) and Wettermark and Ricci (1963) were able to
detect and measure the disappearance rate constant of a colored isomer in an
aqueous solution (A of the colored isomer was 360-410 nm, depending upon
^ . max
the pH) of £-nitrotoluene and 2,4-dinitrotoluene. They suggested the mechan-
ism depicted in Figure 7.
J-H
hv
dark
Nitro Form
Aci Form
Figure 7. Suggested Mechanism to Explain Photochromism of o-Alkylnitrobenzenes
Using deuterium oxide and £-nitrotoluene, Morrison and Migdalof (1965) were
able to show that deuterium was incorporated into the methyl group even when
>290 nm light (Pyrex filter) was used. They concluded that hydrogen abstrac-
tion was definitely taking place. From this data, it is possible to conclude
that £-alkylnitroaromatic compounds are probably quite susceptible to photo-
chemical alteration, since they isomerize to highly colored compounds which
may react further. (For an example of Lbi.s, ,SPP r ho following <} ln< 'uM;ilori of
work by Burlinson e* al_., 1973.)
36
-------
Of course, many nitroaromatic compounds do not have an
abstractable ortho hydrogen. Numerous conditions have been used in studies of
these compounds. A favorite solvent is isopropyl alcohol, which contains the
abstractable hydrogen missing in non-o-alkylnitrobenzenes. Hurley and Testa
(1966, 1967) photolyzed nitrobenzene in isopropanol with 366 nm light. They
concluded that phenylhydroxylamine is generally the initial product, but an
oxidation in air to nitrosobenzene, which then couples with the hydroxylamine
to form azoxybenzene, also takes place. The measured quantum yield was approxi-
_2
mately 10 , and the overall photochemical reduction involved four hydrogen
abstractions. Hashimoto and coworkers (1968) found similar results using
330-340 nm light. However, they found that, by carrying out the reaction in
the presence of hydrochloric acid and a sensitizer, the final reduction products
K*MM> lllO.N?*- . j
were aniline, £-aminophenol, and £-chloroaniline
Hashimoto and Kano (1970, 1972) have studied the photo-
chemical reduction of substituted nitrobenzenes in isopropanol under nitrogen
atmosphere using 316 and 366 nm light. They found that benzenes with electron-
withdrawing substituents yielded aniline derivatives, while the use of benzenes
with electron-donating substituents resulted in the formulation of the hydroxyl-
amine derivative (see Table 11) . They also found a quantitative correlation
between the quantum yield and the Hammett constant. When the experiments were
run in air-saturated isopropanol using 313 nm light, the photochemical reaction
was completely quenched. This result suggests that photoreduction of nitro-
aromatics proceeds by an excited triplet state. Most of the photolysis ex-
periments were conducted in isopropanol. However, with £-nitrobenzonitrile,
other solvents were used and a considerable solvent effect on the quantum
yield was noted (Table 12) .
37
-------
Table 11. Quantum Yield and Products from the Disappearance of Substituted
Nitrobenzenes in 2-Propanol Under Nitrogen Atmosphere (Hashimoto
and Kano, 1972)
XC.H.NO,
64 2
£-N02
£-CN
m-CN
£-COOC2H5
m-COOC2H5
£-COOCH(CH3)2
£-COOH
m-COOH
H
£-CH3
£-OCH3
£-OH
£-NH2
Cone.
x 103
mol/1
1.00
1.03
1.00
1.00
1.00
0.25
1.00
1.17
1.10
1.00
0.25
0.50
0.50
Light
nm
366
313
313
313
313
313
313
313
313
313
313
313
366
Product
p-Nitroaniline
£-Aminobenzonitrile
m-Aminobenzonitrile
Ethyl £-aminobenzoate
Ethyl m-aminobenzoate
Isopropyl £-aminobenzoate
£-Aminobenzoic acid
m-Aminobenzoic acid
Phenylhydroxylamine
p-Hydroxylaminotoluene
£-Hydroxylaminoanisole
— -
Quantum
Yield
0.16
0.4'8*
0.34
0.15
0.11*
0.15
0.12
0.18
0.03
0.07
0.02
0.00
0.004
* Quantum yield for formation of Anilines.
38
-------
Table 12. Solvent Effect for Photoreduction of £-Nitrobenzonitrile (Hashimoto
and Kano, 1972)
3
Cone. (10 mol/1) Solvent Quantum Yield
1.03
1.00
1.00
Isopropanol
Ethanol
Cyclohexanane
0.48
0.11
0.00
Letsinger and McCain (1969) and Wubbels and Letsinger (1974)
have photolyzed nitroaromatic compounds in aqueous solutions which contained high
anion concentrations. Letsinger and McCain (1969) photolyzed (light >290 nm)
-4 -3
4-nitroanisole (10 M) in an aqueous solution of potassium cyanide (4 x 10 M)
that had been well purged with nitrogen. They isolated 2-cyano-4-nitroanisole
and 3,3'dicyano-4,4'~dimethoxyazobenzene. Under similar light conditions but
in 12N hydrochloric acid solution, Wubbels and Letsinger (1974) found that the
nitro group of various nitroaromatics was usually reduced to an amine, and the
ring was substituted with one or more chlorine atoms.
Barltrop and Bunce (1968) studied the photoreduction of
nitroaromatic compounds under a variety of conditions and light sources. One
of the more interesting studies is the photoreduction of nitrobenzene with
f>290 nm light in various solvents (see Table 13). Since the presence of oxygen
was shown to have little effect, the experiments in Table 13 were run without
deoxygenation. In solvents having readily abstractable hydrogen atoms, no
39
-------
Table 13. Solvent Dependence of the Products of the Photoreduction of Nitrobenzene with Light
A >290 nm (Barltrop and Bunce, 1968)
Solvent
Petroleum
Toluene
Ether
Isopropyl alcohol
Isopropyl alcohol
t-Butyl alcohol
50% Toluene/_^-butyl alcohol
Diethylamine
Triethylamine
33% Aq./diethylamine
50% Aq./isopropyl alcohol
50% Ammonia/isopropyl alcohol
Irradiation time (hr.)
5
5
5
5
3
5
5
5
5
2
3
2
Conversion (%)
26
64
72
86
60
39
23
76
97
84
53
58
Aniline
4
<1
44
13
19
—
—
6
14
5
40
16
Azobenzene
<2
—
2
—
—
—
—
20
12
8
—
4
2-Hydroxyazobenzene
<2
—
1
—
—
—
—
31
5
17
~
7
In all cases nitrobenzene (0.5 g) was irradiated in 100 ml. of solvent; percentage yields are based on nitrobenzene consumed.
The absence of a column entry means that this compound could not be detected.
-------
azoxy compounds were detected, although these products have been noted by other
authors. Sandus and Slagg (1972) have suggested that the poor reproducibility
might be attributable to photolysis of the intermediates (e.g., nitroso deriva-
tives), which could take place with 310-360 nm light. Because of this suspected
photosensitivity of the products, Sandus and Slagg (1972) decided to use 254 nm
light for their flash and continuous photolysis studies of nitroaromatics.
They concluded that the aci and radical intermediates that have been observed
are probably due to side reactions and are not related directly to the products.
A number of nitroaromatic compounds used as pesticides have
been tested for photochemical stability. Often the experimental conditions
used attempt to simulate conditions that the pesticide may be exposed to in
nature, but this has not always been the case. In one of the earliest pesti-
cide photochemistry studies, 254 nm light was used to screen the effect of
light on 141 pesticides (Mitchell, 1961) adsorbed on paper (chromatographed
after irradiation). The author found that the dinitrophenol pesticides
(2,4-dinitro-6-sec-amylphenol, 4,6-dinitro-o-sec-butylphenol, 4,6-dinitro-£-
cresol, and 4,6-dinitro-o-cyclohexylphenol) showed little or no degradation,
while pentachloro- and tetrachloronitrobenzenes were almost completely de-
graded. The conditions and results of some other pesticide photolysis studies
have been summarized in Table 14. These nitroaromatic pesticides have very
unusual chemical structures compared to some of the other commercially impor-
tant nitroaromatic compounds and, therefore, extrapolation to simpler structures
is difficult (e.g., 2,6-dinitroaniline herbicides absorb strongly at 350-450 nm).
However, the last two studies are important because they demonstrate that nitro
groups can be converted to nitroso or amino functional groups under simulated
environmental conditions. The observation that azoxybenzene derivatives can be
41
-------
Table 14. Photolysis Studies of Nitroaromatic Pesticides
Ret'erenr.e
Wright ana Warren (1965)
Hanadmad (1967)
Cotnpound(s) Studied
Trifluralin
(oc t Kt 290 nm)
Sunlight and
artificial light
(254 nm)
Reaction Media
Coated on )Uass or
soil
In solution, on TLC
plates or on soil
Results
After 6 hours irradiation on glass,
significant change in the UV
absorption spectrum had occurred.
Degradation on soil very slow.
PCNB degraded with 254 nm light but
would not decompose with > 270 nm light
With sunlight no effect.
Matsuo and Casida (1970)
Bandal and Casida (1972)
Nakagawa and Crosby (1974)
2-sec-Butyl-4 ,6-dinitrophenol
Sunlight
Bean leaves
6-Amino-2-sec-butyl-4-nitrophenol and
other unknowns were formed when sen-
sitizers were present. Isopropyl
carbonate derivative is stable.
Parochetti and Hein (1973)
Brewer et_ al. (1974)
Trifluralin
Fenitrothion (0,0-dimethyl-
0-(3-methyl-4-nitrophenol)
phosphorothioate
Sunlight and
artificial light
(> 290 nm)
Artificial light
(> 290 nm)
Adsorbed on soil
Vapor phase and
in ethanol
No significant loss from photo-
decomposition
4-Nitro-3-methylpheno 1 is the major
product. Extended photolysis de-
graded initial photoproducts . Pho-
tolysis of the vapor for 240 hours
resulted in ^ 1-2% decomposition.
Nitrofen (2,4-dichlorophenyl
£-nitrophenyl ether) and
derivatives
(e.g. £-nitrophenol)
Sunlight and
simulated sunlight
Aqueous suspension
100-200 mg/1 of
deionized water
Isolated 4-nitrocatechol, 2,4-
dichlorophenyl £-aminophenyl ether,
and 4,4'-bis(2,4-dichlorophenoxy) .
azobenzene. £-Nitrophenol is more
photochemically stable than 2,4-
dichlorophenol. It.degraded to 4-
nitrocatechol, hydroquinone, and a
nonvolatile, dark polymer.
Plimmer and Klingebiel (1974)
N-sec-Butyl-4-tert-butyl-2.6-
dinitroaniline
Sunlight and
artificial light
(•-• 290 nm)
Water solution and
methanol TLC
silica plates
Decomposed more slowly than triflura-
lin. Major product in methanol or
water was 4-tert-butyl-2-nitro-6-
nitrosoaniline. Photodecomposition
occurred upon irradiation of the TLC
plates with sunlight or a sunlamp.
-------
formed is also quite significant, although the rate of such intramolecular
reactions would probably be very slow at trace concentrations.
Perhaps the most significant environmental photolysis study
of a non-pesticide is the work on the aqueous photodecomposition of trinitro-
toluene (TNT) by Burlinson jet _al. (1973). These authors photolyzed saturated
aqueous solutions (120-130 ppm) of TNT with Pyrex-filtered (>290 nm) ultraviolet
light to understand why water effluents containing TNT turned pink. (These
effluents are commonly referred to as "pink waters.") From a continuous photo-
reactor where 65% of the TNT had decomposed, the authors were able to extract
20% of the degradation products by benzene extraction. The products isolated
and their yields are listed in Table 15. The azoxy derivatives were isolated
from a static run (30 hours of sunlight with 120 ppm) where 75% of the TNT had
decomposed. Burlinson and coworkers (1973) were unable to isolate and identify
any of the 80% of the degradation products that were water-soluble.
Carrying out the TNT photolysis in hydrogen-donor solvents
resulted in a 47% yield of the four isomeric tetranitroazoxytoluenes. This
illustrates the effect that solvent can have on a photochemical process. With
dioxahe (d_) solvent (a good hydrogen donor), they observed no deuterium ex-
es
change into the recovered TNT. Also, an inverse relationship between photo-
decomposition and deuterium uptake was noted when D^O was used as a solvent.
From this information, as well as from the fact that the photodegradation rate
is pH dependent, the authors concluded that the aci form was probably not in-
volved in the formation of the azoxy derivatives and that the anion (II) in
Figure 8 may be involved in the formation of the major products of aqueous
TNT photolysis.
43
-------
Table 15. Photolysis Products from an Aqueous Solution of TNT (Burlinson
et al., 1973)
1,3,5-Trinitrobenzene
,6-Dinitroanthranil
3-4
2,4,6-Tr inlLrobenzaIdehyde
2,4,6-Trinitrobenzonitril«
2,2',6,6'-Tetranitro-
A,A' ,6,6'-Tetranitro-
2,2' -azoxy toluene.
NO,
2' .A-Dimetljyl-S.S1 ,5,5'-
tetronltro-ONN-
nzoxybenzene f S~\
tetranitro-ONN-
azoxy benzene,
* Found in only trace amounts in "pink water."
44
-------
Burlinson et al. (1973) also noted a significant difference
between the reactivities of trinitrobenzene and TNT. Photolysis of 1,3,5-trinitro-
benzene in aqueous solution produced no photoproducts after six hours of irradiation.
NO,
II
Figure 8. Proposed Mechanism for the Primary Photochemical Step for the
Photodecomposition of TNT in Water (Burlinson et al., 1973)
In summary, although very few of the commercially important
nitroaromatic compounds have been experimentally tested for sensitivity to ultra-
violet light, it is likely that many of the compounds may react photochemically.
Although prediction of products is extremely difficult, conversion of the nitro
group to nitroso, hydroxylamine, amine, and azoxy moieties seems possible based
upon the available information.
45
-------
II. Environmental Exposure Factors
A. Production, Consumption
1. Quantity Produced and Imported
A considerable amount of quantitative information on the
production and sales of nitroaromatic compounds is available from the reports
of the U.S. International Trade Commission (formerly the U.S. Tariff Commis-
sion) (USITC, 1959-1973). The available information is presented in Table 16.
Unfortunately, the production and sales quantities are published by USITC only
when there are three or more producers, no one or two of which may be pre-
dominant. Table 16 covers only a limited number of nitroaromatic chemicals,
but it probably has some information on most of the high volume chemicals,
since those chemicals usually have several manufacturers. Production trends
are more easily visualized from the USITC (1959-1973) information plotted in
Figure 9.
The U.S. International Trade Commission also publishes infor-
mation on the quantity of benzenoid chemicals that are imported into the
United States. The statistics are reported annually, but are based only upon
importation reported through major U.S. customs districts. Therefore, the
quantities reported are somewhat lower than the actual total material imported.
(For 1973, coverage is estimated to be 68% for flavors and perfumes, 78% for
drugs, 79% for pigments, 83% for intermediates, and 94% for dyes). Table 17
presents the published statistics on quantities of nitroaromatic: compoundM
that were imported.
From the available published data, as well as from information
obtained during discussions with the chemical producers, a list of nitroaromatic
47
-------
Table 16. U.S. Production and Sales of Nitroaroinatic Compounds (U.S. International Trade Commission,
1959-1973)
(1000 Ibs)
Nitroaromacic Hydrocarbons
Nitrobenzene P*
S**
£-Nitrotoluene P
S
2,4-Dinitrotoluene P
S
2,4-(and 2,6-) ' Dinitro- P
toluene S
Nitroxylenes, mixed P
S
Chloronit roaromatic
Hydrocarbons
l-Chloro-2-nitrobenzene P
S
l-Chloro-3-nitrobenzene P
S
l-Chloro-4-nitrobenzene P
S
o- and £-Chloronitrobenzene P
mixture (1-chloro and S
2 and 4-nitro)
l-C'nloro-2,4-dinii:ro- P
benzene S
l,4-Dichloro-2-nitro- P
benzene S
1959
172,133
6,897
545
7,031
7,851
1960
162,308
6,171
24,540
10,037
5,324
1961
184,558
6,686
17,177
7,494
6,761
628
1962
199,587
9,095
3,890
4,852
7,217
1,208
276
1963
219,971
9,421
18,879
10,715
6,435
3,920
8,164
1,580
396
1964
239,216
9,513
20,088
9,648
8,063
86,902
8,193
1,499
417
1965
280,341
11,506
28,290
10,536
109,757
8,107
1,659
705
1966
326,853
1.3,612
6,517
36,226
12,315
7,908
121,735
8,535
1,508
793
1967
347,700
12,623
34,226
12,402
101,508
6,257
1,768
623
32
1968
397,937
11,450
17,750
14,623
6,626
2,192
1969
'<84,457
9,860
253,533
20,391
1970
547,680
::96,503
88,854
1971
444,869
16,756
352,746
1972
.".:•; .169
12,622
433,885
1973
308.667
471,237
-p-
oo
* - P = Production
** - S - Sales
-------
Table 16. U.S. Production and Sales of Nitroaromatic Compounds (U.S. International Trade Commission,
1959-1973) (Cont'd)
(1000 Ibs)
VO
Chlo ron i t roaroma tic
Hydrocarbons (Cont'd)
4-Chloro-2-nitrotoluene P*
S**
4-Chloro-3-nitrotoluene P
S
Nltrophenols and Realted
Compounds
£-Nitrophenol P
S
p_-Ni t rophenr.l and sodium P
salt S
2-Amlno-4-nitrophenol P
S
2,4-Dinitrophenol, tech. P
S
p_-(2,i-Dinitroanillno) P
phenol S
Methyl parathlon P
S
Ethyl parathlon P
Nitroanisoles and
Nitroanisldines
4-Nitro-c-anlsidine P
S
5-Nitro-o-anisidine P
S
1939
_—
„_
109
598
41
5,987
7,814
9,180
7,924
133
18
748
I960
76
831
11,794
10,262
7,434
7,518
91
325
19ft1
60
945
18,527
14,265
8,423
7,423
11
131
I9f,2
313
71
13,093
5,618
65
1,053
16,156
12,196
8,786
5,847
144
26
201
1963
696
16,161
132
1,035
15,999
19,174
8,618
83
284
1%'j
396
18,935
98
1,037
33
18,640
21,713
12,786
10,338
73
331
1.9 (if)
—
19,856
11,273
137
935
360
29,111
27,440
16,607
14,198
103
108
'.966
102
20,025
17,920
971
35,862
29,973
19,444
15,536
250
196
15,370
15,145
102
775
33,344
31,919
11,361
14,573
119
1968
33,594
14,451
192
863
38,163
45,178
19,510
!%9
38,837
15,741
104
149
50,572
32,818
1970
32,600
19,312
41,353
39,869
15,259
15,504
1971
16,158
37,226
46,354
1.972
—
51,076
52,438
1973
48,890
52,450
* - P - Production
** - S = Sales
-------
Table 16. U.S. Production and Sales of Nitroaromatlc Compounds (U.S. International Trade Commission,
1959-1973) (Cont'd)
(1000 Ibs)
Nitroanilines and Related
Compounds
m-Nitroaniltne P*
S**
p_-Nitroaniline P
S
2-Chloro-4-nttroanillne P
S
4-Chloro-2-nltroanlllne P
S
2,6-Dichloro-4-nicroaniline P
S
2-Bromo-4,6-dinitroaniline P
S
2-4-Dinitroanlline P
S
4-Nltro-£-toLuidine P
S
5-Nltro-o-tolutdlne P
S
2-Nitro-£-toluidine P
S
3'-Nltroacetanllide P
S
4'-Nitroacetanilide p
S
3' , 4-Dlnitrobenzanillde P
S
1959
201
412
448
279
19
337
86
1,573
706
1960
105
361
312
151
41
12
165
1,291
603
1961
148
426
315
180
56
176
1,152
602
1962
159
8,769
6,922
297
172
219
81
326
234
1,204
581
1963
9,808
6,890
289
267
413
249
172
97
11
300
1,195
633
1964
10,890
7,876
301
226
225
185
358
941
643
1965
12,478
6,883
448
461
259
1,257
822
1966
10,750
389
249
566
391
607
431
206
100
367
183
1,208
1967
9,001
275
221
503
463
187
94
156
192
864
1968
11,029
348
355
491
112
207
111
218
179
15
1969
147
164
66
277
199
16
1970
196
135
99
95
15
277
1971
258
88
137
123
—
1972
626
85
397
1973
944
353
312
—
Ui
o
* - P = Production
** - S = Sales
-------
Table 16. U.S. Production and Sales of Nitroaromatic Compounds (U.S. International Trade Commission,
(1959-1973) (Cont'd)
(1000 Ibs)
Nitroaromatic Acids and
Related Compounds
.m-Nitrobenzoic acid and P*
sodium salt S**
m- and £-Nit robenzoic acids P
S
£-(4-Chloro-3-nitrobenzoyl)- P
benzole acid S
in-Nitrobenzenesulfonic acid P
S'
m-Sit robenzenesalfonic P
acid and sodium salt S
2-Chloro-5-nitrobenzene- P
sulfonic acid and S
sodium salt
2-Chloronitrobenzene- P
sulfonic arid • S
4-Chloro-3-nitrobenzene- P.
sulfonic acid S
2-Amino-5-nitrobenzene- P
sulfonic acid S
2-(£-Aminoanilino)-5- P
nitrobenzenesulfonic acid S
5-Amino-2 (£-arainoanilino)- P
benzenesulfonic acid S
3-Nitro-£-toluenesulfonic P
acid s
1959 '
65C
109
1,472
1,386
270
102
32
42
14
276
1960
157
2,519
1,388
127
163
59
22
90
1961
96
2,011
1,332
245
180
54
9
75
12
1962
106
1,603
1,608
__-
42
89
26
75
1963
259
134
2,092
2,065
190
56
—
24
86
1964
255
93
3,090
2,118
36
33
15
1965
145
2,293
2,397
89
48
21
73
1966
220
3,711
2,705
72
7
87
1967
147
3,090
2,551
368
33
18
4
67
1968
351
3,464
2,289
174
42
81
1969
911
3,081
1,447
49
11
1970
3,654
2,165
39
10
1971
23
1972
—
1973
—
—
* - P = Production
** - S = Sales
-------
Table 16. U.S. Production and Sales of Nitroaromatic Compounds (U.S. International Trade- Commission,
1959-1973) (Cont'd)
(1000 Ibs)
Nitroaromatic Acids and
Related Compounds (Cont'd)
5-Nitro-o-toluenesulfonic P*
acid S**
7-(and 8-)Nitronaphth[ 1 , 2d]- P
oxadiazole-5-sulf onic acid S
3-Sitro-l ,5-napthalene- P
disulfonic acid S
4-Amino-4'-nitro-2,2'- P
stilbenedisulfonic acid S
4,4'-Dinitrostilbene-2,2'- P
disulfonic acid S
m-Nitrobenzenesulfonyl P
chloride S
4-Chloro-3-nitrobenzene- P
sulfonyl chloride S
4-Chloro-3-nitrobenzene- P
sulfonamide S
Miscellaneous Nitroaromatic
Compounds
4'-(£-Nitrophenyl)- P
acetophenone S
Dinitrobutylphenol, P
ammonium salt S
1959
3,730
2,256
.76
89
I960
3,399
1,967
177
167
1961
3,894
497
2,352
129
139
1962
4,870
1,184
2,885
165
130
1963
5,403
913
128
3,423
23
141
164
L9(>4
6,680
758
207
4,159
235
258
1965
8,429
1,084
223
6,449
248
275
1W>
1.1,261
969
51
9,376
500
320
85
70
1967
10,419
278
145
11,443
553
420
58
66
1968
6,735
676
200
11,319
390
372
1969
12,911
551
201
14,682
487
556
1970
9,025
264
10,161
426
431
42
1971
7,164
10,953
345
503
1972
8,017
9,230
507
1973
7,955
245
743
Cn
to
* - p = Production
** - S - Sales
-------
Nitrobenzene
100
.1—Chloro—2—Nitrobenzene
10
0.1
0.01
2,4- and 2,6-Dinitrotoluene
Methyl Parathion ~
^^
'p-Nitrophenol and Sodium Salt
Ethyl Parathion
•»- "p—Nitroaniline
1— Chloro—2.4—Dinitrobenzene
1-Chloro-3-Nitrobenzene
'•-,,2—Nitro—p—Toluidine
2,4—Dinitrophenol, Technical -
\x/5-Nitro-o-Toluidine
romo^-4,6~0initroaniline
I
i |__. j. I i 1-
1959 1961 1963 1965 1967 1969 1971 1973
Production Trends of Nitroaromatic Compounds
Figure 9. U.S. Production Trends of Nitroaromatlc Compounds
53
-------
Table 17. Imports of Nitroaromatlc Chemicals (U.S. International Trade
Commission, 1967-1973)
PfimpnnnH
2-(p-Aminoanilino)--5-nitrobenzene-
sulfonic acid
2-Amino-3-chloro-5-nitrobenzonitrile
2-Amino-4-chloro-5-nitrophenol
2-Amino-4-chloro-6-nitrophenol
2-Amino-6-chloro-4-nitrophenol
2-Amino-4,6-dinitrophenol
2-Amino-N-ethyl-5-nitrobenzenesulfonanilide
2-Amino-5-nitrobenzenesulfonic acid
2-Amino-5-nitrobenzenesulfonic acid,
ammonium salt
2-Amino-5-nitrobenzenesulfonic acid,
sodium salt
2-Amino-5-nitrobenzoic acid
2-Amino-5-nitrobenzonitrile
2-Amino-6-nitrobenzothiazole
2-Amino-5-nitro-N-(phenethyl)benzenesulfonamide
2-Amino-4-nitrophenol
2-Amino-4-nitrophenol, sodium salt
2-Amino-5-nitrophenol
4-Amino-4'-nitro-2,2'-stibenedIsulfonic acid 39.8
2-Bromo-4,6-dinitroaniline
l-Bromo-2-nitrobenzene
4-^-Butyl-2,6-dimethyl-3,5-dinitroacetophone 26.8
2-sec-Butyl-4,6-dinitrophenol
6-j:-Butyl-3-methyl-2,4-dinitroanisole
5-_t-Butyl-2,4,6-trinitro-m-xylene
2-Chloro-l,4-dibutoxy-5-nitrobenzene
2-Chloro-l,4-diethoxy-5-nitrobenzene
2-Chloro-4,6-dinitroaniline
l-Chloro-2,4-dinitrobenzene
4-Chloro-5-nitro-2-aminophenol
2-Chloro-4-nitroaniline
2-Chloro-5-nitroaniline
4-Chloro-2-nitroaniline
4-Chloro-3-nitroaniline
4-Chloro-3-nitroanisole
5-Chloro-2-nitroanisole
l-Chloro-2-nitrobenzene
(1000 Ibs)
1967 1968 1969 1970 1971 1972 1973
30.1
1.9
0.7
8.6
ide
11.6
39.8
26.8
66.9
127.0
8.9
15.2
55.2
0.3
43.9
3.5
3.0
0.7
2.2
1.8
14.3
0.4
0.5
30.3
21.2
28.9
22.8
122.5
156.2
1.1
13.8
23.3
49.6
25.5
28.0
0.7
14.8
4.4
0.1
11.5
4.3
138.1
12.8
46.7
10.3
15.5
85.4
90.0
1.1
6.3
21.4
88.2
5.8
54.8
144.7
29.3
1.5
1.3
0.8
1.3
11.3
2.2
0.6
30.0
0.8
48.4
107.3
26.2
47.9
0.1
23.1
55.3
163.4
1.1
4.3
12.8
132.3
162.3
377.4
0.1
0.2
.67.3
4.6
0.4
16.8
40.2
24.8
141.5
23.8
155.1
0.7
37.5
83.6
35.9
1.5
5.7
67.0
353.6
50.1
1.5
0.3
15.5
4.8
4.4
2.1
2.2
43.0
129.7
125.8
29.9
148.2
49.8
77.2
1.6
25.9
118.8
0.6
37.4
48.7
143.3
409.5
11.9
0.6
124.1
88.6
2.8
1.2
51.1
2.1
3.8
83.9
149.9
118.0
90.1
34.7
92.5
42.6
433.2
84.4
49.4
2.2
71.8
170.2
20.0
541.0
1.3
4.4
0.5
0.2
315.4
54
-------
Table 17. Imports of Nitroaromatic Chemicals (U.S. International Trade
Commission, 1967-1973) (Cont'd)
Compound
2-Chloro-A-nitrobenzoic acid
4-Chloro-3-nitrobenzoic acid
2-Chloro-4-nitrophenol
4-Chloro-3-nitrotoluene
4-Chloro-<*, <*, <*-trifluoro-3-nitrotoluene
2,6-Dichloro-4-nitroaniline
1,3-Dichloro-4-nitrobenzene
l,4-Ditnethoxy-2-nitrobenzene
2,4-Dinitroacetanilide .
2,-4-Dinitroaniline
m-Dinitrobenzene
3,-5-Dinitrobenzoic acid, tech
3,-5-Dinitrobenzoyl chloride, tech
Dinitrobutylphenol
4,6-Dinitro-o-cresol
2,4-Dinitro-6-methylphenol
2,4-(and2,6)-Dinitrophenol
4,4'-Dinitro-2,2'-stilbenedisulfonic acid 434.5
2,4-Dinitrotoluene
2,4'-Dinitro-4-trifluoromethyldiphenyl ether
2-(Methylsulfonyl)-4-nitroaniline
m-Nitroaniline
o-Nitroaniline
j)-Nitroaniline
2-Nitro-£-anisidine
4-Nitro-o-anisidine
5-Nitro-o-anisidine
Cj-Nitroanisole
m-Nitrobenzaldehyde
m-Nitrobenzenesulfonic acid, sodium salt
m-Nitrobenzoic acid
o-Nitrobenzoic acid
2_- Nitrobenzoic acid
m-Nitrobenzoyl chloride
£-Nitrobenzoyl chloride
(1000 Ibs)
1967 1968 1969 1970 1971 1972 1973
0.6
22.0
129.4
11.0
0.1
42.9
2.3
434.5
19.9
55.6
20.2
13.0
11.0
9.3
2.3
252.1
219.8
1.7
13.2
0.1
1.0
55.0
284.8
15.9
56.3
116.0
20.0
19.2
4.4
42.9
197.9
. 7.2
183.4
2.2
2.1
0.2
4.4
105.8
29.9
0.05
441.6
39.9
7.2
112.7
75.9
32.0
. 15.0
3.3
21.0
165.8
1.2
15.5
29.9
20.9
16.7
0.2
2.2
273.9
49.9
6.4
732.8
147.2
82.6
88.2
75.4
98.1
30.1
8.5
34. r>
114.3
6.1
29.9
7.2
30.3
80.2
0.5
1.3
10.3
3.3
2.2
358.8
10.1
29.9
0.2
19.7
312.8
117.2
278.6
53.0
6.0
J.n.2
9.9
99.9
7.5
112.8
3.3'
9.9
679.7
89.9
657.7
217.9
628.5
70.2
97.4
24.2
27.0
% . *\
5.0
120.0
270.4
0.7
9.4
238.9
30.9
2.2
10.4
60.4
0.9
596.9
30.1
0.2
146.6
2.2
311.9
191.6
2963.6
69.8
345.3
Vil.'\
J.O
237.4
108.2
3.4
0.1
232.9
55
-------
Table 17. Imports of Nitroaromatic Chemicals (U.S. International Trade
Commission, 1967-1973) (Cont'd)
(1000 Ibs)
Compound
4-Nltro-m-cresol
4-Nit rodipheriylamine
5-Nitro-l-diazo-2-naphthol-4-sulfonic acid 33.6
3-Nitro-£-phenetidine
4-Nitro-£-phenetidine
5-Nitro-£-phenetidine
5-Nitro-£-phenetidine
jn-Nitrophenol
£-Nitrophenol
£-Nitrophenol
2-Nitro-£-phenylenediamine
4-Nitro-m-phenylenediamine
4-Nit ro-o-pheiiy lenediamine
l-(m-Nitrophenyl)-5-oxo-2-pyrazoline-3-
carboxy.lic acid
£-Nitrophenylphosphate, disodium salt
£-Nitrotoluene
2-Nitro-m-toluic acid
3-Nitro-o-toluic acid
3-Nitro-£-toluic acid
2-Nitro-£-toluidine
4-Nitro-£-toluidine
5-Nitro-£-toluidine
£-Nitro-£-xylene
Pentachloronitrobenzene
Trinitrotoluene
1967 1968 1969 1970 1971 1972 . 1973
33.6
319.3
6. A
0.1
200.9
33.1
15.2
30.0
49.8
17.7
0.8
50.1
0.4
9.9
20.0
33.1
3.8
0.2
11.6
0.6
0.6
0.2
18.0
1.1
31.5
132.4
ioo.o
124.3
58.3
5.5
187.6
0.5
0.1
8.2
13.2
47.7
31.6
2.5
19.1
534.3
0.4
0.1
2.9
.
134.4
4.6
62.1
2.0
4.0
21.6
1610.3
0.6
1.1
0.5
4.6
0.2
4.4
1.8
334.1
7.4
407.1
0.04
459.6
16.7
7.4
1.5
19.1
2237.0
3.2
0 ^
4.2
3.4
0.06
0.6
50.9
2.4
56
-------
compounds that are produced annually in over 500,000 Ibs was developed. The
compounds are listed in descending order of production volume in Table 18.
In some cases, the information may not be exact, but the relative magnitudes
of production volumes are believed to be accurate. From Table 18 it is
apparent that only about a half dozen nitroaromatic compounds are produced
in truly large scale. (Austin (1974) suggests 50 million Ibs/year as a
reasonable breaking point between large scale and modest production.) Further-
more, some of the large production compounds are related by synthesis to other
large volume nitroaromatic chemicals. For example, 50% of £-chloronitrobenzene
is consumed in the production of £-nitrophenol, and _p_-nitrophenol is in turn
consumed (87% of the total) in the production of methyl and ethyl parathion.
2. Producers and Production Sites
The Chemical Index contains a complete list of commercial nitro-
aromatic compounds with producers and production sites. Table 19 contains in-
formation on producers and plant capacities and locations only for the major
nitroaromatic compounds listed in Table 18.
3. Production Methods and Processes
a. General
Although nitroaromatic compounds may be produced by a
number of synthetic steps, the one common reaction that they all undergo is
direct nitration with a combination of nitric and sulfuric acids (mixed
acid). This step may occur in a variety of positions in the synthesis se-
quence (see Section I-B-1, p. 21 for synthesis strategies). Both batch and
continuous operations are used for the nitration step and the procedure
used is dependent upon the quantity produced. Production methods for some
57
-------
J
1
oo
s*
Table 18. Production Volumes of Major Commercial Nitroaromatic Compounds (U.S. International Trade
Commission, 1959-1973, 1967-1973; Chemical Marketing Reporter, 1969, 1972, 1973 a,
1974 a, b; Industry Sources)
Compound
Nitrobenzene
2,4-(and 2,6)Dinitrotoluene
2,4,6-Trinitrotoluene
l-Chloro-4-nitrobenzene
£-Nitrophenol and sodium salt
l-Chloro-2-nitrobenzene
0-0-Dimethyl-o,£-nitrophenylphosphorothioate
Unethyl parathion)
S s °^Trifluoro-2,6-dinitro-N,N-
dipropyl-£-toluidine (Trifluralin)
£-Nitrotoluene
0-0-Diethyl-£,£-nitrophenylphosphorothioate
(Parathion)
o-Nitrophenol
£-Nitroaniline
Ji,
Approximate Volume
millions of pounds
655,000
(demand, 1974)
471,237
(production, 1973)
432,000
(production, late 1973)
110,000
(production)
60-100,000
\
60,000
48,890
(production, 1973)
25,000
(production, 1972)
20-25,000
Most Recent
Statistics
Same
107,000 (1974)
Reference
CMR (1974b)
USITC (1959-73)
Rosenblatt et al.
(1973)
Industry sources
CMR (1974a)
35,000 demand (1972) Industry sources
CMR (1972)
34,226
(production, 1967)
17,750
(production, 1968)
15,259
(production, 1970)
10-15,000
^14,000
11,029
(production, 1968)
10,500
(demand, 1973)
Industry sources
USITC (1959-73)
USITC (1959-73)
Lawless et al. (1972)
Industry sources
USITC (1959-73)
USITC (1959-73)
Industry sources
Industry sources
USITC (1959-73)
CMR (1969)
-------
Table 18. Production Volumes of Major Commercial Nitroaromatic Compounds (U.S. International Trade
Commission, 1959-1973, 1967-1973; Chemical Marketing Reporter, 1969, 1972, 1973 a,
1974 a, b; Industry Sources) (Cont'd)
VO
Compound
I,3-Dinitrobenzene
p-Nitrotoluene
4,4'-Dinitrostilbene-2,2'-disulfonic acid
5-Nitro-o-toluenesulfonic acid
l-Chloro-3-nitrobenzene
l-Chloro-2,A-dinitrobenzene
1-Nitronaphthalene
o-Nitroaniline
ra-Nitrobenzenesulfonic acid and sodium salt
1,2-Dichloro-4-nitrobenzene
2-sec-Butyl-4,6-dinitrophenol
2-Chloro-4-nitroaniline
o-Nitroanisole
£-Nitroanisole
2,4-Dinitrophenoi
2-Bromo-4,6-dinitroaniline
Approximate Volume
millions of pounds
-^12,000
10-12,000
9,230
(production, 1972)
7,955
(production, 1973)
7,908
(production, 1966)
6,626
(production, 1968)
6,'2 90
(production, 1972)
6,000
3,654
(production, 1970)
3,000-3,600
3,000
2,500-3,000
2,500-3,000
750-1,500
1,000
944
(production, 1973)
Most Recent
Statistics
541
(import, 1973)
863
(production, 1968)
Reference
Starr (1972)
Industry sources
USITC (1959-73)
USITC (1959-73)
USITC (1959-73)
USITC (1959-73)
Industry sources
Industry sources
USITC (1959-73)
Industry sources
Lawless et^ al. (1972)
Industry sources
USITC (1967-73)
Industry sources
Industry sources
Industry sources
USITC (1959-73)
USITC (1959-73)
-------
Table 18. Production Volumes of Major Commercial Nitroaromatic Compounds (U.S. International Trade
Commission, 1959-1973, 1967-1973; Chemical Marketing Reporter, 1969, 1972, 1973 a,
1974 a, b; Industry Sources) (Cont'd)
Compound .
m-Nitrobenzoic acid and sodium salt
2-Nitro-£-toluidine
1,4-Dichloro-2-nitrobenzene
4-Chloro-3-nitrobenzenesulfonamide
4-Chloro-2-nitrotoluene
2,4-Dinitroaniline
2,6-Dichloro-4-nitroaniline
2-Chloro-5-nitrobenzenesulfonic acid and
sodium salt
7- (and 8)Nitronaph[1,2]oxadiazole-5-
sulfonic acid
Approximate Volume
millions of pounds
911
(production, 1969)
864
(production, 1967)
700-800
743
(production, 1973)
696
(production, 1963)
>679
(import, 1972)
607
(production, 1966)
500-600
551
(production, 1969)
Most Recent
Statistics
623
(production, 1967)
396
(production, 1964)
368
(production, 1967)
Reference
USITC (1959-73)
USITC (1959-73)
USITC (1959-73)
USITC (1959-73)
.USITC (1959-73)
USITC (1967-73)
USITC (1959-73)
Industry sources
USITC (1959-73)
USITC (1959-73)
3,5-Dinitrobenzoic acid
Industry sources
-------
Table 19. Major Nitroaromatic Compound Producers, Capacities, and Plant Locations
Compound
N.i trobenzene
.2,4-(and 2,6)Dinitrotoluerie
2,4,6-Trinitrotoluene (TNT)
l-Chloro-4-nitrobenzene
£-N.itrophenol and sodium salt
l-Chloro-2-nitrobenzene
Man_ujE a c t u rer
Allied Chemical
American Cyanamid
DuPont
First Chemical
Mobay
Monsanto
Rubicon
DuPont
Mobay
government-owned
contractor-operated
facilities
DuPont
Monsanto
DuPont
Martin Marietta
Monsanto
Northern Chem
Searle
American Color &
Chem.
DuPont
Monsanto
Moundsville, WV
Bound Brook, NJ
Willow Island, WV
Gibbstown, NJ
Beaumont, TX
Pascagoula, MS
New Martinsville, WV
Sauget, IL
Geismar, LA
Deepwater, NJ
Cedar Bayou, TX
New Martinsville, WV
Radford, VA
Newport, IN
Chattanooga, TN
Joliet, IL
Deepwater, NJ
Sauget, IL
Deepwater, NJ
Sodyeco, NC
Anniston, AL
Sauget, IL
Franklin, NJ
Norwood, OH
Lock Haven, PA
Deepwater, NJ
Sauget, IL
Annual
Capai- ity
(1Q6 Ibs)
55
85
. 60
200
310
135
135
10
75
108
180
360
432
45
(90 by 1977)
95
15
1
24
12
Reference
Chemical Marketing
Reporter (1974b)
SRI (1975)
Nay, 1972
Forsten (1973)
Rosenblatt et al.,
(1973)
Chemical Marketing
Reporter (1974a)
Anon. (1975a)
Anon. (1975b)
Chemical Marketing
Reporter (1972)
SRI (1975)
SRI (1975)
(see 1-chloro-
4-nitrobenzene)
-------
Table 19. Major Nitroaromatic Compound Producers, Capacities, and Plant Locations (Cont'd)
Compound
a b
Methyl and ethyl parathion
Trifluralin
£-Nitrotoluene
£-Nitrophenol
£-Nitroaniline
ui--Dinitrobenzene
o-Nitrotoluene
4,4'-Dinitrostilbene-2, 2' -
disulfonic acid
5-Nitro-o_-toluenesulfonic acid
l-Chloro-3-nitrobenzene
l-Chloro-2,4-dinitrobenzene
Manufacturer
Hercules
Kerr-McGee
Monsanto
Stauffer
Vicksburg Chem.
Eli Lilly
DuPont
First Mississippi
Martin-Marietta
Monsanto
American Color &
Chem.
Monsanto
Universal Oil Prod.
DuPont
DuPont
First Mississippi
American Cyanamid
Ciba-Geigy
GAF
Toms River
American Cyanamid
DuPont
GAF
Toms River
GAF
Martin-Marietta
Plant Location
Plaquemlne, LA
Hamilton, MS
Anniston, AL
Mt. Pleasant, TN
Vicksburg, MS
Lafeyette, IN
Deepwater, NJ
Pascagoula, MS
Sodyeco, NC
Sauget, IL
Lock Haven, PA
Sauget, IL
McCook, IL
Deepwater, NJ
Deepwater, NJ
Pascagoula, MS
Bound Brook, NJ
Mclntosh, AL
Rensselaer, NY
Toms River, NJ
Bound Brook, NJ
Deepwater, NJ
Rensselaer, NY
Toms River, NJ
Linden, NJ
Sodyeco, NJ
Annual
Capacity
(IP6 Ibs)
15a
(not operating)
173 .
5°a'£
30a'b
Reference
SRI (1975)
2.0
10.0
3.0
SRI (1975)
SRI (1975)
SRI (1975)
USITC (1959-73)
SRI (1975)
SRI (1975)
SRI (1975)
SRI (1975)
SRI (1975)
-------
Table 19. Major Nitroaromatic Compound Producers, Capacities, and Plant Locations (Cont'd)
ON
Compound
1-Nitronaphthalene
£-Nitroaniline
m-Nitrobenzenesulfonic acid and
salts
1,2-Dichloro-4-nitrobenzene
2-sec-Butyl-4,6-dinitrophenol
2-Chloro-A-nitroaniline
o-Nitroanisole
£-Nitroanisole
2-Bromo-4,6-din itroaniline
m-Nitrobenzoic acid and sodium salt
2-Nitro-£-toluidine
2,4-Dinitrophenol
Manufacturer
DuPont
Monsanto
American Cyanamid
GAF
USM
Blue Spruce
DuPont
Blue Spruce
Dow Chemical
Vicksburg Chem.
Chemetron
DuPont
DuPont
Monsanto
DuPont
American Color &
Annual
Capacity
Plant Location (106 Ibs)
Deepwater, NJ
Sauget, IL
Bound Brook (K only)
Linden, NJ (Na on^.y)
Greenville, SC (Na only)
Edison, NJ
Deepwater , NJ
Edison, NJ
Midland, MI
Vicksburg, MS
Huntington, WV
Deepwater, NJ
Deepwater, NU
St. Louis, MO
Deepwater, NJ
Lock Haven, PA
Reference
Industry sources
SRI (1975)
SRI (1975)
SRI (1975)
Industry Sources
SRI (1975)
SRI (1975)
SRI (1975)
SRI (1975)
SRI (1975)
Chem.
Martin-Marietta
Toms River
Bofors
Salisbury
Sterling Drug
Sherwin-Williams.
Martin-Marietta
Sodyeco, NC
Toms River, NJ
Linden, NJ
Charles City, IA
Cincinnati, OH
Chicago, IL
Sodyeco, NC
SRI (1975)
SRI (1975)
SRI (1975)
-------
Table 19. Major Nitroaromatic Compound Producers, Capacities, and Plant Locations (Contrd)
Compound
1,4-Dichloro-2-nitrobenzene
4-Chloro-3-nitrobenzenesufonamide
4-Chloro-2-nitrotoluene
2,4-Din itroaniline
2,6-Dichloro-4-nitroaniline
2-Chloro-5-nitrobenzenesulfonic
acid and sodium salt
7-(and 8)Nitronaph[1,2]oxodiazole-
5-sulfonic acid
3,5-Dinitrobenzoic acid
Manufacturer
DuPont
Mob ay
GAF
Inmont
Salisbury
Toms River
Plant Location
Deepwater, NJ
Bayonne , N J
Rensselaer, NY
Hawthorne , NJ
Charles City, IA
Toms River, NJ
Annual
Capacity
(106 Ibs)
Reference
SRI (1975)
SRI (1975)
Industry sources
American Color &
Chem.
Synalloy Corp.
American Color &
Chem.
Martin-Marietta
GAF
Kewanee Oil
Upjohn
DuPont
Toms River
GAF
Mobay
Toms River
Ashland Oil
Bofors
Salisbury
Lock Haven, PA
Spartanburg, SC
Lock Haven, PA
Sodyeco, NC
Rensselaer, NY
Louisville, KY
North Haven, CT
Deepwater, NJ
Toms River, NJ
Rensselaer, NY
Bayonne, NJ
Charleston, SC
Toms River, NJ
Great Meadows, NJ
Linden, NJ
Charles City, IA
SRI (1975)
SRI (1975)
SRI (1975)
SRI (1975)
Industry Sources
SRI (1975)
SRI (1975)
-------
of the large production nitroaromatic compounds are reviewed in detail in
the following sections, while production methods for the less important com-
mercial chemicals are briefly outlined in Section I-B-1 (see Figure 6, p. 26).
b. Nitrobenzene (Matsuguma, 1967a; Processes Research, Inc.,
1972)
Nitration of benzene can be carried out in either a batch
or continuous process. The reaction vessels, constructed of cast iron or
steel, are jacketed and generally have external cooling coils for maintaining
temperature control of the strongly exothermic reactions. Emergency "drown"
tanks containing water are also provided, so that a reaction that has gone
out of control can be quenched.
The batch equipment is normally sized for 1000-1500 Ib
quantities of benzene and operates on a 2-4 hour time cycle. A typical run
begins by charging the nitrator with benzene and a heel of spent acid,
followed by slow addition of the mixed acid (53-60% H2S04, 32-39% HNC>3, and
8% HLO) under the surface of the benzene. The reaction temperature is main-
tained at 50-55°C by adjusting the rate of feed, the rate of heat exchange,
and the amount of agitation. Near the end of the reaction the temperature
is usually raised to 90°C to promote completion. The nitrobenzene is removed
from the spent acid by gravity separation. (About 0.5% of the yield of nitro-
benzene is lost due to incomplete separation; Matsuguma, 1967a.) The spent
acid, which is drawn off from the bottom of the separator, is either recovered
or used to start, subsequent runs. The crude nitrobenzene can be used directly
in the manufacture of aniline (^ 97% of the nitrobenzene produced is used
directly for aniline synthesis). However, if pure nitrobenzene is required,
the product is washed with water and dilute sodium carbonate and then distilled.
65
-------
In newer plants, which usually use a continuous process,
the sequence of operation is essentially the same. The main differences are
that smaller reaction vessels, lower nitric acid concentrations, and higher
reaction rates are used. For comparable production capacity, a 30-gal stain-
less steel continuous nitrator can be used instead of the 1500-gal batch
nitrator. Because a high speed (600 rpm) agitator is used, a reaction time of
only 15-20 minutes is required. Typical yields from the continuous reactor
are 96-98% of theoretical, compared to 95-98% for batch process nitration.
A flow diagram for a representative nitrobenzene plant is presented in Figure 10.
Two other methods which could be used for the production
of nitrobenzene have been explored: 1) continuous vapor phase nitration (this
eliminates the use of sulfuric acid) and 2) tubular reactors for reaction of
aromatic hydrocarbons with mixed acid (yields up to 99.3% are possible). Whether
these processes have reached commercial scale yet is unknown.
c. Dinitrotoluene (Processes Research, Inc., 1972)
As with nitrobenzene, dinitrotoluene can be produced by
batch or continuous process. The starting material for dinitrotoluene is
mononitrotoluene, either 2-nitrotoluene or 4-nitrotoluene, although toluene
itself is sometimes used. If 2-nitrotoluene is used, the product will contain
the 2,6-dinitrotoluene isomer. The continuous process usually consists of
several reactors joined in series. The raw materials are added only to the
first reactor with the successive kettles providing additional reaction time.
The mixed acid that is used in this process has an approximate composition of
72% H2S04, 17% HN03, and 11% H^O (made from 50-60% HNC>3 and 93% H2S04) and
66
-------
CRUDE HIT ROBE tire Ne
Figure 10. Nitrobenzene Process (Processes Research, Inc., 1972)
67
-------
the exothermic reaction is maintained at 75-85°C. The overall yield for the
reaction is approximately 96%.
The product is removed from the spent acid in a decanter
and the spent acid is recycled. In the Meissner process , three washing and
neutralization steps are used. A flow sheet for the washing steps and the
nitrators is presented in Figure 11. Most of the product that is formed goes
directly to a reduction step for the formation of diaminotoluene, but some
material may be distilled if high purity 2,4-dinitrotoluene is required.
d. Chloronitrobenzene Process
Chloronitrobenzenes are produced by nitration of chloro-
benzene in equipment very similar to that used for the previously-described
nitration processes. The reaction is easily carried out, even though the
chlorine substituent slightly deactivates the ring to electrophilic substitution
reactions.
A typical plant operates with a batch process, using
2640-gal reactors into which are fed 5500 Ibs of spent acid and 10,000 Ibs of
chlorobenzene, followed by 15,600 Ibs of mixed acid (53% H2SOA» 35% HN03>
and 12% HO).
The crude Chloronitrobenzene (process yield is not
available) obtained from the nitration process contains about 34% o-chloro-
nitrobenzene, 65% £-chloronitrobenzene, and 1% m-chloronitrobenzene (Matsuguma,
1967a). The isomers are separated by a combination of crystallization and
distillation. The crude material is first cooled to 16°C, where 50% of the
para-isomer crystallizes out as pure product. The mother liquor is then
68
-------
bEC.ANTER
1
Ol
I
DECANrER
PRODUCT^
^HiQ
Figure 11. 2,4-Dinitrotoluene Process (Processes Research, Inc., 1972)
69
-------
fractionally distilled at reduced pressure. The flow and processing of the
various cuts are illustrated in Figure 12.
e. Trinitrotoluene Processes
Trinitrotoluene can be prepared by a countercurrent con-
tinuous flow process using toluene as a starting material. The usual practice
is to nitrate toluene stepwise to mono-, di-, and then trinitrotoluene.
Between the three steps, the nitrated toluenes and the mixed acids flow in
opposite directions, so that the third stage receives the strongest acid,
which becomes weaker in the second stage and weaker still in the first stage.
A flow diagram for the countercurrent continuous process is presented in
Figure 13.
There are a few differences between the TNT process and
the previously described nitration processes. The final TNT product has
to have a high degree of isomer purity. (Unsymmetrical isomers cause random
explosivity.) In order to remove the undesirable unsymmetrical isomers
(non-2,4,6-isomers), a sodium sulfite (sellite) washing is used after the
base wash. Sulfite reacts with the unsymmetrical isomers, solubilizing them
in the wash water. Another difference is that the amount of 93% sulfuric
acid produced in the acid concentrator exceeds what can be used in the process,
so this becomes a by-product.
Nay (1972) (see also Rosenblatt et al., 1973) reported
that a more automated continuous countercurrent process is presently being
used to produce TNT at the Radford Army Ammunition Plant. A major difference
is that no "red water" (sellite water) is released as waste. It is re-
cycled as much as possible and then routed for incineration of combustibles
70
-------
Figure 12. Monochloronitrobenzene Process (Processes Research, Inc., 1972)
-------
to
»!*.
&L.
V
?A
^
T,
j ».
i
i
/)
.1
oec ANT
Ht0
li
DEC AWE*.
OECANTPR
, r/vr
PRODUCT
Figure 13. Continuous Countercurrent Trinitrotoluene Process (Processes Research, Inc., 1972)
-------
and oxidation of sodium sulfite to marketable sodium sulfate. With the TNT
process, 100 Ibs of finished TNT requires about 47 Ibs of toluene, 210 Ibs
of oleum (H-SO,), 125 Ibs of nitric acid, 6 Ibs of sodium sulfite, 1 Ib of
soda ash, and 650 Ibs of water.
Recently, a low temperature TNT process has been reported
by Haas et^ a±. (1975). The basic feature consists of low-temperature (-8°C)
dinitration, followed by higher temperature (90°C) trinitration.
f. Nitrophenol Processes (Matsuguma, 1967b)
Because of the presence of a hydroxyl group on the benzene
ring, phenols can be nitrated readily; however, since they are also readily
oxidized under .nitration conditions, direct nitration is not used commercially
to produce nitrophenol. Instead, the commercial process uses hydrolysis of
chloronitrobenzenes with aqueous sodium hydroxide at elevated temperatures.
For example, when £-chloronitrobenzene is heated for four hours at 160°C with
15% aqueous sodium hydroxide, good yields of ^-nitrophenol are obtained.
Generally, only o- or £-chloronitrobenzene is used to make nitrophenols by
hydrolysis because the chlorine substituent has been activated by the nitro
group. (This is not the case with m-chloronitrobenzene.)
4. Market Prices
Reported prices of nitroaromatic compounds vary considerably
and appear to be generally related to the quantity produced. Table 20 compares
the price and production volume of a number of nitroaromatic compounds. The
compounds in the range of ten cents per pound are those produced in very high
quantities. The lower-priced chemicals appear.to be simpler chemical
entities, suggesting less complex (fewer synthesis steps), and, therefore,
73
-------
Table 20. Comparative Prices and Production Volumes of Some Nitroaromatic
Chemicals (U.S. International Trade Commission, 1959-1973)
Total Range
of Prices
Production Range
Compound
Nitrobenzene
l-Chloro-2-nitrobenzene
£-Nitrophenol
p_-Nitroaniline
m-Nitrobenzenesulfonic acid
and sodium salt
1,4-Dichloro-2-nitrobenzene
2,4-Dinitroaniline
4-Chloro-2-nitroaniline
2-Chloro-4-nitroaniline
2-Nitro-p_-toluidine
2-Amino-5-nitrobenzenesulfonic
acid
5-Nitro-o-toluidine
Dinitrobutylphenol, ammonium st
2,6-Dichloro-4-nitroaniline
4-Nitro-o-anisidine
Reported ($/lb)
0.11-0.06
0.06-0.09
0.42
0.42-0.43
0.34-0.43
0.56
0.72-0.79
0.78-0.84
0.88-0.93
1.03-1.25
1.22
1.32-1.60
Lt 1.57-1.80
1.13-1.74
2.00-2.38
Time Range
1959-72
1963-69
1962
1962, 1965
1959-70
1960
1967-71
1959-68
1963, 1968
1959-65
1961
1959-65
1966-67
1963-66
1959-62
Reported (1000 Ibs
172,123-551,169
17,177-34,226
13,092-18,935
8,769-12,478
1,472-3,711
276-793
164-207
172-566
275-448
864-1,573
23-72
99-397
58-85
19-607
73-144
74
-------
cheaper production processes. Table 21 contains recent market prices for
nitroaromatic chemicals.
5. Market Trends
Nitroaromatic compounds are used mostly as chemical intermediates
for dyes, pigments, Pharmaceuticals, rubber chemicals, photographic chemicals,
and agricultural chemicals. These markets appear to be fairly stable, and,
as a result, nitroaromatic chemical production and consumption has, in general,
remained constant or increased (see Figure 9, p. 53 for production trends).
During 1963-1973, nitrobenzene experienced an average growth
rate of 10.6% per year, and a 7% increase per year through 1978 is projected
(Chemical Marketing Reporter, 1974 b). Most of this growth is attributed to
the growth of aniline, the major (97%) application; the market for aniline
continues to grow. Major applications for aniline include isocyanates (40%),
rubber chemicals (35%), dye stuffs and intermediates (6%), hydroquinone (6%),
drugs (4%), and miscellaneous (9%) (Anon., 1974). The total market for ani-
line, which was 535 million Ibs in 1974, is projected to grow at 9.5% per
year through 1977 (Chemical Marketing Reporter, 1973 b).
Dinitrotoluene should also look forward to a growth market
because of its use in the production of toluene-2,4-diisocyanate. Toluene
diisocyanate accounted for 67% of the total isocyanate production in 1970
(Dean, 1971). From 1965 to 1970, the United States isocyanate production
grew by an average of 21% per year and a 12-15% annual growth rate for
1970-1975 was projected. Isocyanates are used almost exclusively to produce
polyurethane polymers.
75
-------
Table 21. Recent Market Prices of Nitroaromatic Chemicals (Chemical Marketing Reporter, 1974 c,
1975 a, b)
Chemica 1
2-Chloro-A-nitroaniline
paste, delivered, East 100% basis
powder, delivered, East 100% basis
4-Chloro-2-7nitroaniline
powder, delivered, East
4-Chloro-2-nitrophenol, technical
paste, drums, freight allowed
4-Chloro-2-nitrotoluene, technical
solid, drums, freight allowed
6-Chloro-2-nitrotoluene, technical
solid, drums, freight allowed
2,6-Dichloro-4-nitroaniline
drums, 10,000 Ibs or more, works
2,4-Dinitroaniline
drums, delivered
Dinitroaniline, orange toner
chemically pure, bags, delivered •, East
m-Dinitrobenzene, 89°, technical
drums, truckload
2,4-Dinitrochlorobenzene
47°C, f.o.b., Charlotte, NC
2,4-Dinitrophenol, drums
2,4-Dinitrotoluene, drums, carload
truckload, works
tanks, works
Methyl parathion, technical, 80%
drums, freight allowed, East
Musk, synthetic ambrette
drums, 100 Ib lots
Musk, synthetic, xylol, drums
1.00 Ib lots
m-Nitroaniline
crystalline, drums, freight allowed
paste, drums, freight allowed, 100%
Price ($/lb)
November A, 1975 April 21, 1975
Low High
Low
October 20, 1975
Low High
0.95
1.05
0.86
0.5
0.99
0.40
1.38
1.20
2.85
0.36
0.53
0.90
0.24
0.225
0.48
3.20
1.08
1.40
1.33
0.95
3.10
0.50
0.95
1.05-
0.86
0.75
0.99
0.40
1.38
1.20
2.85
0.36
0.53
1.04
0.24
0.225
0.85
3.20
1.08
1.40
1.33
, — —
0.95
3.10
0.95
1.05
0.86
0.75
0.99
0.40
1.38
1.20
2.85
0.36
0.53
1.04
0.24
0.225
0.92
5.90
2.50
.1.40
1.33
0.95
3.00
1.00
6.00
2.60
-------
Table 21. .Recent Market Prices of Nitroaromatic Chemicals (Chemical Marketing Reporter, 1974 .
1975 a, b) (Cont'd)
•Chemical
November 4, 1975
Low High
o-Nltroaniline
flake, drums, truck load, works 0.40
molten, tanks, freight, works
orange toner, bags, freight allowed 1.90
£-Nitroaniline, drums, carlot truckload; 30,000 Ibs min.,
works '.-..• 0.52
o-Nitroanisole, technical, tanks, works —
£-Nitroanisole, technical, solid, drums, freight allowed 0.72
Nitrobenzene, double-distilled, tanks, works 0.19
£-Nitrobenzoic acid, drums, carlot, truckload, works 0.50
o-Nitrochlorobenzene
drums, carlot, freight allowed 0.39
tanks, same basis 0.34
£-Nitrochlorobenzene
drums, carlot, truckload, works 0.39
tanks, same basis 0.34
2-Nitro-p-cresol, technical, drums, truckload,
freight allowed . 0.71
£-Nitrophenol, drums, f.o.b., works 0.45
tanks, same basis 0. 43
drums, less truckload,.freight allowed 0.47
m-Nitrotoluene, technical, drums, freight allowed 0.60
£-Nitrotoluene, drums, carlot, freight allowed 0.16
tanks, freight allowed 0.14
£-Nitrotoluene, technical, drums, carlot, works 0.27
tanks, works 0.20
2-Nitro-£-toluidine, drums, f.o.b., works 1.25
Parathion, ethyl, drums, freight allowed 0.50
Picric acid
pure paste, 300 Ib drums, dry basis, f.o.b. Charlotte, NC 2.00
tech. paste, 300 Ib drums, dry basis, f.o.b.
Charlotte, NC 1.40
0.22
0.53
Price ($/lb)
April 21, 1975
Low t High
0.74
0.84
0.75
0.72
0.19 0.22
0.50
0.49
0.42
0.43
0.38
0.71
0.45
0.43
0.47
0.60
0.14
0.14
0.27
0.20
1.25
0.87
2.00
1.76
October 20, 1975
Low . High
0.77
0.74
1.90
0.84
0.75
0.72
0.19 0.22
6.50
0.49
0.42
0.43
0.38
0.71
0.45
0.43
0.47
0.60
0.16
0.14
0.27
0.20
1.25
0.87
2.00
1.76
-------
Other major nitroaromatic compounds have experienced considerable
growth and are projected to continue that trend. Table 22 summarizes past
growth patterns and predictions of future growth. Markets for £-chloronitro-
benzene (50% to £-nitrophenol) and p_-nitrophenol (87% to parathions) are very
dependent upon parathion consumption, which in turn is dependent upon cotton
planting, and hence upon natural conditions. No growth was projected for £-
nitroaniline, because most of the end-uses for the compound are under attack
from other chemicals (Chemical Marketing Reporter, 1969). Inorganic pigments
or other light-fast materials, for example, have virtually replaced pigments
made from £-nitroaniline.
Table 22. Market Trends of Major Nitroaromatic Chemicals (Chemical
Marketing Reporter, 1969, 1972, 1973 a, 1974 a, b)
Compound
£-Chloronitrobenzene
£-Nitroaniline
Nitrobenzene
£-Nitrophenol
Parathions
Historical Growth
2% (1964-73)
2.1% (1962-68)
10.6% (1963-73)
11.5% (1962-71)
10.5% (1962-72)
Projected Growth
3.5% (through 1978)
no growth (through 1973)
7% (through 1978)
3% (through 1976)
3% (through 1977)
78
-------
B. Uses
1. Major Uses
Although some nitroaromatics are used as explosives (e.g., TNT),
the major applications of nitroaromatic compounds are as chemical intermediates
for dyes, pigments, Pharmaceuticals, rubber chemicals, photographic chemicals,
and agricultural chemicals. The applications of the major nitroaromatic com-
pounds are listed with approximate volumes, when available, in Table 23. The
large volume agricultural intermediates (50% of l-chloro-4-nitrobenzene and
£-nitrophenol) are somewhat unusual in that the nitro group is not used as a
means of introducing an amine function, as is frequently the case with most of
the other chemical intermediate applications.
Amination by reduction (Shreve, 1963) of the nitro functional
group consumes considerable quantities of nitroaromatic intermediates (see
Table 23). Reductive amination is a very old commercial process which can be
traced back to 1847 when aniline was first manufactured commercially from
nitrobenzene (Kouris and Northcott, 1963). The aromatic amines that are
formed were initially consumed in the dye and pigment industry, but now a
major portion of the production is consumed in the manufacture of such com-
pounds as isocyanates (for production of polyurethanes) and rubber chemicals
(aniline-based antioxidants and thiazole accelerators). The chemistry and
synthesis approaches for the various chemicals derived from nitroaromatic com-
pounds have been reviewed in the section on chemistry (Section I-B-1, p. 21).
This section will discuss briefly some of the commercial reduction processes.
Reduction of nitrobenzene to aniline was first accomplished
by the Be'champ process employing iron turnings and hydrochloric acid. The
79
-------
Table 23. Uses of Major Nitroaromatic Chemicals
Compound
Nitrobenzene
Application or
Chemical Product
Aniline
Other (chemical intermediate in
dyes such as benzidine and
explosives; Friedel-Crafts
solvent; solvent in petroleum
refining; mild oxidation agent
CO
o
2,4-(and 2,6)Dinitro-
toluene
2,4,6 Trinitrotoluene (TNT)
l-Chloro-4-nitrobenzene
j>-Nitrophenol and
sodium salt
l-Chloro-2-nitrobenzene
Approximate Volume of
Nitroaromatic Compound
Consumed, % of
millions of pounds Total
635,000
20,000
^470,000
^50,000
432,000
Toluene diisocyanate
Toluene diamine
Produce 2,4-dinitrotoluene for
smokeless powder
Major ingredient for composition B
military explosive
High explosive and propellant
component
Chemical intermediate for
2,4,6-trinitrobenzoic acid
p-Nitrophenol
p-Nitroaniline
Miscellaneous agricultural chemicals
(excluding parathions)
Phenacetin
p-Aminophenol
Rubber chemicals
Other
Total
Ethyl and methyl parathions 52-87,000
All other 8,13.000
Total 60-100,000
Chemical intermediate for _o- ^60,000
chloroaniline, o-nitroaniline,
£>-anisidine, ^-phenetidine,
o-aminophenol, and others
55
19
11
3
5 . 5
11
5.5
97
3
100
50
17
10
3
5
10
_5
100
87
13_
100
100
Reference
CMR (1974b)
Matsuguma (1967a)
Gilbert (1969)
Lindner (1965)
Lurie (1964)
Olah and Cupas (1966)
Nelson (1968)
USITC (1959-73)
Austin (1974)
Thirtle (1968)
Small and Rosenblatt (1974)
Rosenblatt et^ al. (1973)
Rinkenbach (1965)
Lindner (1965)
Dressier (1968)
CMR (1974a)
Industry sources
CMR (1972)
Industry sources
Matsuguma (1967a)
Wooster (1963)
Kouris and Northcott (1963)
-------
Table 23. Uses of Major Nitroaromatic Chemicals (Cont'd.)
Compound
Methyl parathion
Trifluralin
£-Nitrotoluene
oo
Parathion
jj-Nitrophenol
JJ-Nitroaniline
1,3-Dinitrobenzene
Application or
Chemical Product
3,3*-Dichlorobenzidine
Cotton insect poison
Pesticide
5-Nitro-o-toluenesulfonic acid
Others (p_-toluidine, other dye
intermediates, TNT)
Total
Pesticide
Dye intermediate
Approximate Volume of
Nitroaromatic Compound
Consumed, % of
millions of pounds Total
2,800 ^5
48,890
25,000
^5,000
15-20.000
20-25,000
15,259
10-15,000
Rubber antioxidant 5,600
Gasoline additives 2,800
Dyes and pigments 2,800
Pharmaceutical and veterinary use 980
Agricultural chemicals 420
Miscellaneous 1,400
Total Vi.4,000
Intermediate for m-phenylenediamine ^12,000
Possible TNT replacement
Cathodic material in batteries
40
20
20
7
3
10
100
VLOO
Reference
Back calculation assuming
100% yield
USITC (1959-73)
CMR (1973a)
Lawless et al. (1972)
Plimmer (1970)
Matsuguma (1967a)
and back calculations from
production data for other
compounds (assume 100%
yield)
USITC (1959-73)
Industry sources
Matsuguma (1967b)
Shreve (1963)
CMR (1969)
o-Nitrotoluene
c>-Toluidine and other dye
intermediates
10-12,000
Industry sources
Kouris and Northcott (1963)
Thirtle (1968)
Starr (1972)
Russell (1971)
Almerini (1966)
Doe and Wood (1968)
Matsuguma (1967a)
-------
Table 23. Uses of Major Nitroaromatic Chemlcala (Cont'd)
oo
Approximate Volume of
Nitroaromatic Compound
Compound
Application or
Chemical Product
Consumed,
millions of pounds
% of
Total
4,4'-Dinitrostilbene-2,2'- Key intermediate for stilbene dyes
disulfonic acid
5-Nitro-o-toluenesulfonic
acid
l-Chloro-3-nitrobenzene
4,4-Dinitrostilbine-2,2'-disulfonic
acid (optical brightener
intermediate)
_m-Chloroaniline, 2,2'-dichloro-
benzidene, and other dye
intermediates
9,230
7,955
7,908
l-Chloro-2,4-dinitrobenzene Chemical intermediate for azo dyes, 6,626
sulfur blacks, fungicides, rubber
chemicals, and explosives (e.g. 2,4-
dinitroaniline, 4-nitro-2-anisidine,
4-chloro-3-anisidine, 4-chloro-l,3-
phenylenediamine, and picric acid)
2,4-Dinitrophenol 1,100
1-Nitronaphthalene
o-Nitroaniline
in-Nitrobenzenesulf onic
acid and sodium salt
l,2-Dichloro-4-nitro-
benzene
2-sec_-Butyl-.i, 6-dinitro-
phenol
ct-Naphthylamine 6,290
Known as Azoic Diazo Compound 6 6,000
(C.I. 37025); used to prepare a
few azo and anthraquinone dyes
Dye intermediate 3,654
Intermediate for 2-Chloro-4-
nitroaniline
Pesticide
3,000-3,600
3,000
VLOO
100
16
100
Reference
Schwander and Dominguez
(1969)
Schwander and Dominguez
(1969)
Matsuguma (1967a)
Matsuguma (1967a)
Wolfson (1967)
Kouris and Northcott
(1963)
Back calculation assuming
100% yield
Industry sources
Treibl (1967)
Kouris and Northcott
•(1963)
Bannister and Olin (1965)
Industry sources
Wooster (1963)
Lawless et al. (1972)
-------
Table 23. Uses of Major Nitroaroma-tie Chemicals (Cont'd)
00
u>
Compound
2-Chloro-4-nitroaniline
•^.
o-Nitroanisole
£-Nitroanisole
2,4-Dinitrophenol
2-Bromo-4,6-dinitro-
aniline
m-Nitrobenzoic acid and
sodium salt
2-Nitro-p_-toluidine
l,4-Dichloro-2-nitro-
benzene
;4-Chloro-3-nitrobenzene-
sulfonamlde
4-Chloro-2-nitrotoluene
2,4-Dinitroaniline
Application or
Chemical Product
Dye intermediate
Anisidine intermediate
Anisidine intermediate
Approximate Volume of
Nitroaromatic Compound
Consumed, % of
millions of pounds Total
2,500-3,000
2,500-3,000
750-1,500
Chemical intermediate for sulfur 1,000
dyes, azo dyes (2,4-diamino-
phenol, 4-nitro-2-aminophenol);
photochemicals, pest control agents,
wood preservatives, and explosives.
Polymerization inhibitor in styrene
production
Dye intermediate 944
Chemical intermediate for azo dyes 911
Dye intermediate 864
Mostly 2,5-dichloroaniline 700-800
Other dyes (4-chloro-2-nitro-
aniline, 4,4l-dichloro-2-amido-
phenyl ether, 4-chloro-2-nitro-
phenol, and 4-chloro-2-nitroanisole)
Chemical intermediate 743
Azo dye intermediate (e.g., 4-chloro- 693
2-toluidine)
Intermediate, toner, azo dye >679
intermediate
Reference
Industry sources
Industry sources
Industry sources
Matsuguma (1967 b)
Coulter et al. (1969)
Stenger and Atchison
(1964)
Duncker (1964)
Bannister and Olin (1965)
Ehrich (1968)
Johnson et^ al. (1963)
Industry sources
Matsuguma (1967a)
Wooster (1963)
USITC (1959-73)
Matsuguma (1967a)
Kouris and Northcott (1963)
Ehrich (1968)
Matsuguma (1967a)
-------
Table 23. Uses of Major Nitroaromatic Chemicals (Cont'd)
Compound
Application or
Chemical Product
2,6-Dichloro-4-nitroaniline Fungicide
Dye intermediate
Pigment intermediate
Synthesis of diarylamines
2-Chloro-5-nitrobenzene-
sulfonic acid and
sodium salt
7-(and 8)Nitronaph[l,2]- Chemical intermediate
oxadiazole-5-sulfonic acid
3,5-Dinitrobenzoic acid
00
Reagent for identification
of alcohols
Explosives ingredient
Approximate Volume of
Nitroaromatic Compound
Consumed,
millions of pounds
607
500-600
551
^500
% of
Total
Reference
Cappellini and Stretch
(1962)
McMillan (1972)
Industry sources
Kehe (1965)
USITC (1959-73)
Duncker (1964)
Pristera et al. (1960)
-------
oxidation of the iron to the ferrous or ferric ion results in reduction of the
nitro group to an aromatic amine by the following equation:
FeClo
RN02 + 3Fe + 4H20 *+ RNH2 + Fe(OH)2 + FeO + Fe(OH)3 + (H)
The Be"champ process may still be used in some small batch reactions, but has
been replaced in most large scale reductions by catalytic hydrogenation.
Practically all nitroaromatic compounds can be reduced by
catalytic hydrogenation using either vapor or liquid (usually alcoholic solu-
tion) phases (Shreve, 1963). However, hydrogenation is not selective enough
for partial reductions of compounds containing more than one nitro group, and,
in those cases where partial reduction is required, other reducing agents have
to be used (see Section I-B-1). Perhaps a typical hydrogenation plant is the
fluid-bed catalytic vapor phase hydrogenation plant operated by American Cyanamid
at Willow Island, WV, which produces aniline from nitrobenzene (see Figure 14).
The feedstock consists of nitrobenzene containing less than 10 ppm nitrothio-
phene. The nitrobenzene is vaporized, mixed with three times the theoretical
amount of hydrogen, and passed over the catalyst (copper on silica). After
cooling with condensation, the condensed aniline, aniline-water (water gener-
ated by the reaction), and excess hydrogen are separated. The crude aniline,
which contains less than 0.5% nitrobenzene and about 5% water, is crudely dis-
tilled. The product is dehydrated and distilled to a purity of about 98%.
Reduction of nitroaromatic compounds under basic conditions
results in the formation of hydrazobenzene derivatives, which are easily con-
verted to benzidine derivatives (see Section I-B-1). Sizable quantities of
nitroaromatic compounds are consumed at benzidine and benzidine-derivative
production plants.
85
-------
Hydrogen
Figure 14. Continuous Fluid-Bed Vapor Phase Reduction of Nitrobenzene
(Shreve, 1963)
1. Nitrobenzene vaporizer 7. Aniline-water settler and decanter
2. Reactor with fluidized catalyst bed 8. Crude aniline still
3. Cooling tubes 9. Reboiler for crude aniline still
4. Catalyst filters 10. Condenser
5. Product condenser 11. Aniline-finishing still
6. Hydrogen recycle compressor 12. Reboiler for aniline-finishing still
(Reprinted with permission from John Wiley & Sons, Inc.)
Table 24 lists the manufacturers of some large volume aromatic
amines that are derived from nitroaromatic compounds. Some other aromatic
amines that have been produced by reduction, but still maintain a nitro sub-
stituent,have been discussed in Section I-B, p. 21).
2. Minor Uses
There are a sizable number of nitroaromatic compounds that are
produced and consumed in small commercial quantities. Information on their
uses is not plentiful or quantitative. The available information, which has
been taken mostly from the Kirk-Othmer Encyclopedia of Chemical Technology, is
tabulated in Table 25. As with the major nitroaromatic compounds, the major
use of the small volume nitroaromatics is as chemical intermediates, with
nitration used for introduction of amine functional groups.
86
-------
Table 24. Large Volume Aromatic Amines Produced by Reduction of Nitroaromatic Compounds (SRI, 1975; Dean, 1971)
Nitroaromatic Compound
Nitrobenzene
Aromatic Amine
Aniline
2,4-(and 2,6-)Dinitro-
toluene
Toluene diisocyanate
(made from toluene—
2,4-diamine)
oo
Toluene-2,4-diamine
Company
American Cyanamid
DuPont
First Mississippi
Mobay
Rubicon
Allied
BASF Wyandotte
DuPont
Mobay
Olin-General Tire
Olin
Rubicon
Union Carbide
Air Products
American Cyanamid
DuPont
GAF
Olin
Rubicon
Union Carbide
Location
Bound Brook, NJ
.Willow Island, WV
Beaumont, TX
Gibbstown, NJ
Pascagoula, MS
New Martinsville, WV
Geismar, LA
Moundsville, WV
Geismar, LA
Deepwater, NJ
New Martinsville, WV
Cedar Bayou, TX
Ashtabula, OH
Lake Charles, LA
Geismar, LA
Institute, WV
Pasadena, TX
Bound Brook, NJ
Deepwater, NJ
Rensselaer, NY
Lake Charles, LA
Ashtabula, OH
Brandenburg, KY
Rochester, NY
Geismar, LA
Institute and South
Charleston, WV
Capacity
(millions
of pounds)
60
50
200
130
100
100
55
70
40
170
100
100
40
90
30
55
-------
Table 25. Uses of Minor Nitroaromatic Chemicals
Chemical
N-Acetyl-4,4'-dinitrodiphenylamine
Use
Intermediate, precursor of 4,4'-diaminodi-
phenylamine
00
CD
2-(£-Aminoanilino)-5-nitrobenzenesulfonic Intermediate
acid
2-Amino^-5-nitrobenzenesulfonic acid Intermediate
6-Amino-4-chloro-5-nitrophenol
2-Amino-4-chloro-5-nitrophenol
2-Amino-4-chloro-6-nitrophenol
2-Amino-6-chloro-4-nitrophenol
2-Amino-4,6-dinitrophenol
(picramic acid)
2-Amino-5-nitrophenol
4-Amino-2-nitrophenol
2-Amino-4-nitrophenol
4-Amino-5-nitrophenol
6-Amino-4-nitro-l-phenol-2-sulfonic acid
4-Amino-6-nitro-l-phenol-2-sulfonic acid
2-Amino-6-nitro-l-phenol-4-sulfonic acid
Azo dye intermediate, antiamebic agent
Intermediate for production of azo, acid,
and mordant dyes
Dye intermediate for mordant dyes
Azo dye intermediate in production of
mordant dyes
Diazo base for azo dyes, explosive
Dye intermediate for azo and oxidation dyes
Dye intermediate, fur dye, hair dye (blond),
Oxidation Base 25CCI 76555 (fur dye)
Dye intermediate, hair dye (reddish)
Hair dye
Intermediate for azo dyes
Dye intermediate (Mordant Red 80(CI 26565))
Intermediate for azo dyes (wool and anodized
aluminum)
Reference
Thirtle (1968)
USITC (1959-73)
USITC (1959-73)
Morse (1963)
Elslager (1969)
Morse (1963)
Morse (1963)
Morse (1963)
Morse (1963)
Morse (1963)
Morse (1963); Orton
(1969); Markland (1966)
Markland (1966)
Tucker and Schwartz (1971,
Morse (1963)
Morse (1963)
Morse (1963)
-------
Table 25 . Uses of Minor Kitroaronatic Chemicals (Cont'd)
Chemical
4-Amino-4' -nitro-2,2' -stilbenedisulfonic
acid
Ammonium picrate
2-Bromo-6-chloro-4-nitroaniline
2-sec-Butyl-4,6-dinitrophenol
alkanolamine salt
ammonium salt
isopropanolamine salt
triethanolamine salt
2-sec-Butyl-4,6-dinitrophenyl-3,3-
dimethylacrylate (Binapacryl)
co N-Butyl-N-ethyl-a, <*,«- t'rif luoro-2,6-
dinitro-p-toluidine (Benefin)
6-jtert-Butyl-3-methyl-2,4-dinitroanisole
(Musk ambrette)
N-sec-Butyl-g-nitroaniline
1-tert-Butyl-3,4.5-trimethyl-2.6-
dinitrobenzene (Musk tibetene)
5-tert-Butyl-2,4,6-trinitro-m^xylene
(Musk xylene)
2-Chloro-3,5-dinitrobenzenesulfonic acid
2-Chloro-3,5-dinitrobenzotrifluoride
Use
Stilbene dye intermediate
Explosive
Intermediate
Pesticides
Insecticide
Pesticide
Perfume material
Intermediate in preparation of N>N'-di-(sec-
butyl) -p_-phenylenediamine
Perfume material
Perfume material
Azo dye intermediate used to prepare 6-amino-
4-nitro-l-phenol-2-sulfonic acid
Used in preparation of 2'-fluoro-3,5-dinitro-
benzotrif luoride
Reference
Schwander and Domin-
guez (1969)
Rinkenbach (1965)
USITC (1959-73)
SRI (1975)
USITC (1959-73)
SRI (1975)
Industrial sources
Thirtle (1968)
Industrial sources
Industrial sources
Morse (1963)
Barbour et al. (1966)
-------
Table 25 . Uses of Minor Nitroaromatic Chemicals (.Cont'd)
vo
o
Chemical
6-Chloro-2,4-dinitrophenol
4-Chloro-2,6-dinitrophenol
4-Chloro-2-nitroaniline
4-Chloro-3-nitrobenzenesulfonic acid
4-Chloro-3-nitrobenzenesulfonyl chloride
2-Chloro-4-nitrobenzoic acid
2-Chloro-5-nitrobenzoic acid
5-Chloro-2-nitrobenzoic acid
2-Chloro-5-nitrobenzotrifluoride
4-Chloro-5-nitrobenzotrifluoride
o-(4-Chloro-3-nitrobenzoyl)benzoic acid
4-Chloro-6-nitro-l-phenol-2-sulfonic acid
6-Chloro-2-nitro-l-phenol-4-sulfonic acid
Use
Azo dye intermediate (used in commercial
preparation of 2-amino-6-chloro-4-nitrophenol)
Azo dye intermediate (used in commercial
preparation of 2-amino-4-chloro-6-nitrophenol)
Dye intermediate
Dye intermediate (commercial synthesis of
2-amino-l-phenol-4-sulfonic acid)
Intermediate
Intermediate for dyes and medicinal chemicals,
especially acridine derivatives
Azo dye intermediate
Dye intermediate
Used in preparation of 2-fluoro-5-nitro-
benzotrifluoride
Used in preparation of 4-fluoro-5-nitro-
benzotrifluoride
Intermediate
Dye intermediate (synthesis for 6-amino-4-
chloro^l-phenol-2-sulfonic acid)
Dye intermediate (preparation of 2-amino-
6-chloro-l-phenol-4-sulfonic acid)
Reference
Morse (1963)
Morse (1963)
Wooster (1963)
Morse (1963)
Elliott and Bannister
(1968)
USITC (1959-73)
Duncker (1964)
Duncker (1964)
Duncker (1964)
Harbour et^ al. (1966)
Barbour et ai. (1966)
USITC (1959-73)
Morse (1963)
Morse (1963)
2-Chloro-4-nitrotoluene
Dye intermediate
Matsuguma (1967a)
-------
Table 25. Uses of Minor Nitroaromatic Chemicals (Cont'd)
Chemical
2-Chloro-6-nitrotoluene
4-Chloro-3-nitrotoluene
Diaminotrinitrobenzene
2-Diazo-A,6-dinitrophenol
2,6-Dibromo-4-nitroaniline
2,5-Dichloro-l-nitrobenzene
2,4-Dichloro-l-nitrobenzene
Dichloronitrobenzoic acid, isometric
mixture
2,5-Dichloro-3-nitrobenzoic acid
4,6-Dichloro-2-nitrophenol
2',5-Dichloro-4'-nitrosalicylanilide
(Niclosamide)
2,4-Dichlorophenyl-4-nitrophenyl ether
(Nitrofen)
Use
Dye intermediate
Intermediate
Spacecraft propellant
Initiating explosive, especially in electric
blasting caps and detonators
Pesticide
Dye intermediate
Intermediate (azo dyes, fungicides, rubber
chemicals, and explosives)
Pesticide
Intermediate for synthesis of amiben
Dye intermediate (commercial preparation of
2-amino-4,6-dichlorophenol)
Antitapeworm chemotherapy
Molluscacide
Pesticide
Reference
Matsuguma (1967a)
USITC (1959-73)
Industry sources
Matsuguma (1967b)
Rinkenbach (1965)
SRI (1975)
Matsuguma (1967a)
Matsuguma (1967a)
SRI (1975) ,
Plimmer (1970)
Morse (1963)
Mrozik (1967)
Metcalf (1968)
SRI (1975)
2,6-Diiodo-4-nitrophenol
Used in dog hookworm therapy
Mrozik (1967)
-------
Table 25 • Uses of Minor Nitroaroinatic Chemicals (Cont'd)
Chemical
N,N-Dimethyl-o-nitroaniline
N,N-Dimethyl-m-nitroaniline
4,6-Dinitro-2-aminophenol
Dinitroanilines
_o- (2,4-Dinitroanilino) phenol
£- (2,4-Dinitroanilino)phenol
2,4-Dinitroanisole
3',4-Dinitrobenzanilide
2,4-Dinitrobenzenesulfonic acid
2,4-Dinitrobenzenesulfonic acid,
sodium salt
2,2'-Dinitrobenzidine
3,3'-Dinitrobenzidine
4,4'-Dinitrodiphenylamine
3,5-Dinitrobenzoyl chloride
4,6-Dinitro-o-sec-butylphenol
ammonium salt
triethanolamine salt
Use
Intermediate for preparation of N,N-dimethyl-
j)-pheny lened iamine
Intermediate (preparation of N,N-dimethyl-m-
phenylenediamine)
Hair coloring (reddish)
Herbicides
Intermediate
Intermediate
Explosives ingredient
Intermediate
Dye intermediate (preparation of
2,4-diaminobenzenesulfonic acid)
Surface active agent
Reference
Thirtle (1968)
Thirtle (1968)
Markland (1966)
Plimmer (1970)
USITC (1959-73)
USITC (1959-73)
Pristera e£ al. (1960)
USITC (1959-73)
Thirtle (1968)
SRI (1975)
Dye intermediate for Mordant Yellow 21 Lurie (1964)
Formerly a dye intermediate for Sulfur Brown 13 Lurie (1964)
Preparation of 4,4'-diaminodiphenylamine Thirtle (1968)
Reagent for identifying alcohols Duncker (1964)
Pesticide Mitchell (1961)
SRI (1975)
Dinitro capryl phenyl crotonate
Pesticide
SRI (1975)
-------
Table 25. Uses of Minor Nitroaromatic Chemicals (Cpnt'd)
VO
u>
Chemical
2,4-Dinitro-o-cresol
4,6-Dinitro-o-cresol
and sodium salt
4,6-HDinitro-oj-cyclohexylphenol
2,4-Dinitrodiazobenzene
4,4'-Dinitrodiphenylamine
4,6-Dinitro-2-(l-methylheptyl)phenyl
crotonate (Karathane)
4,6-Dinitro-2-methylphenol
2,6-Dinitro-l-phenol-4-sulfonic acid
Dinitroresorcinol
2,4-Dinitroresorcinol
2,4'-Dinitro-4-trifluoromethylcliphenyl
ether
2,2', 4,4' , 6,6'-Hexanitrodiphenylamlne
4-Hydroxy-3-nitrobenzenesulfonic acid
3-Hydroxy-3'-nitro-2-naphthanilide
N- (2-Hydroxy-5-nitrophenyl)glycerine
2-Iodo-3-nitrobenzoic acid
Lead 2,4-dinitroresorcinate
Use --
Herbicide
Molluscacide
Pesticide
Pesticide, molluscacide
Dye intermediate
Intermediate (4,4f-diaminodiphenylamine)
Fungicide (against powdery mildew on some
fruits, flowers, and shrubs)-
Leather fungicide
Dye intermediate
Explosive
Explosive for detonators, caps, and igniters
Pesticide
Explosives ingredient
Dye intermediate
Dye intermediate
Dye intermediate
Hair dye (blond)
Plant growth regulator
Explosive
Reference
McNeil (1965)
Metcalf (1968)
SRI (1975)
Metcalf (1968)
Johnson et^ al. (1963)
Thirtle (1968)
Gearhart (1965)
Turner (1966)
Morse (1963)
Rinkenbach (1965)
Rinkenbach (1965)
Matsuguma (1967b)
SRI (1975)
Pristera ^t al. (1960)
Matsuguma (1967a)
Elliott and Bannister
(1968)
SRI (1975)
Markland (1966)
Duncker (1964)
Rinkenbach (1965)
-------
Table 25 . Uses of Minor Nitroaromatic Chemicals (Cont'd)
VD
•e-
Chemical
Lead picrate (trinitrophenolate)
Lead styphnate (2,4,6-trinitro-
resorcinate)
2-(1-Methyl-n-heptyl)-4,6-dinitrophenyl
crotonate (Dinocap)
3'-Nitroacetanilide
5-Nitro-4-amino-l,3-dimethylbenzene
N-Nitro-1-aminonaphthalene
4-Nitro-2-aminophenol
4-Nitroaniline-3-sulfonic acid
(6-nitrometanilic acid)
4-Nitro-3-anisidine
4-Nitro-o-anisidine
5-Nitro-j>-anisidine
Nitrobenzaldehydes
£-Nitrobenzaldehyde
o-Nitrobenzenesulfonyl chloride
2-Nitrobenzidine
3-Nitrobenzidine
Use
Reference
Initiator (too dangerous for practical use Rinkenbach (1965)
because of very high sensitivity to impact)
Initiating explosive (relatively poor)
Pesticide
Intermediate
Dye intermediate
Azo dye intermediate
Azo dye intermediate
Intermediate (ammonolysis to 4-nitro-m-
phenylenediamine)
Azo dye intermediate
Azo dye intermediate
Azo dye intermediate
Limited use in dye field
Azo dye intermediate, indigo and its
derivatives
Intermediate in manufacture of orthanilic
acid
Formerly a dye intermediate
Formerly a dye intermediate
Rinkenbach (1965)
SRI (1975)
USITC (1975)
Kouris and Northcott
(1963)
Johnson et^ al. (1963)
Morse (1963)
Thirtle (1968)
Matsuguma (1967a)
Matsuguma (1967a)
Matsuguma (1967a)
Deinet and DiBella (1964)
Matsuguma (1967a)
Gilbert (1969)
Lurie (1964)
Lurie (1964)
-------
Table 25. Uses of Minor Nitroaromatlc Chemicals (Cont'd)
Chemical . Use
VO
Ui
£-Nitrobenzoic acid
p-Nitrobenzyl bromide
Manufacture of procaine, p-aminobenzoic acid,
and esters of £-hydroxybenzoic acid
Reagent in qualitative organic analysis
6-Nitro-l-diazo-2-naphthol-4-sulfonic acid Azo dye intermediate
4-Nitrodiphenylamine-2-sulfonic acid Dye intermediate
Nitronaphthalenesulfonic acids
j>-Nitrophenacyl esters
jh-Nitrophenol
6-Nitro-l-phenol-2,4-disulfonic acid
4'-(£-Nitrophenyl)acetophenone
2-Nitrophenylamine
4-Nitrophenylarsonic acid
2-Nitro-£-phenylened iamine
4-Nitro-o-phenylenediamine
5-Nitro-m-phenylenediamine
4-Nitro-m-phenylenediamine
N- (p_-Nitrophenyl) glycine
m-Nitrophenylhydroxylamine
Intermediates in preparation of
naphthylaminesulfonic acids
Used to identify fatty acids
Dye production (anisidine)
Azo dye intermediate
Intermediate
Propellant component
Control of histomoniasis (blackhead) in
turkeys and chickens
Hair dye (reddish, light brown)
Hair dye (reddish, dark/medium brown)
Hair coloring (reddish)
Intermediate
Hair dye
Dye intermediate in manufacture of
4-amino-2-nitrophenol
Reference
Duncker (1964)
Stenger and Atchison
(1964)
Johnson et^ _al. (1963)
Thirtle (1968)
Treibl (1967)
Elam (1965)
Kouris and Northcott
(1963)
Morse (1963)
USITC (1959-73)
Lindner (1965)
Shor and Magee (1970)
Markland (1966)
Markland (1966)
Markland (1966)
Thirtle (1968)
Markland (1966)
Morse (1963)
-------
Table 25 . Uses of Minor Nitroaromatic Chemicals (Cont'd)
vo
O\
Chemical
Nitropyridines
4-Nitropyrogallol
m-Nitrotoluene
3-Nitro-p_-toluenesulfonic acid
5-Nitro-o-toluenesulfonic acid
Pentachloronitrobenzene
1,1,3,3,5-Pentamethyl-4,6-dinitroindan
Picramic acid, sodium salt
Picramic acid (2-amino-4,6-dinitrophenol)
1,2,4,5-Tetrachloronitrobenzene
Tetryl (N-methyl-N-nitro-2,4,6-
trinitroaniline)
3-Trifluoromethyl-4-nitrophenol
2,4,6-Trinitroaniline
1,3,5-Trinitrobenzene
Use
Amebicides
Preparation of benzenetetrol
Preparation of m-nitrobenzaldehyde,
m-toluidine
Intermediate
Dye intermediate, stilbene dyes
Pesticide
Perfume material
Photographic chemical
Explosive, diazo base for azo dyes
Pesticide
High explosive (booster charge)
Lamprey larvicide
(Explosive) Hair dye (reddish); in nitro-
benzene used to extract cesium from
fission product waste; used to extract
potassium salt from sea water
Explosive
Preparation of 2,4,6-trinitroaniline
Vulcanizing agent for natural rubber
Reference
Elslager (1969)
Dressier (1968)
Matsuguma (1967a)
USITC (1959-73)
Zweidler (1969)
SRI (1975)
SRI (1975)
USITC (1959-73)
Morse (1963)
SRI (1975)
Rinkenbach (1965)
Metcalf (1968)
Markland (1966)
Davis (1964)
Mcllhenny (1967)
Rinkenbach (1965)
Kouris and Northcott (1963)
Barnhart (1968)
2,4,6-Trinitrobenzoic acid
Synthesis of phloroglucinol
Dressier (1968)
-------
Table 25 . Uses of Minor Nitroaromatic Chemicals (Cont'd)
Chemical
2,4,6-Trinitrochlorobenzene
2,4,6-Trinitrophenol (picric acid)
Use
Preparation of trinitroaniline (picramide)
Dye intermediate, explosive, analytical
reagent, germicide, fungicide, staining
agent and tissue fixative, tanning agent,
photochemical, Pharmaceuticals, process
material for oxidation and etching of
iron, steel, and copper surfaces
Naphthalene picrate
2,4,6-Trinitroresorcinol (styphnic acid), Initiating explosive
lead salt
Reference
Kouris and Northcott
(1963)
Matsuguma (1967b)
Morse (1963)
Thiessen (1967)
Matsuguma (1967b)
vo
-------
3. Possible Alternatives to Use
Chemical intermediate uses are the major application of the
nitroaromatic compounds and, therefore, by varying the synthesis approach to
the desired final product, alternatives to nitroaromatics may be possible. In
many cases, the nitro functional group is used to introduce an amine group.
There are two major commercial ways of introducing amines:
(1) amination by ammonolysis and (2) amination by reduction (Shreve, 1963).
Reductive amination usually requires a nitro substituent, but ammonolysis can
be used to substitute an amine group for a number of other functional groups.
Ammonolysis can be broadly defined as "the cleavage of a bond by the addition
of ammonia" (Wooster, 1963). The reaction is presently used to produce nitro-
anilines from chloronitrobenzenes (e.g.,£-nitroaniline from £-chloronitrobenzene)
(see Figure 6, p. 26, for further examples). Substitution of -NH? for chlorines
located ortho or para to nitro groups (or carboxylic groups) is possible with
relatively mild (175 C, 530-580 psi) conditions because the chlorine atoms
are "labilized" (Wooster, 1963) by the nitro group (see Section I-B-1 for
mechanism). However, other ammonolysis processes which require more rigorous
conditions have been commercially used to produce aromatic amines.
Aniline has been produced from chlorobenzene using ammonolysis
and a copper catalyst. Because the chlorine has not been "labilized", a catalyst
is required along with temperatures of 200 to 210 C and pressures of 850 to
950 psi. For the process to be economically competitive to nitration-
reduction, the ammonolysis plant should be located near large-scale (therefore,
inexpensive) production of chlorine and chlorinated products (Wooster, 1963).
The crude product from the autoclave is a complex mixture containing aniline,
98
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ammonia, chlorobenzene, phenol, diphenylamine, and copper and ammonia com-
pounds. Although a complex product results, the process has been adapted to
continuous operation.
Ammonolysis may in many cases provide an alternative to the
nitration-reduction approach to aromatic amines. The production of aniline
from chlorobenzene and o-phenylenediamine from ^-dichlorobenzene has reached
commercial stages, although £-phenylenediamine from p_-dichlorobenzene does
not appear to be a technically satisfactory process yet (Wooster, 1963).
Chloroanilines from bromochlorobenzenes are technically feasible because the
bromine is more readily replaced than the chlorine. The application of
ammonolysis to toluene and xylene derivatives will probably be dependent upon
the reaction conditions. For example, p_-chlorbtoluene can be converted to
p_-chlorobenzonitrile rather than p_-toluidine by vapor phase ammonolysis using
the proper catalysts. Substitution of ammonolysis for nitration-reduction will
be dependent upon catalyst and chemical engineering developments which will
determine the economics of the alternative processes.
A variation of ammonolysis, commonly referred to as the Bucherer
reaction, may also be an important alternative to nitration-reduction. The
B.ucherer reaction consists of the conversion of naphthols to naphthylamine
*•
derivatives, using sulfite catalysts. The suggested mechanism is depicted in
Figure 15. The reaction is generally effective for replacement of naphthol
and resorcinol, but not phenol, hydroxyl groups.
99
-------
NH.HSO
4
NH0 NH,
*• •* -J
Figure 15. Mechanism of Bucherer Reaction (Wooster, 1963)
The Bucherer reaction occurs also between hydroxyl groups and primary amines;
this has been used commercially for many years to produce N-phenyl-2-naphthyl-
amine from aniline and 3-naphthol. Up until about 1973, the corresponding
a-compound, N-phenyl-l-naphthylamine, was produced by the reaction between the
product of nitration-reduction of naphthene (1-naphthylamine) and aniline.
However, N-phenyl-l-naphthylamine is now produced by the Bucherer reaction
with a-naphthol and aniline. Although this has resulted in reduced necessity
for the production of a-naphthylamine, the amine is still produced in signi-
ficant quantities by nitration-reduction for other applications.
100
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C. Environmental Contamination Potential
1. General
The major source of environmental release of nitroaromatic com-
pounds appears to be from production plants and from by-product manufacturing
plants. The release from these plants will depend upon the reaction processes,
effluent treatment, and disposal procedures used, and these are likely to vary
considerably for different compounds and at plants of different companies. Very
little exact information on treatment and disposal procedures is available for
individual plants, and the effluent air and water monitoring data are inadequate
for quantitating release. For the vast majority of the numerous nitroaromatic
compounds, the quantities of material released can only be estimated. However,
from the available information, it can be concluded that the major potential source
of contamination is from chemical plants and not from final product use. (An ex-
ception to this is contamination from the use of nitroaromatic pesticides).
2. From Production and Uses
It is frequently difficult to divide nitroaromatics into produc-
tion and uses, since the use of one nitroaromatic compound may be the production
of another. Many of these processes are carried out in aqueous media (e.g.,
hydrolysis of £-chloronitrobenzene to j>-nitrophenol) or are worked up with water
(e.g., water wash after nitration step or aqueous drawn tanks that are used to
quench nitration reactions that are out of control). These water solutions can
be a major source of environmental contamination if not properly treated. A
number of nitroaromatic compounds have been detected in wastewater effluents from
a number of chemical plants (see Table 31, p. 128). The compounds detected in
effluents so far include nitrobenzene, chloronitrobenzenes, nitrophenols
101
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and -cresols, and nitrotoluenes. Similar compounds have been found in drinking
water and raw river water.
, /ua_
/ No nitroaromatic compounds have been detected in air samples
(Table 31, p. U38JI.(/ This lack ofTdetection is difficult to explain since many
of the nitroaromatic compounds have appreciable vapor pressures and are probably
released to the atmosphere during production and use, as well as transport,
storage, and disposal.
Rosenblatt et_ al. (1973) and Small and Rosenblatt (1974) have con-
ducted a detailed survey of nitroaromatic munitions wastes which included
estimates of quantities released (calculated from average concentration and
quantity of effluents) and estimates of concentrations to be expected downstream
from the plants. These estimates are tabulated in Table 26. Only estimates of
2,4,6-trinitrotoluene (TNT) and dinitrotoluenes (DNT) have been reported but other
nitroaromatic compounds are probably present. The effluents and treatment processes
used are described in Section II-D-5, p. 108.
Release estimates for other commercial nitroaromatic compounds are
not available, but they would be extremely useful in providing an assessment of
environmental hazard.
3. From Transport and Storage
No information was available that would allow an intelligent
estimate of losses of nitroaromatics during transport and storage. In general,
the higher volume chemicals seem more likely to suffer larger losses due to spills
and accidents than the smaller commercial products. It is probable that some losses
do occur with all the chemicals, but the quantity lost is unknown.
4. From Disposal
Incineration, land burial, and, in the past, ocean dumping have
been used to dispose of nitroaromatics. It is unlikely that incineration results
in any significant release of nitroaromatics, but land burial may allow leaching
into ground water or evaporation into the atmosphere if a proper site is not
chosen. Ocean dumping of munition wastes is no longer practiced.
102
-------
o
OJ
Table 26. Estimates of Trinitrotoluene and Dinitrotoluene from Army Munitions Plants (From Rosenblatt
et al., 1973 and Small and Rosenblatt, 1974)
Plant Location
TNT Production
Volunteer, TN
Effluent
Discharge
to Compound
Tennessee R. TNT
DNT
Quantity
Released
(Ibs/day)
210
550
Maximum
Concentration in
Downstream Rivers
or Water Supplies
(nw/A)
0.014
(Chattanooga)
0.022
Radford, VA
Joliet, IL
Stroubles Creek
into New River
Illinois R.
Load, Assemble, and Pack Plants
Kingsport, TN Holston R.
Burlington, IA
Baraboo, WI
Shink R. into
Mississippi R.
TNT
DNT
TNT
DNT
TNT
TNT
DNT
105
40
61
530
150
1
(Chattanooga)
0.025
(Kanawha R.)
0.002
(Ohio R.)
0.01
(Kanawha R.)
0.006
(Peoria)
0.030
(Peoria)
0.038
(Morristown)
< 0.001
(Keokuk)
0.0006
(Muscoda)
-------
5. Potential Inadvertent Production of Nitroaromatics in Other
Industrial Processes as a By-Product
Nitration processes rarely produce pure isomers of a desired
product. Any given process may produce a variety of undesired by-products which
may be a major source of environmental contamination if not properly treated.
With TNT, a major pollution problem was the "red water" generated in the Sellite
process used to remove impurities. £-Chloronitrobenzene, a by-product of
£-chloronitrobenzene, is another example of a by-product that has become an en-
vironmental contaminant (Council on Environmental Quality, 1971). Nitroaromatics
may also be formed in non-nitration processes. For example, Knowles et al.
(1974, 1975) have identified ^-nitrophenols in smoked bacon. These compounds
were postulated to have formed from the oxidation of the nitroso derivative.
6. Potential Inadvertent Production in the Environment
No information in the literature examined suggested that nitro-
aromatic compounds are formed in the environment. From chemical considerations,
it would appear that these compounds could be produced: (1) by the oxidation of
natural or man-made aromatic amines, and (2) by the reaction between NO in highly
X
polluted air and aromatic hydrocarbons with activating substituents.
D. Current Handling Practices and Control Technology
1. Special Handling in Use
Toxic body levels of many of the nitroaromatic compounds can be
reached by skin absorption, inhalation, or ingestion; considerable caution must
be exercised in handling these materials. The following discussion is based upon
safety data sheets developed for nitrobenzene, j>-nitroaniline, and dinitrotoluene
(Manufacturing Chemists Association, 1966 a, b, 1967) but is generally appli-
cable to other nitroaromatics.
104
-------
Nitroaromatic compounds should be handled only in well-ventilated
areas, and air monitoring is recommended during handling of some of the more
volatile compounds. Personnel should wear protective clothing and respirators
i
where necessary, and food should not be consumed in the handling areas. With
poly-nitror-compounds, explosions may occur if the compounds are heated, and,
therefore, engineering controls are necessary to prevent localized heating (Bateman
et al., 1974).
2. Methods for Transport and Storage
Liquid nitroaromatics, such as nitrobenzene, are shipped in drums,
tank trucks, or tank cars. Nitrobenzene is classified by the Department of Trans-
portation as a Poisonous Liquid, Class B, and as such, must be packed in specified
containers when shipped by rail, water, or highway. There are also regulations
regarding nitrobenzene loading, handling, and labeling.
Solids, such as 2,4-dinitrotoluene, may be shipped in fiber or
metal drums. (The latter should be used if the product is melted before use).
Shipping solid material in a molten state in tank cars or tank trucks is a common
practice. With dinitrotoluene, the temperature of the unloading operations should
be kept between 75° to 90°C, and localized overheating should be prevented. Pumps
should not be used for unloading because of the hazard of explosion from heat
generated by friction inside the pump.
Nitroaromatic compounds may be stored in shipping containers or
in bulk storage containers. Storage tanks should be kept away from all sources
of fire and excess heat. Outdoor storage is preferable, but if that is not possible,
the storage tank should be equipped with a vent that terminates outdoors. If
nitrobenzene is stored outdoors, precautions should be taken to prevent freezing,
105
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which could cause tank rupture. Dinitrotoluene storage tanks should be kept at
temperatures less than 90 C and should be protected by deluge sprinkler systems.
Occasionally, tank trucks or cars and related equipment need to
be repaired or cleaned; the tanks are drained, then washed with a hot, detergent
wash (dinitrotoluene) or steamed (nitrobenzene and j3-nitroaniline).
3. Disposal Methods
In general, it is recommended that all local, state, and federal
regulations concerning waste disposal of nitroaromatics be determined and com-
/
plied with (Manufacturing Chemists Association, 1966 a, b, 1967). Small amounts
of nitroaromatics may be burned in an open field or in a properly designed chem-
ical waste incinerator. With dinitrotoluene, care should be taken to ensure that
no material goes to the incinerator in confined containers in order to prevent
explosions. Forsten (1973) has reported that incineration of TNT has been suc-
cessfully accomplished. Large quantities of liquid dinitrotoluene may be well
diluted with fuel oil and burned safely in a liquid chemical incinerator.
Nitroaromatic wastes may also be buried in a landfill set aside for toxic wastes.
Such landfills should be located "where the water will not seep into underground
water courses used as a source of drinking water, onto farmland or into streams
and other bodies of water" (Manufacturing Chemists Association, 1967).
Waters from tank or spill clean ups may contain nitroaromatic
wastes and may be treated in a variety of ways. Dinitrotoluene aqueous wastes
should be cooled and settled to recover the bulk of the chemical for disposal by
the methods described above. -When adequate assimilative capacity is available,
the remaining aqueous liquor may be discharged to a receiving stream or municipal'
sewage with regulatory approval. If permitted by regulatory authorities, nitrobenzien
106
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waste may be disposed of by dilution to less than 1% slurry and washed into a
sewer connected to a municipal treatment plant.
Nitroaromatic munitions (e.g., TNT and tetryl) have been dumped
at sea by scuttling old Liberty Ships (Hoffsommer and Rosen, 1972); since 1964
at least 18,342 tons of ammunition and explosives have been dumped (Council on
Environmental Quality, 1970). In 1973, no explosives were disposed of by ocean
dumping (Cox, 1975).
4. Accident Procedures
If a nitroaromatic chemical contacts the skin of a worker, the
contaminated clothing should,be removed and the affected area washed with soap
and water. Ingestion of nitroaromatics should be treated by inducing vomiting,
and gastric lavage should be performed as soon as possible. Individuals exposed
to nitroaromatic vapors should be removed from exposure and kept under obser-
vation until a physician arrives. If the patient becomes cyanotic, oxygen may
be administered.
Spills should be cleaned up immediately. If the material is a
solid, it should be shoveled up, taking care to protect personnel with dust
respirators and rubber gloves. The area should be promptly washed after major
removal of the spilled material.
Fire fighting procedures for the nitroaromatics vary for different
compounds. Nitrobenzene and p_-nitroaniline fires can be extinguished with water,
carbon dioxide, or chemical foam. Both compounds produce noxious fumes, but the
combustion products of p_-nitroaniline are particularly toxic and hazardous.
Water is also effective with fires of unconfined dinitrotoluene, but with con-
fined dinitrotoluene, no attempt should be made to fight the fire because of the
107
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explosion hazard. The surrounding area should be evacuated for protection of
personnel and fire fighting capabilities should be supplied by remotely con-
trolled systems.
Two case studies of explosions from a dinitrotoluene pipeline
(Bateman et^ al^., 1974) and a nitroaniline reactor (Vincent, 1971) have been
IMWA&A&
reported. Both cases seem to have resulted from rather unique circumstances.
5. Current Control Technology
With the exception of the nitroaromatic compounds used as ex-
plosives, which are manufactured at government-owned contractor-operated plants
(Forsten, 1973), very little information is available about air and water pollu-
tion control technology that is used with individual nitroaromatic compounds
during manufacture or use. As noted in the section on disposal, wastes may be
incinerated, buried in a landfill, sent to a municipal water treatment plant,
etc., depending upon local, state, and federal regulations. Many of the
nitroaromatic compounds are not very volatile and scrubbers are frequently used
on nitration reactor vents (Process Research Inc., 1972), thus resulting in en-
vironmental release of nitroaromatic compounds into water effluents. However,
many of the large volume nitroaromatic compounds (e.g. nitrobenzene, dinitro-
toluene) have relatively high vapor pressure which could result in sizable
vapor releases.
Considerable information is available on the water treatment
procedures used with nitroaromatic munitions wastes. Although the wastes from
these plants are atypical (the last nitration step for TNT uses 109% sulfuric
acid and 98.5% nitric acid and the isomer purity requirements are much more
stringent), the techniques may be somewhat similar to those used with other
nitroaromatics.
TNT is the military explosive produced in the largest quantities.
Water effluents from its production and use are unique in that they have even
108
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received common names. Following nitration, crude TNT is washed with water to
remove acid. These washings, which are termed "yellow water", are returned to
early stage nitrators, or incinerated if not recycled. Spent acid is sent to be
recovered. A major pollution problem involved with TNT production is an effluent
termed "red water", which is produced during the aqueous sodium sulfite washing
step (Sellite) that is used to remove non-2,4,6-TNT isomers (about 5% of crude
product). "Red water" contains approximately 25% solids - 9% organics, 10.6%
sodium sulfite, 0.6% sodium sulfate, 3.5% sodium nitrite, and 1.7% sodium nitrate
(Rosenblatt, 1973). In the past, "red water" was disposed of by dumping into
a convenient stream (Forsten, 1973). Presently, it is concentrated by evapor-
ation and either sold to paper plants for its sulfur content or incinerated
(Rosenblatt et^ a±. , 1973). Recently, scientists at the Picatinny Arsenal have
suggested that ammonium sulfite be substituted for sodium sulfite (Anon., 1975 c)
in order that the washing can be mixed with spent acid and collected in a spent
acid recovery system. (Sulfur is recovered as sulfur dioxide and reused, ammonia
is; converted to nitrogen, and production is increased by 8%).
"Pink water" is generated both in manufacturing plants and load,
assemble, and pack plants (LAP's). In manufacturing plants "pink water," so
named because of its pink color under neutral or basic conditions especially
when exposed to sunlight, can be generated in:(l) Mahon fog filter (anti-air
pollution systems) (Siele and Ribaudo, 1971), (2) nitrator fume scrubber dis-
charges, (3) "red water" distillates (from concentration step), (4) finishing
building hood scrubber and wash-down effluents, and (5) possibly spent acid re-
covery wastes (Rosenblatt £t al., 1973). These effluents contain 2,4,6-TNT and
other isomers, as well as less-nitrated isomers (e.g., dinitrotoluene) and other
109
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by-products. Carbon adsorption was for a long while considered economically un-
attractive because the carbon could not be regenerated because of the explosion
hazard. Now carbon adsorption treatment of "pink water" may be used with a tolu-
ene leach regeneration step with the toluene then used as a feedstock. "Pink
water" also results from shell washout operations in LAP's; it contains mostly
pure 2,4,6-TNT. The general practice for disposing of TNT wastes from LAP's is
to use evaporation ponds, although some plants use activated charcoal (Rosenblatt
e± ad. , 1973). Nay (1972) has studied the biodegradability of TNT wastes with
activated sludge pilot plants; he concluded that TNT oxidized more slowly than
it was transferred to the biomass; therefore, contact stabilization should be
used if biological systems were used to treat TNT. It is unknown whether bio-
logical treatment processes are currently being used.
Tetryl (2,4,6-trinitrophenylmethylnitramine) is no longer being
manufactured, but, when it was produced at Joliet, Illinois, wastewaters from
the process were routed to two parallel drainage ditches (Small and Rosenblatt,
1974). Wastewaters from trinitroresorcinol production are routed to lagoons
and, once every few years, the sludge is removed to a landfill (Small and
Rosenblatt, 1974).
110
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E. Monitoring and Analysis
1. Analytical Methods
Since a variety of functional groups are found on the commer-
cially important nitroaromatic compounds, numerous analytical approaches are
possible. Gas chromatography appears to be by far the most popular technique,
but liquid and plasma chromatography and mass spectrometry have also been fre-
quently used. The first two subsections of this section will review a variety
of methods that have been used both for commercial product analysis and for
trace analysis of explosive and pesticide nitroaromatics. The third section
will review methods that have been used for trace analysis of non-pesticide or
non-explosive chemicals and/or have actually been used for environmental moni-
toring.
a. Explosives
A large number of nitroaromatic compounds find application
as explosives. Because of military and security considerations and the fact
that TNT (trinitrotoluene) production processes cause many environmental prob-
lems, analytical methods for these nitroaromatic compounds are well developed.
Most of the compounds that fall into the category of explosive materials are
polynitroaromatic hydrocarbons, although other functional groups, such as
chlorine or hydroxyl groups, are not uncommon. The methods and their applica-
tions have been summarized in Table 27. Many of the methods are probably appli-
cable only to analysis of the explosive formulations (e.g., spot test, infrared
and nuclear magnetic resonance spectrometry), but many of the techniques have
been used or have great potential for environmental monitoring. Electron capture
detectors respond similarly for nitroaromatics and chlorinated aromatics; since
111
-------
the electron capture detector is one of the most sensitive detectors known,
very small concentrations of nitroaromatic compounds can be detected by gas
chromatography or by thin layer chromatography with electron capture detection.
Hoffsommer and coworkers (1972) were able to detect compounds at parts per
trillion levels in sea water; a similar procedure was capable of measuring
5 ppt in soil (Hoffsommer, 1975). The strong absorption of nitroaromatics
in the ultraviolet wavelength region makes high pressure liquid chromatography
with UV detection quite attractive for trace analysis; Doali and Juhasz (1974)
suggest sensitivities well into the nanogram range. A relatively new analy-
tical development, plasma chromatography, appears to be applicable to de-
tection of nitroaromatics in water or in air samples when only picogram quan-
tities are isolated (Karasek and Denney, 1974; Wernlund, 1973). Mass spectro-
meters, alone or combined with gas chromatographs, provide a very specific
technique for determining nitroaromatics at very low concentrations (ppb - Wall
and Gage, 1973; Karasek, 1974).
b. Pesticides
The methods used to analyze the nitroaromatic pesticides
are similar to the techniques for the explosives. Gas chromatographic techniques
for dinitrophenols and £-nitrophenol (parathion degradation product) are widely
used, especially with electron capture detection. Since this review is not as
concerned with pesticides as it is with other commercial nitroaromatics, these
techniques are only briefly reviewed in Table 28, and monitoring data for pesti-
cides have not been covered in Section II-E-2.
112
-------
Table 27. Analytical Methods for Nitroaromatic Explosives
Technique
Reference
Colorimetry
Spot test
Spot test
U>
Color test
Infrared spectroscopy
Infrared spectroscopy
Gas chromatography
Paper chromatography
MacKay et al.
(1958)
Coldwell
(1959)
Sawicki and
Stanley
(1960)
Hackett and
Clark (1960)
Pristera et al.
(1960)
Kite (1961)
Parsons et al.
(1961)
Colman (1962)
'lypc^ qf_ Sample
TNT in air
Explosives
Polynitro-
aromatic
compounds
Explosives in-
cluding TNT,
tetryl, picric
acid, and some
mononitrotoluenes
Explosives and ex-
plosives ingredients
Red water wastes
from munitions plant
Products of nitra-
tion .of toluene
14 Substituted tri-
nitrobenzenes in 10
partition systems
Isolation and/or
Cleanup Method"
Bubbling of air through
peroxidt-free echylene-
glycol tnonoethyl ether
Organic nitrates and
nitramines are dissolved
in acetone and reacted
with diphenylamlne when
U.V. induced
Results and Comments
Sample dissolved in ethanol,
reduced to nitroso compound,
color developed with penta-
cyanoaminoferrate
Extraction with MEK/butanol
(80/20). Separation by column
chromatography
Spotted on paper impregnated with
fprmamide or heavy mineral oil
Violet color obtained by adding alkali to
the TNT in the ether solution, sensitive to
2 ug/mjt of ether (used 2-8 ml of ether in
sampler when sampling approximately 1.5
cubic feet of air).
Method.distinguished between nitrates and
nitramines, could differentiate between TNT,
tetryl, picrite, and picric acid. Sensitiv-
ity not reported.
Blue or green color was developed with
cyclopentadiene methylene group (fluorine)
and alkaline. Positive for aromatic com-
pounds with two or three electron-attracting
groups in the meta position. Identification
limit dinitrobenzene (0.1 ug), 2,4-dinitro-
chlorobenzene (0.05 ug)* 2,4-dinitrotoluene
(0.02 ug), 2,4-dinitroaniline (0.01 ug), and
picric acid (0.05 ug).
Test was used for rapid identifications in
an explosives laboratory. Limits of detec-
tion were from 10 - 100 ug.
Compilation of infrared spectrograms of 68
compounds, mainly for qualitative use.
Detection limits not reported.
Rp's were determined for tetryl, picric
acid, and picramide among others with var-
ious solvent systems. Results simplify se-
lection of solvent system for separation
of compounds in a mixture.
-------
Table 27. Analytical Methods for Nitroaromatic Explosives (Cont'd)
Technique
Reference
_Sampjle__
Isolation and/or
Cleanup Method
Results and Comments
Thin layer
chromatography
Spot test
Gas chromatography
with flame ionization
detection
Gas chromatography
with thermal con-
ductivity detection
Nuclear magnetic
resonance spectro-
metry
Gas chromatography
with flame ionization
detection
Yasuda (196A)
Yasuda (1970)
Amas and Yallop
(1966)
Rowe (1966)
Gehring and
Shirk (1967)
Gehring (1970)
Dalton et al.
(1970)
Crude trinitro-
toluene
Tetryl (N-methyl-
N,2,4,6-tetranitro-
aniline) and related
compounds
Dinitro-and trinitro-
aromatics in indust-
rial blasting explo-
sives
2,4,6-Trini trotoluene
in castable explosives
Trinitrotoluene and .
dinitrotoluene iso-
mers in crude and
refined TNT
Impurities in crude TNT
from the trinitration
step, red oil exudate
and extracts thereof
Organic phases of the
continuous TNT process
(mono- , di- , and tri-
nitrotoluenes)
Dissolve 5 -10 mg sample in
acetone alcohol solution
Two dimensional TLC was used to separate and
identify TNT impurities. Silica gel G/zinc
TLC plates were used. Spots were developed
by p_-diethylaminobenaldehyde (0.25%) and
HCil (0.25 N).
Two dimensional TLC was used•to separate and
identify components of production grade
tetryl and thermal decomposition product
of tetryl (see above). Detection limit 0.5
to 1.0 ug.
Test using color developed with tetramethyl
ammonium hydroxide was developed for for-
ensic use. Limits of identification were
4 ug for m-dinitrobenzene (color not speci-
fied) , 2 yg for 2,4-dinitrotoluene (blue),
and 1 ug for a-trinitrotoluene (dark red).
The method required high purity external
standards and frequent instrument cali-
bration. The lowest detectable amount is
0.02%, requires high-purity isomers for
preparation of internal standards (Dalton
et al_. , 1970).
The purpose of the work was to define TNT
nitration and purification processes.
Limit of detection was 0.03%.
None, or ether extraction if the
organics were in the acid phase
The need for frequent instrument calibra-
tion was avoided by using predetermined
flame ionization detector responses to
calculate relative percentages. Lowest
concentration report 0.01%.
-------
Table 27. Analytical Methods for Nitroaromatic Explosives (Cont'd)
Technique
Thin layer
chroma tography
Reference
KohlbecK et al.
• (1970)
Type of Sample
TNT nitrator samples
and TNT product
Isolation and/or
Cleanup Method
Results and Comments
Two dimensional TLC separations were per-
formed on samples -made under varying con-
ditions for the purpose of improving the
continuous TNT process.
Thin layer
chromatography and ga
chromatography with
electron capture
detection
Gas chromatography
with electron
capture detection
Thin layer
chromatography and
gas chromatography
with electron capture
detection
Gas chromatography with
electron capture de-
tection
Thin layer
chromatography of
charge-transfer
complex formed vitn
amines
Thin layer
chromatography and
spectrophotometry
Gas chromatography -
flame ionization de-
tection (slight modi-
fication of Dalton et al.
1970)
Hoffsommer (1970)
Hoffsommer and
Rosen (1971, 1972)
Hoffsoramer e_t al.
(1972)
Hoffsommer and
Rosen (1973)
Parihar et al.
(1967)
Parihar et al.
(1971)
Nay (1972)
Nay et al. (1974)
10 Nitro compounds,
including TNT
TNT, RDX (not a
nitroaromatic), and
tetryl in sea water
TNT, RDX, tetryl, and
ammonium perchlorate
In sea water, sedi-
ment , and ocean floor
fauna
TNT, RDX, and tetryl
in sea water
2,4,6-Trinitrochloro-
benzene, 1,3,5-tri-
nltrobenzene
6 Binary mixtures con-
taining tetryl, TNT,
picramide, etc.
TNT waste waters
Benzene extraction of sea water
Sea water-benzene extraction.
Sediment and fauna-benzene ex-
traction, cleanup on TLC
Ether extraction
Quantitative analysis of nitro compounds in
micro- to picogram range. 1,3,5-trinitro-
benzene was used as a standard. The detec-
tor is very sensitive; 1 x 10 & g of TNB
overloaded it.
Method capable of detecting TNT, RDX, and
tetryl at 2, 5, and 20 parts per trillion,
respectively, was used to monitor ocean
dumping areas (no explosives were found in
the samples).
Detection levels were as above but somewhat
less sensitive with analysis of marine
fauna (47 - 740 ppt). No explosives were
found in the samples.
The method was used to determine progress of
hydrolysis of these compounds in sea water.
TLC plates were impregnated with various
amines. The complex that formed was
chromatographed. Procedure was able to re-
solve up to 2-3 pg.
Use of charge-transfer complexes of the
compounds allow much better resolution.
Up to 1.5 Ug could be measured.
The method did not detect soluble nitro-
aromatic salts from the sellite process,
or hexanitrodibenzyl.
-------
Table 27. Analytical Methods for Nitroaromatic Explosives (Cont'd)
Technique
Colo rime eric
measurements
Reference " Type of Sample
,, TNT waste waters
Isolation and/or
Cleanup Method
Dilution where necessary
Results and Comments
Color of waste waters was used as an in-
dication of nitrobody concentration; a
Colorimetric
measurements
High speed liquid
chromatography
Negative ion mass
spectrometry
Spano et al. (1972)
Yinon et al. (1972)
Thin layer
chromatography with
e thylene diamine
spot development
High pressure liquid
chromatography using
me thylene chloride
solvent
Chandler et al.
(1972a)
Chandler et al.
(1972b)
TNT in-waste waters Filtration
Aqueous effluents Water sample placed directly on
from TNT finishing XAD-2 resin
processes
TNT in trace quantities
TNT by-products from Dilution with water and acetone
nitration vessel
Hexanitrobibenzyl
(HNBB) and 3-methyl-
2',4,4*,6,6'-penta-
ni t rod iphenyIme thane
(MPDM) in TNT
potassium chloroplatinate color standard was
used.
a-TNT by Silas-Mason method (Standard
Methods of Water Analysis. American Public
Health Association, 1965 and 1971) was
determined by measuring at 425 mu the color
of the complex between a-TNT and diethy1-
aminoethanol; TNT was report as mg/fc
(concentration range 1-50 mg/C.).
The work was done to study a-TNT wash waters
as they were exposed to sunlight and neu-
tralized. The concentration of pure a-TNT
in the comparison sample was 100 ppm.
Method was considered for possible use in
TNT detector. Negative ion mass spectro-
metry was more specific than positive
(focus on NOj peak). At 20°C and equili-
brium the TNT concentration would be 1 part
in 106, which is well within the detection
range. Mass spectrometer leak detectors
have been reported to be capable of
measuring picogram (10 12) quantities.
Method is capable of measuring all major
oxidation products from continuous TNT
nitration process. No detection limit
reported.
In samples from continuous TNT process,
HNBB varied from 0.1 - 0.5% and MDPM from
0.1 - 0.3%. Maximum concentrations found in
batch process were 0.06% MDPM and 0.01%
HNBB. Structures of MDPM and HNBB were
assigned from NMR and IR spectra and TLC
retention times.
-------
Table 27. Analytical Methods for Nitroaromatic Explosives (Cont'd)
Technique
Thin layer
chromatography and
gas chromatography
with electron
capture detection
Liquid chromatography
with ultraviolet
absorbance and differ-
ential refractometer
detection
Reference
Burlinson et al.
(19 7 3)"
Walsh e_t £l. (1973)
Type of Sample
TNT and photoprodurts
In water
Standard nitrotoluene
solutions and TNT
waste water
Isolation and/or
Cleanup Method
Benzene extraction
Water samples were used directly
Results and Comments
TLC. methods indicated 8 photodecomposition
of TNT in irradiated aqueous solutions of
TNT (pink water). Sainplc initially con-
tained 600 pptn TNT.
TNT waste waters were studied for waste
abatement purposes. The range of TNT
measurements was 1 - 100 ppm. Quantities
of less than 1 ppm were easily detectable.
Plasma chromatography
Wemlund (1973)
TNT in river water
Portable vapor
detection systems
(tested sensitivity
and specificity)
Ion Mobility Spectrometer
(version of plasma
chromatography)
Bioluminescent Sensor
System
Mass Spectrometer
Model 58 Explosive
Detector
Wall and Gage (1973) TNT
Direct sampling of atmosphere
Selective detection of nanogram and lower
quantities of TNT in river water (Karasek
and Denney, 1974).
Ni emits beta particles which ionize mole-
cules. The rate of migration in an electric
field is characteristic of the molecule. TNT
was not measured.
Marine microorganisms luminesce when exposed
to vapors of certain compounds. Light change
is measured. Threshold concentration level
in air for TNT was 30 ppb. False detections
due to other compounds or changes in humid-
ity were noted (poor specificity). Very
portable system.
Portable quadrupole mass spectrometer with
three stage membrane separator. Threshold
level for TNT 25 ppb. Most specific method.
Consists of a membrane and an electron cap-
ture detector. Threshold concentration for
TNT was 0.2 ppb (most sensitive of all in-
struments tested). Specificity is imparted
by different recovery times following de-
tection. At elevated temperatures required
for TNTjrecovery times for different com-
pounds are very similar. Therefore, the
method is not very specific.
-------
Table 27. Analytical Methods for Nitroaromatic Explosives (Cont'd)
oo
Technique
Reference
Type of Sample
Isolation and/or
Cleanup Method
Results and Comments
Electron capture
detector
Plasma chromatography
or gas chromatography
using plasma chromato-
graph detector
High speed liquid
ch romatography (U.V.
and differential re-
fractometer detection)
Automated 2 channel
colorimetric analysis
Gas chromatography
(flame ionization
detection) and
colorimetric method
Cline et al. (1974)
Karasek and Denney
(1974)
Doali and Juhasz
(1974)
Hess et al. (1975)
Hess et al. (1975)
TNT in air
Mixtures of explosives
Nitro compounds (esp.
trinitrotoluene and
related compounds)
Direct sample of atmosphere
Direct injection - no preconcen-
tration
Water sample
TNT manufacturing
water effluent
GC samples extracted with benzene
Two sample streams. One passes through a
decomposition chamber which allows differ-
entiation between explosive (thermally un-
stable) and other background vapors.
Method provided rapid detection and identi-
fication of picogram quantities of TNT and
related compounds. Identification is pro-
vided by characteristic positive and nega-
tive mobility spectra.
The method could separate TNT'and tetryl,
a mixture of toluene, p_-nitrotoluene, di-
nitrotoluene, and trinitrotoluene, etc.
Sensitive well into the nanogram range with
U.V. detector.
One channel measures the color of nitro com-
pounds with strong base (Meisenheimer com-
plexes); the response here is attributable
to the total of all nitro compounds present.
In the other channel, 15% potassium hydrox-
ide hydrolyzes nitro groups prior to re-
action with a color reagent; the method is
selective for trinitro compounds and is
sensitive to 1 ppm TNT (reproducibility at
1 ppm is + 10%).
GC method operating range 1-80 ppm.
Authors compared the two techniques and
concluded that the automated colorimetric
technique is more useful for routine work
but is slightly less accurate than the GC
method.
-------
Table 28. Methods Used for Analysis of Nitroaromatic Pesticides and Related Compounds
v£>
Technique
Paper chromatography
Paper chromatographic
separation plus quanti-
tative determinations
Reference
Karlog (1957)
Erne (1958)
Type of Sample
Parathion and p_-
nitrophenol in
organic tissue
material
Parathion and p_-
nitrophenol in
biological materials
Isolation Method
A solvent extract of
an acid extract was
purified by column
chromatography
Solvent extractions of
acidified sample; alumina
column cleanup
Results and Comments
Tt was recomnsnded that the spots
should contain 5 - 100 ug of p_-
nitrophenol. Quantitation of p_-
nitrophenol was determined by a U.V,
spectrophotometer.
P_-Ni trophenol was determined spectro-
photometrically. The determination
was sensitive to about 1 ug/g.
by other methods
Comparison of methods:
Colorimetry (1)
Polarography (2)
Microcoulometric gas
chromatography (3)
Gas chromatography with
electron capture detection
Gas-liquid chromatography
with electron capture
detector
Osciliopolarography com-
bined with thin layer
chromatography
Gas chromatography with
electron capture and
flame ionization detectors
Gas-liquid chromato-
graphy - thermal con-
ductivity detection
Klein and
Gajan (1961)
Carey (1963)
Duggan e_t^ jal^
(1966)
Hearth et al.
(1968)
Hrivnak and
Stota (1968)
Cliffoxd and
Watkins (1968)
Pentachloronitrobenzene
in vegetables
Tetrachloronitroanisole
(TCNA) on vegetables
and grains
Pentachloronitrobenzene
and 1,2,4,5-tetrachloro-
3-nitrobenzene in total
diet composite samples
Parathion, paraoxon, and
p_-ni trophenol in processed
peaches
Dlnitrophenols and other
substituted phenols
Mixture of dinitroalkyl
phenols
Ethanol petroleum ether
extract, used as is for (2)
and purified by column
chromatography for (1) and
(3)
Benzene extract, column
chromatography cleanup
(1) could measure down to 0.1 ppm
(+0.008 ppm); (2) down to 0.02 ppm
(+ 0.008 ppm); and (3) down to
0.1 ppm.
Low concentration analyzed 0.013 ppm.
Sensitive to 0.001 ppm.
Methylene chloride extraction Accurate in the 0.5 - 2.0 ppm range
Use of polar polyester type columns
prevented tailing (usually a problem
with phenols) and allowed separation.
With the electron capture detector,
only a few nanograms were necessary
for analysis.
GLC with DEGA/phosphoric Results indicate that DEGA modified
acid on Chromosorb W or G with 0.4% phosphoric acid is effec-
tive in separating dinitroalkyl
phenols. Limit of detection not
reported.
-------
Table 28. Methods Used for Analysis of Nitroaromatic Pesticides and Related Compounds (Cont'd)
Tgghjiique
Thin layer (adsorption
and reversed-phase) and
gas-liquid chromatography
Gas chromatography
Gas chromatography with
electron capture detection
High speed liquid
chroraatography with
polarographic detector
Gas chromatography with
electron capture or alkali
flame ionization detection.
Gas chromatography with
electron capture detection
Gas chromatography with
ele c tron cap t ure de te c t ion
Automated gas-liquid
chromatography with electron
capture detection
^Reference
t. Li f ford _«c, _al.
(1969)
Clifford and
Watkins (1970)
Cranmer (1970)
Koen et al.
(1970)
Newsom and
Mitchell (1972)
Shafik et al.
(1973)
Bradway and
Shafik (1973)
Allen and Sills
(1974)
De Vos e_t al.
(1974)
Type of Sample
Isolation Method
Results and Comments
Homologous series of sub-
sti tuted dinitrophenols
Nitrophenols and nitro-
anisole
£-Nitrophenol (PNP) in urine
Mixture of parathion,
methyl parathion, and
£-nitrophenol
N,N-diethyl-2,4-dinitro-
6-1ri fluorome thy1-ro-
phenylenedlamine (dinitra-
mine) in soil forage, and
crops
Nitrophenols in urine
3-Tri fluorome thy1-4-
nitrophenol (TFM) in fish
Pentachloronitrobenzene
tetrachloronitrobenzene,
and 2 ,6-dichloro-4-nitro-
aniline in lettuce
Sample is hydrolyzed and PNP is
extracted
Methanol extraction, methylene
chloride partitioning, Florisil
column cleanup
Hydrolyze sample, extract
ethyl ether, cleanup on silica
gel column
Hexane-ether extraction, parti-
tioning, and methyl ether der-
ivatization
Extraction with ethyl acetate -
no cleanup
Method is sensitive to 50 ppb for
£-nitrophenol in urine. The tri-
methylsilyl ether derivative is made
in the GC column by injecting PNP with
hexamethyldisilazane.
— 8
Concentrations of 10 mol/2. can be
determined with a 2% standard devia-
tion. Polarograph detector requires
that the solvent system have a high
conductivity.
Sensitive down to 0.01 ppm.
Limits of detection 0.01 - 0.05 ppm.
Suitable recoveries at 0.01 - 2.00 ug/g
fish muscle.
Extracts were diluted with hexane and
analyzed automatically. The system was
reportedly suitable for screening large
series of sample with accuracy equal
to that of manual analysis; 1-3 ppm
added to lettuce at extraction stage
were recovered 100 +5%,
-------
Table 28. Methods Used for Analysis of Nitroaromatic Pesticides and Related Compounds (Cont'd)
Technique
Reference
Type of Sample
Isolation Method
Results and Comments
Gas chromatography with
electron capture detector
and flame photometer
Sherma and
Shafik (1975)
Gas chromatography with
electron capture detection
Gas chromatography with
flame ionization
detection
Olson et al.
(1975)
Klus and Kuhn
(1975)
Parathions in pesticide
multiresidues in air
Dlnitramine in fish
Nitrophenols in various
samples
Methylene chloride extraction
from ethylene glycol trapping
solvent, then fractionation on
silica gel column
Extraction, Florisil cleanup
Acid-base partitioning, ether
extraction, methylation with
diazomethane
In multiresidue method developed for
National Air Monitoring Program,
-fractions from silica gel fraction-
ation were gas chromatographed. The
phosphate compounds were analyzed by
flame photometry; the others, by
electron capture method. For parathion,
50 and 207 ng added to 100 mJZ. of
ethylene glycol were recovered 93 and
90%, respectively; 40 and 162 ng of
methyl parathion, 97 and 87Z, respec-
tively.
Limit of detection 0.01 mg/fc.
Easily measures 100 ^g of various
nitrophenols.
-------
c. Miscellaneous Nitroaromatic Analytical Methods and
Monitoring Studies
The analytical methods reviewed in this section have been
applied more frequently in actual environmental monitoring situations; they
are summarized in Table 30. Gas chromatography with flame ionization and/or
electron capture (Zielinski et_ al_. , 1967 b, uses both for qualitative purposes)
has been quite popular for laboratory studies, but its application to monitoring
has been infrequent. The relationship between chemical structure and electron
capture sensitivity was studied by Zielinski et_ al. (1967 a). Their results,
summarized in Table 29, demonstrate that most nitroaromatics exhibit large responses
Table 29. Relative Electron Capture Sensitivities of Nitroaromatic
Compounds (Zielinski et al., 1967 a)
Compound Group
Chlorobenzenes
Chloronitrobenzenes
Fluoronitrobenzenes
Dinitrobenzenes
Nitroanilines
Miscellaneous
Compound
£-Dichlorobenzene
2 , 3-Dichloronitrobenzene
m-Chloronitrobenzene
2 , 5-Dichloronitrobenzene
2,4,5-Trichloronitrobenzene
o-Chloronitrobenzene
3,4-Dichloronitrobenzene
2,4-Dichloronitrobenzene
_p_-Chloronitrobenzene
o-Fluoronitrobenzene
j>- Fluor oni tr obenzene
m-Fluoronitrobenzene
m-Dinitrobenzene
o-Dinitrobenzene
o-Nitroaniline
m-Nitroaniline
o-Bromonitrobenzene
2-Nitro-4-chloroaniline
Relative
Sensitivity
1.88
2.34
2.21
2.12
2.10
1.66
1.13
. 1.11
1.00
0.74
0.685
0.206
1.63
1.29
0.302
0.260
].22
0.214
122
-------
Table 30. Miscellaneous Nitroaromatlc Analytical and Monitoring Techniques
to
Technique
Carbon filter
chloroform extracts
(CCE) and infrared
analysis
Colorimetrie
method with
air sampler
Midget impinger
cold trap
collection -
U.V. analysis
Gas chromatography
with flame
ionization (FI)
detection
Gas chromatography
with FI
References
Middleton and
Lichtenberg
(1960)
Hands (1960)
Linen and
Chars ha
(1960)
Habboush and
Norman (1962)
Selucky et al.
(1967)
Type of Sample
£-Chloronitro-
benzene in the
Mississippi River
Nitrobenzene in
air
Nitrobenzene in
air
Mixtures of
isomers of
disubstituted
benzenes
Nitrobenzene in
air of nitrobenzene
plant
Isolation and
Cleanup Method
CCE cleaned up
chromatographlcally
Concentration in
air sampler con-
taining cellusolve
Cold trap impinger
was used for
collection
Celite 545 con-
centration tubes
were used
Results and Comments
Several thousand gallons pass
through the sampler in 10 days.
£-chloronitrobenzene was
detected 105 and 1020 miles
above the mouth of the
Mississippi River.
Zinc amalgam and hydrochloric
acid were used to reduce the
aniline which was then diazotized
with a-naphthol salt. With
a 6-liter sample, concentrations
of 0.5 to 2 ppm v/v could be
determined.
Collection efficiencies for
1 and 5 ppm of nitrobenzene
in air were 85% to 95% at
-21°F and -116°F.
The column packing materials
that were capable of
separating ortho, meta, and
para isomers of nitrotoluenes,
fluoronitrobenzenes, chloro-
nitrobenzenes, bromonitro-
benzenes, and nitroanisoles
were reported.
With the concentration tube.
10 mg of nitrobenzene per m^
of air could be measured.
-------
Table 30. Miscellaneous Nitroaromatic Analytical and Monitoring Techniques (Cont'd)
technique
Gas chromatography
with electron capture
(EC) detection
References
Zielinski e£ al.
(1967a)
Type of Sample
Chloro-, bromo-,
nitrobenzenes
Isolation and
Cleanup Method
Results and Comments
Relationship between structure
and sensitivity in electron
capture analysis was
determined.
Gas chromatography
with dual EC-FI
Zielinski et al.
(1967b)
Chloroni t robenzene
isomers
The ratio of the electron
capture/flame ionization
response was used for
qualitative purposes. No
detection limit noted.
Thin-layer chromato-
graphy (UV light
development of
spots)
Gas chromatography
Berei and Vasaros
(1967)
Habboush and
Tameesh (1970)
Nitrobenzene and
chloronitrobenzene
isomers
Fluoro-, chloro-, and
bromonitrobenzenes;
nitroanisoles; and
nitrotoluenes
(), m, and j>-chloronitro-
benzene could be separated.
Retention times on various
packing materials were
presented.
Preparative gas
chromatography
and identification
by 1R and mass
spectrometry
Same as above
Colorimetric
Method
Friloux (1971)
U.S. EPA (1972)
Tiwari and Pande
(1972)
Nitrobenzene, chloro-
nitrobenzene, and
dinitrotoluene in
New Orleans finished
water
Number of nitroaromatics
m-dini t robenzene, ?. ,4,6-
trinitrobenzene in nitro-
phenol l-chloro-2,4-
dini trobenzene
Carbon chloro-
form extract (CCE)
Same as above
No concentrations
were noted.
Because recoveries were
not determined, quantitation
was not possible.
Sensitive for 100-600 pg
-------
Table 30. Miscellaneous Nitroaromatic Analytical and Monitoring Techniques (Cont'd)
Technique
Colorimetric
method
Gas chromatograph-
mass spectrometry
Gas chromatograph-
mass spectrometry
Plasma chromatography
Hollow fiber probe
with mass spectrometry
Gas chromatography
with FI detection
References
Kurenko (1972)
Burnham et al.,
(1972)
Webb et al.,
(1973)
Karasek and
Kane (1974)
Westover et al.
(1974)
Austern et al.,
(1975)
Type of Sample
p_-, £-nitrotoluene
in air
p_-nitrophenol and
4,6-dinitro-2-
aminophenol in
water
Industrial water
effluents
Halonitrobenzenes
Nitrobenzene in
air or water
samples
Nitrobenzene in
wastewater
Isolation and
Cleanup Method
Preconcentration
by absorbers
Preconcentration
by macroreticular
resin bed (XAD
resins)
Solvent extraction
Extraction in
Freon
Results and Comments
Capable of detecting con-
centrations of 0.75 mg/m^
Recovery for p_-nitrophenol
was 100% at 0.2 ppm; for
4,6-dinitro-2-aminophenol
recovery was 43% at 0.4
ppm. Procedure allowed
detection of aromatic hydro-
carbons down to 1 ppb.
201 samples allowed apparent
detection limits of 0.1 ug/l
while with 1 £ samples the
detection limits were 2 ug/l.
Sample size was on the order
of 10~7 to 10~ g. Isomers
could be identified by
mobility spectra.
Capable of continuous
monitoring at ppm levels.
Limit of detection -1 ppm
(methanol) to 10 ppb
(chloroform) in water.
Minimum detectable
quantity of nitrobenzene
was 0.7 ng. Recovery from
spiked raw and treated
wastewaters was 100% at
0.317 mg/1 for nitrobenzene.
-------
with electron capture detectors. (Minimum detectable quantity with electron
capture detectors = 10 g, Karasek, 1975.).
Techniques for monitoring nitroaromatics in air (Hands, I960;
Linch and Charsha, 1960; Selucky et_ al., 1967; and Westover £t a^. , 1974) seem
more suitable to occupational or effluent monitoring applications than to ambient
analyses, since most of the techniques are sensitive only in the ppm range. None
of the techniques noted in Table 30 have actually been used for ambient air moni-
toring. Because of the strong electron-capturing characteristics of most nitro-
aromatics, both gas chromatography with electron capture detection or plasma
chromatography, which is also dependent upon electron capture, could be used for
ambient air monitoring (e.g., see TNT detection in air, Karasek, 1974, and Karasek
and Denney, 1974), especially if some preconcentration step were used (see Sherma
and Shafik, 1975).
In contrast to the lack of data in ambient air monitoring,
nitroaromatic compounds have been detected in raw river water and finished drinking
water (Middleton and Lichtenberg, 1960; Friloux, 1971; U.S. EPA, 1972). Unfor-
tunately, all of these researchers have used a carbon chloroform extraction (CCE)
preconcentration step. Although this technique makes qualitative analysis easier
(larger sample), it precludes quantitative analysis unless recovery studies are
undertaken. In a study conducted on the lower Mississippi River (U.S. EPA, 1972),
quantitation was not attempted; therefore, the following section (II-E-2) does not
report concentrations. Burnham &t_ a^. (1972) have developed an XAD resin pre-
concentration step for drinking water that would allow quantitation (they used
GC-MS), but so far this procedure has not been used extensively in field surveys.
126
-------
With industrial wastewaters, the concentrations of contam-
inants is usually much higher, so smaller samples can be used. The most compre-
hensive study of wastewaters (Webb et al., 1973) used solvent extraction of a 1 or
20 liter sample, combined with GC-MS, to give a detection limit of 0.1 yg/fc for
the larger sample.
In summary, there are a number of analytical methods that are
specific and sensitive enough for trace analysis of nitroaromatics in ambient
environmental samples. However, as will be noted in the following section, appli-
cation of these techniques in monitoring studies has been infrequent.
2. Monitoring Studies
The available monitoring data on nitroaromatic compounds are
summarized in Table 31. Ambient monitoring (including monitoring of drinking water)
is presented in the top portion of the table, while effluent monitoring is in the
lower part of the table. The lack of air monitoring data in the literature
available for this report is very noticeable. It is difficult to attribute this
lack to a particular cause, since many of the nitroaromatic compounds have appre- '
ciable vapor pressures and are probably released to the atmosphere in sizable
quantities. The. available water monitoring data is more extensive than the air
monitoring data, but, considering the number of nitroaromatic compounds, it would
seem to be far from adequate. Only six compounds have been detected at ambient
levels, and none of the studies have provided quantitative data. Whether or not
other nitroaromatic compounds are present in river or drinking water is unknown.
However, there have been a number of monitoring studies that have had the capa-
bility to detect nitroaromatic compounds that have reported that detectable
amounts were not present. These studies are summarized in Table 32. Perhaps the
127
-------
Table 31. Ambient or Effluent Monitoring of Nitroaromatic Compounds
Reference
Analytical Method
Monitoring Site
_gnd_Ty]p_e_ qf_ ^Samp le
Chemical
Concentration
(ppm)
OO
Middleton and
Lichtenberg (1960)
Friloux (1971)
Borodin and
Kuchinskaya (1971)
U.S. EPA (1972)
U.S. EPA (1975)
Golubeva (1957)
Kite (1961)
Jenkins and Hawkes
(1963)
Carbon chloroform
extract (CCE) with
infrared spectro-
metry
CCE
Unspecified
CCE
Colorimetric
Solvent extraction,
column chromatography,
infrared spectrometry
Unspecified
Cape Cirardeau, MO
(1020 milos frum mouth
of Mississippi River)
New Orleans (105 miles
from mouth)
New Orleans drinking water
Drinking water for the city
of Tomsk, USSR
Carrollton drinking water
plant, New Orleans
Jefferson Parish #2, raw
water, New Orleans
Rubicon Chem. (nitro-
benzene producer -
75 mill. Ibs capacity)
Drinking water
Sewage of a petroleum
refinery
Picatinny Arsenal, raw red
water wastes, Dover, NJ
Fison's Pest Control Ltd.,
waste water, Harston,
Cambridge, U.K.
o-Chloronitrobenzene
Chloroni trobenzene
Dinitrobenzene
Nitrobenzene
Nitrobenzene
m~ Chloronitrobenzene
Nitrobenzene
2,6-Dinitrobenzene
Nitrobenzene
Nitroanisole*
Nitrobenzene
4,6-Dinitro-2-aminophenol
m-Chloronitrobenzene
2,6-Dinitrotoluene
Nitrobenzene
Nitrobenzene
2,4-Dinitrotoluene-5-sulfonic
acid
2,4-Dinitrotoluene-3-sulfonic
acid
2,4-Dinitrotoluene
3,5-Dinitrobenzenesulfonic
acid
Dinitro-o-cresol
0.004 - 0.037
0.001 - 0.002
0.2 - 0.3
-------
Table 31. Ambient or Effluent Monitoring of Nitroaromatic Compounds (Cont'd)
Reference
Karelin e^ al. (1964)
Kurmeier (1964)
Papov (1965 a, b)
Trifunovic et al.
(1971)
Nay (1972)
Walsh e_t al. (1973)
Zetkin £t al. (1973)
Webb et al. (1973)
-
Analytical Method
Unsper.i f ied
Unspecified •
Unspecified
Unspecified
Gas chromatography-
flame ionization
Liquid chromatography
Unspecified
Solvent extraction of
water effluent, GC-MS
Monitoring Site
and Type of Sample
Kuibyshev petroleum processing
plant
TNT manufacturing plant waste-
water effluent
Water effluent from sulfur
dye production
Water effluent from parathion
production
Radford TNT Plant, water
effluent, Radford, VA
TNT finishing plant wastewater
m-Chloronitrobenzene production
was tewaters
Specialty chemical plant
Explosives (DNT) plant
" " " raw was te
" " " pond effluent
TNT plant raw effluent
Explosives (DNT) plant
Chemical companies lagoon after
steam stripping
II M
II II
Paper mill's five-day lagoon
TNT plant's raw effluent
DNT plant's raw effluent
n n
Chemical company's lagoon after
steam stripping
DNT plant's raw effluent
TNT plant's raw effluent
Chemical
Ni t robenzene
Dinitrotoluene
Nitrotoluene
2 ,4-Dini tro-1-chlorobenzene
p_-Nitrophenol
2-Nitrotoluene
4-Ni trotoluene
2,4-Dinitrotoluene
2, 6-Dini trotoluene
2,4,6-Trinitrotoluene (TNT)
Trinitrobenzoic acid
2,4-Dinitrotoluene
TNT
m- , o- , p_-Chloronitrobenzene
4 ,6-Dinitro-£-cresol
2,4-Dinitrotoluene
2 ,6-Dinitrotoluene
n n
n n
3,4-Dinitrotoluene
Nitrobenzene
2-Nitro-p_-cresol
o-Nitrophenol
o-Ni trotoluene
n n
n n
m-Ni trotoluene
£-Nitrotoluene
n n
2,4,6-Trinitrotoluene
Con cent rat
(ppm)
0.32 - 16
0.12 - 9.
trace - 39
3.39 - 56.
101 - 143
0.80
1.5 - 1.8
18
190
150
0.02
0.68
40
0.11
9.3
1.4
0.15
7.8
0.04
8.8
0.7
ion-
2
3
g/*
* Compound was dropped from I Sept., 1975 List of Organic Compounds Identified in Drinking Water (U.S. EPA, 1975)
-------
Table 32. Monitoring Studies Reporting No Detectable Quantity of Nitroaromatic Compounds
Reference
Analytical Method
Compounds the
Analytical Method
Could Detect
Monitoring Site
and
Type of Sample
Sensitivity
of the Method
OJ
o
Kleopfer and
Fairless (1972)
Burnham et al.
(L972~
CCE concentration,
GC-FI, MS, 1R, NMR
XAD resin concentra-
tion, GC-MS, GC
Hoffsommer and Solvent extraction,
Rosen (1971, 1972) GC-EC
Hoffsommer et a_l.
(1972)
U.S. EPA (1974)
CCE
_p_-Nitrophenol
4,6-Dinltro-2-
aminophenol
2,4,6-Trinitrotol-
uene (TNT), methyl-
2,4,6-trinitrophenyl-
nitramine (tetryl)
Method detected a
number of nitro-
aromatics in
earlier study
(U.S. EPA, 1972)
Tap water
Well water
Ames, IA
Sea water, 200 miles
off coast of Florida,
45 miles west of
San Francisco
Sea water, sediment,
and ocean floor
fauna; 85 miles west
of Flattery, WA, 172
miles south-southwest
of Charleston, SC
Drinking water in
New Orleans area
ppb range
ppb-ppt range
-------
most significant study was the 1974 duplication (U.S. EPA, 1974) of an earlier
study of New Orleans drinking water (U.S. EPA, 1972). The early study had de-
tected several nitroaromatics, while the later study reported no nitroaromatic
compounds. In summary, a number of nitroaromatic compounds have been detected
in river and drinking water and in various effluents, but the available infor-
mation is so sparse that it is difficult to determine whether nitroaromatic
compounds are widespread environmental contaminants.
131
-------
132
-------
III. Health and Environmental Effects
A. Environmental Effects
1. Persistence
a. Biological Degradation, Organisms and Products
The biological transformation and particularly the micro-
bial transformation of nitroaromatic compounds has received a fair amount of
attention. The interest has arisen in recent years primarily because nitro-
aromatics are increasingly being used as inhibitory agents (e.g., pest control
agents) or in the synthesis of inhibitory agents. The existence of naturally
occurring ,biological nitro compounds (e.g., chloramphenicol, 3-nitropropionic
acid, etc.) (Ehrlich et^ al. , 1948; Carter and McChesney, 1949; Hirata et al.,
1954) suggests the possible existence of organisms able to decompose nitro-
aromatic compounds (Cain, 1958; Gundersen and Jensen, 1956).
An extensive review of the literature has revealed that the
environmental fate related information is available for the following groups
of compounds: nitrobenzenes and chloronitrobenzenes, nitrobenzoic acids, nitro-
phenols and related compounds, nitrotoluenes, and nitroanilines. Information is
reviewed below for each of these categories.
(i) Nitrobenzenes and Chloronitrobenzenes
A number of researchers have examined the blodegrad-
ability of unsubstituted and halogen-substituted nitrobenzenes. A summary of
the conditions employed in the biodegradation studies by various investigators
is presented in Table 33. In one of the earlier studies, Alexander and
Lustigman (1966), while studying the effect of chemical structure on the micro-
bial degradation of substituted benzenes," found that nitrobenzenes were quite
133
-------
Table 33. Summary of the Degradation Studies with Unsubstituted and Halogen Substituted Nitrobenzenes
CO
Reference
Alexander and
Lustigman, 1966
Bringmann and
Kuehn, 1971
Chambers et al. ,
1963
Test Chemicals Gone. Used
Nitrobenzene, 5-10 mg/£
o , m , and p-
dinitrobenzene
1,3,5-trinitro- 118-146 mg/5,
benzene, m-dinitro-
benzene, nitro-
benzene
Nitrobenzene, m, 100 mg/d
and p-dinitrobenzene,
< w /I>-T— ^ +~- 1 r \
Source of
Microorganisms
Soil (Iliagara
silt loam)
Combined action
of Azotobacter
agilis , and micro-
organisms in acti-
vated sludge
Microorganisms in
soil, compost, or
/Duration
of
the Test
64 days
. 170-210
min
Criteria for
Test Chemical
Alteration
Loss of UV
absorbancy
Estimation of
nitro- reduced
metabolites
Oxygen con-
sumption in Wa
Malaney, 1960
Moore, 1949
Villanueva, 1960
1,3,5-trinitrobenzene
Nitrobenzene
Nitrobenzene
500 mg/2.
0.1% v/v
o_, in, and j>-
dinitrobenzene,
1,3 , 5-trinitro-
benzene
250 mg/X.
mud from a catalytic
cracking plant waste
lagoon, adapted to
degrade phenol
Aniline-acclimated 8 days
activated sludge
Two Nocardia sp., -
enriched from soil
on pyridine
Pure culture of 16 days
Nocardia V.
Oxygen con-
sumption in Warburg
Growth on nitro-
benzene as sole
source of carbon,
nitrogen, and
energy
Growth
-------
Table 33. Summary of the Degradation Studies with Unsubstituted and Halogen Substituted Nitrobenzenes
(Cont'd)
Reference
Test Chemicals
Cone. Used
Source of
Microorganisms
Duration
of
the Test
Criteria for
Test Chemical
Alteration
Bielaszczyk e't
al., 1967
_p_-chloro-
nitrobenzene
and 2,4-dinitro-
chlorobenzene
Ludzack and
Ettinger, 1963
c)-chloronitro-
benzene
Stationary con-
ditions:
100 mg/X, £-nitro
20 mg/5, dinitro
Continuous flow
conditions:
9.2 mg/X, _p_-nitro
and 1. 7 mg/fc
dinitro (concen-
tration reduced by
half after 3 days)
Two aeration columns:
92 mg/5. £-nitro and
17 mg/J. dinitro
21.1 mg/fc
Arthrobacter
simplex, Fusartub
sp., Trichoderma
viride, Spreptomyces
coelicolbr, isolated
from soil and in-
dustrial waste con-
taining nitro-
chlorocompounds
8-13 days
Estimation of
nitro and amino
groups
Settled sewage
added weekly
175 days
CO production
-------
difficult to degrade; both mono- and di-substituted (o_, m, or 2) nitrobenzenes
persisted for more than 64 days (the criterion of persistence was the unchanged
U.V. absorbancy [due to intact benzene ring] of the solution). The assay tech-
nique employed in this study required the use of a small amount of soil in-
oculum and precluded the addition of growth factors and supplemental organic
compounds in order to minimize interferences. The recalcitrant nature
of the nitro-
substituted benzenes revealed by this test could thus be due to the unsuit-
ability of the test conditions. The biological resistance of di- and trinitro-
substituted benzene (o-, m-, and _p_-dinitrobenzene, and 1,3,5-trinitrobenzene)
is, however, also suggested from the fact that these compounds failed to serve
as sole carbon or nitrogen source for growth of Nocardia V (Villanueva, 1960).
The culture of Nocardia _V was able to reduce one nitro group of £-dinitro-
benzene to an amino group resulting in the formation of £-nitroaniline; the
enzyme responsible for this reaction has been extracted and purified from a
Nocardia culture by Villanueva (1960, 1964). The selection of Nocardia V_ for
studying utilization of nitro compounds was based on the reported evidence that
some species of genus Nocardia are able to utilize a variety of aromatic compounds
for growth (Sergey's Manual of Determinative Bacteriology, 1948; Moore, 1949;
Cain, 1958). Azim and Mohyuddin (1957) have reported that nitrobenzene was not
utilized as a source of nitrogen by Azotobacter vinelandii, an organism which
fixes atmospheric nitrogen. The authors attributed this to the inability of
nitrobenzene-N to be reduced to NH_.
The number of chemicals entering the environment is
enormous. When a synthetic chemical enters the environment, one or a group of
indigenous populations possessing requisite enzymes or which can adaptively
136
-------
synthesize necessary enzymes frequently multiply and make use of the intro-
duced substrate. It is likely that requisite acclimation may also occur when
an organism in the environment comes in contact with a chemical which has the
same basic configuration as the chemical to be degraded. With this view in
mind, a number of researchers have determined whether a culture .highly adapted
to a compound representing a basic chemical configuration similar to nitro-
benzenes is capable of degrading nitro-substituted benzenes. Malaney (1960)
examined the ability of an aniline-acclimated activated sludge microflora to
oxidize nitrobenzene. They found that under their experimental conditions,
the endogenous oxygen uptake rate exceeded the oxygen uptake in the presence
/ 0
of the test chemical. This le^ad the author to conclude that nitrobenzene e>
under the above conditions is poorly oxidized or not oxidized at all. A
decrease in endogenous oxygen uptake due to the presence of nitrobenzene
could also be due to its inhibitory action on oxidative enzymes. The ability
of phenol-adapted bacteria to degrade nitrobenzene,, m- and jv-dinitrobenzene
and 1,3,5-trinitrobenzene, has been examined by Chambers et^ al. (1963). Micro-
organisms present in soil compost, or mud from a catalytic cracking plant
waste lagoon, were subjected to preliminary adaptation on phenol by techniques
such as soil percolation, activated sludge aeration, primary enrichment in
flask, or enrichment in batch-type fermenters. Further adaptation was carried
out by subculturing the microorganisms periodically on mineral salts medium
containing phenol as the only source of carbon. Cell suspensions adapted in
this manner exhibited very low levels of oxidative activity on nitro substituted
benzene compounds, particularly on the mono nitro-substituted benzene; in that
case;, the endogenous level of oxygen consumption was higher than that observed
137
-------
iesc
Time
(min)
170
210
180
180
Endogenous
(cell alone)
53
57
65
65
Cells & Test
Compound
32
102
97
127
K.ai_O.U UJ.
Endogenous to
Test Compound
>Endog.
1.8
1.5
2.0
in the presence of nitrobenzene as was observed by Malaney (1960) with
aniline-acclimated activated sludge. The resistance of the nitrobenzenes
to degradation seemed to decrease as the number of nitro groups on the ben-
zene ring increased (Table 34).
Table 34. Oxidation of Nitro-Substituted Benzenes by Phenol-Adapted Culture
(Chambers et al., 1963)
Compound
Nitrobenzene
m-Dinitrobenzene
Pj-Dihitrobenzene
1,3,5-Trinitrobenzene
Contrary to the reports that nitrobenzenes are dif-
ficult to degrade, Moore (1949) reported the isolation of two Nocardia sp.
from soil which used nitrobenzene as a sole source of carbon, nitrogen, and
energy. The organisms employed by Moore (1949) in studying degradation of
nitrobenzene were isolated from pyridine enrichment cultures.
The biological removal of 1,3,5-trinitrobenzene,
m-dinitrobenzene and nitrobenzene in a two-stage model waste water purifier
has been reported by--Brigmann and Kuehn (1971). The system consisted of an
aerator (1st stage) which was inoculated with Azotobacter agilis, and 2nd
stage overflow basin which was inoculated with activated sludge. The authors found
practically complete removal of the mono nitro compounds in the aeration stage.
In the case of trinitrobenzene, complete removal was not achieved until after
138
-------
passage through the 2nd stage. No information concerning the nature of the
metabolites formed or the extent of removal due to adsorption on the bio-
logical material was revealed in this study.
Nitrochloro-substituted benzene compounds of com-
mercial significance which have been examined for their biodegradability
include j>- and o-nitrochlorobenzene, and dinitrochlorobenzene. Bielaszczyk
et al. (1967) have reported isolation of microorganisms which reduced nitro-
chloro-compounds to the corresponding amino compounds. The organisms were:
Arthrobacter simplex, Streptomyces coelicolor, Fusarium sp. and Trichoderma
viridis which were isolated from soil, and a species of Arthrobacter simplex
isolated from industrial waste containing nitrochloro-compounds. A mixture
of the above microorganisms was considerably more effective than individual
microorganisms in reducing nitro-compounds. Under continuous flow conditions
involving feeding, aeration, settling and reflux, and containing a mixture of
jj-nitrochlorobenzene and 2,4-dinitrochlorobenzene inoculated with Arthrobacter
simplex, the reduction of the nitrochlorobenzene reached 61-70% and dinitro-
chlorobenzene was reduced quantitatively. When two aeration columns were used,
one with Arthrobacter, the other with Arthrobacter, Fusarium, Trichoderma and
Streptomyces, a reduction up to 90% of nitrochlorocompounds after 10 days was
observed. The reduction of nitrochlorobenzene gave rise to p_-chloroaniline
and some undefined products. After reduction of dinitrochlorobenzene, nitro-
anilines and some unidentified products were formed.
The fate of £-chloronitrobenzene in Ohio River water
supplemented by weekly addition of settled sewage to provide nitrogen, trace
nutrients, and new organisms, has been studied by Ludzack and Ettinger (1963).
139
-------
Biodegradation was assessed from the evolution of (XL. The authors found no
degradation of nitrochlorobenzene after incubation periods as long as 175 days.
In the case of £-chloronitrobenzene, the environmental
stability determination can also be based to some extent on the available
monitoring data. This compound has been shown to travel long distances in
surface water as evidenced by the fact that the reduction in concentration of
the compound observed along 1450 Km of the Mississippi River could be totally
explained by dilution factors (Kramer, 1965). These findings suggested that
o-chloronitrobenzene remained unaltered, at least during the time period re-
quired for transport down 1450 river kilometers.
In summary, the available information regarding the
environmental fate of nitro- and chloronitro-substituted benzenes tends to
suggest that these compounds will not degrade at appreciable rates by micro-
organisms in the environment. The nitro-group of certain nitro-substi-
tuted benzenes has been shown to be reduced by microorganisms, and it is
likely that such modification may occur in the environment also; however, the
one compound (o-chloronitrobenzene) that has been monitored in the environment
does not appear to undergo such reduction. Other pathways of degradation of
these compounds in the environment have not been well studied. The resistance
of nitrobenzenes to extensive microbial degradation is supported by the fact
that very infrequently have researchers succeeded in enriching microbial pop-
ulations from natural mixed cultures, which will utilize nitro-substituted
benzenes as the sole source of carbon or nitrogen.
(ii) Nitrobenzoic Acids
The microbial metabolism of nitrobenzoic acid re-
ceived much attention after the suggestion that some of these compounds may
140
-------
be intermediates in the reduction of nitrates by green plants and micro-
organisms. The studies dealing with biodegradation of substituted and un-
substituted nitrobenzoic acids have been summarized in Table 35.
(a) Mono Nitrobenzoic Acids
Microbial Degradation
From the inability of phenol adapted bacteria
to oxidize a-, m~ and p-nitrobenzoic acid, Chambers e± al. (1963) concluded
that nitrobenzoic acids are resistant to biodegradation. Unlike the results
of Chambers et ajl. (1963), a number of researchers have reported the isolation
of microorganisms capable of growing on nitrobenzoates which may be suggestive
of the biodegradable nature of these chemicals. Cain (1958) isolated Nocardia
erythropolis from £-nitrobenzoate enrichment and IJ. opaca from o-nitro-
benzoate enrichment, both of which were capable of utilizing the respective
nitrobenzoic acids as sole sources of carbon, nitrogen, and energy. The en-
richment cultures were set up using garden soil, or water from polluted
streams as the source of natural mixed cultures. The presence of m-isomer
inhibited the oxidation of o- and ^-nitrobenzoic acid. However, m-nitro-
benzoic acid was found to be oxidized to some extent by organisms grown on
either _p_-nitrobenzoate or ^-nitrobenzoate. None of the isolates were able
to utilize hydroxy substituted (2-, 3-position) p-nitrobenzoic acid. After
continued efforts, Cain and his coworker (Cartwright and Cain, 1959 a) suc-
ceeded in isolating a Nocardia sp. (referred to as Nocardia Ml), which was
capable of metabolizing the meta-isomer of nitrobenzoic acid. Since nitro-
benzoates supported good growth only under alkaline conditions, Cain (1958)
suggested that the substrate was assimilated in the dissociated form (pK values
141
-------
Table 35. Summary of the Studies Dealing with Biodegradation of Substituted and Unsubstituted
Nitrobenzoic Acids
Reference
Alexander and
Lustigman, 1966
Cain, 1958
Cain et al. , 196#
Test Chemical Concn. Used
o-, m-, and £- 5-10 ppm
Nitrobenzoic acid
o-, m-, and £- ' 0.5-10 ppm
Nitrobenzoic acid
2,4-Dinitrobenzoic acid
3-Hydroxy-4-nitrobenzoic
acid
£-Nitrobenzoate, 2- 2-10umoles/3 mi
chloro-, 2-bromo-,
2-iodo-, 3-fluoro-
Source of
Microorganisms
Soil (Niagara
silt loam)
Species of Nocardia
and Pseudomonas
isolated from soil
and polluted stream
water
Nocardia
erythropolis grown
on p-nitrobenzoates
Duration of
the Test
64 days
7-16 days
Up to 2 hours
Criteria for
Test Chemical
Alteration
Loss of UV
absorbancy
Growth; forma-
tion of NH-,
arylamine, etc
Oxygen uptake,
assay of
metabolites
Cartwright and Cain,
1959a
NJ
4-nitrobenzoate
o-, m-, and jj-
Nitrobenzoic acid
1000 ppm
Nocardia opaca
Nocardia Ml
Nocardia erythropolis
Uptake of oxygen,
output of CO ,
and production of
either ammonia or
nitrite
Cartwright and Cain,
1959b
o^-, ^-Nitrobenzoic
acid
4.0 mM
Nocardia erythropolis, 24 hours
]N. opaca, Nocardia Ml,
and Pseudomonas
fluorescens isolated
from soil
Reduction to the
corresponding
arylamine
Chambers e^ al., 1963
Durham, 1958
c>-, m-, £-Nitro-
benzoic acid,
2,5-dinitrobenzoic
acid, 3,4-dinitro-
benzoic acid, 2,4,6-
trinitrobenzoic acid
£-Nitrobenzoic acid
60-100 ppm
Growth: 0.1-
0.2%; oxygen
Uptake: 4 mM
Microorganisms in
soil, compost, or
mud from catalytic
cracking plant waste
lagoon, adapted to
degrade phenol
Pseudomonas
fluorescens
3-3.5 hours
Oxygen uptake
Growth: 20 hours;
Oxygen uptake:
2 hours
Growth,
oxygen uptake
-------
Table 35. Summary of the Studies Dealing with Biodegradation of Substituted and Unsubstituted
Nitrobenzoic Acids . (Cont'd)
Reference
Germanier and Wuhrmann,
1963 (Abstract)
Ke et al. , 1959
Smith et al. , 1968
Symons et^ al. , 1961
Tsukamura, 1954
(Abstract)
Villanueva, 1960
Test Chemical
£-, and o-Nitro-
b.enzoic acid
o-Nitrobenzoic acid
£-Nitrobenzoic acid;
2-fluoro-, 2-chloro-,
2-bromo-4-nitro-
benzoate
o-, m-, and £-Nitro-
Na benzoate, 3,5-
dinltro-Na benzoate,
2,4,6-trinitro-Na
benzoate
£-Nitrobenzoic acid
o-, m-, £-Nitro-
Concn. Used
mM Concns.
(actual concn.
not specified)
Growth: 0.1%;
Oxygen uptake:
2 mM
10. gm/fc
C.O.D. of
125 mg/4
0.015%
0.05% (w/v)
Source of
Microorganisms
Pseudomonas strain
isolated from soil
Flavobacterium
from soil
Nocardia
erythropolis
grown on
£-nitrobenzoate
Nitrobenzoate-
adapted activated
sludge
Mycobacterium
tuberculosis
Nocardia V
Criteria for
Duration of Test Chemical
the Test Alteration
4 Days Growth and forma-
tion of NH ,
NO ~, and NO ~
Growth: 18- Growth, oxygen
20 hours ; uptake
oxygen uptake :
2.5 hours
Growth : Growth ,
4 days; oxygen uptake
Oxygen uptake:
2 hours
6 Hours, oxygen Oxygen uptake,
uptake; 24 hours, nitrogen
nitrogen release, C.O.D
release, up to reduction
54 days in case
of di- and tri-
nitro substituted
compounds
Formation of
arylamine
16 Days Growth
benzole acid
-------
for £- and £-nitrobenzoic acid, 4.23 and 5.62 respectively, Cain, 1958).
Based on these findings, the author proposed that the fate of nitrobenzoic
acids in the natural environment will be dependent on the pH value of the
environment.
The ease with which an organism can be enriched
on a chemical from a natural population of microorganisms can sometimes pro-
vide a fairly good indication of the relative biodegradability of the chem-
ical in the environment. For example, Cartwright and Cain (1959 a) experienced
a considerable amount of difficulty in isolating organisms capable of metabo-
lizing the meta-isomer of nitrobenzoic acid, although microorganisms utilizing
o- and £-nitrobenzoate were isolated with ease. This may be interpreted to
mean that the a- and £-isomer would disappear from the environment far more
easily than the meta-isomer. This conclusion agrees with the results of
a number of studies with pure and mixed cultures and with natural populations.
For example, in a study where soil served as the source of microorganisms, the
meta-isomer persisted for over 64 days whereas o_- and p_-isomers were found to
be. degraded rapidly (Alexander and Lustigman, 1966). The relative rates of
o-, m-, and £-nitrobenzoic acid degradation by activated sludge were determined
by Symons et al. (1961); the a- and £-position compounds were metabolized
almost at the same rate, with the meta-position compound again being the
slowest (Table 36). Of the 34 strains of soil bacteria belonging to the
Pseudomonas group tested for their ability to metabolize various isomers of
nitrobenzoic acid, some were capable of utilizing £-nitrobenzoic acid, but
none of the 34 was active on a- or m-nitrobenzoate (Kameda et^ al., 1957)
(Table 37). Villanueva (1960) studied the ability of nitroaromatic compounds
144
-------
Table 36. Time to Reach 100 mg/X, Level of Soluble C.O.D. Based on
Projected Curves (Symons et^ a^., 1961)
Compound
Time Required for Degradation
(Days)
£-Nitro Na benzoate
, £-Nitro Na benzoate
m-Nitro Na benzoate
5.2
5.5
46.0
* Initial COD concentrations were in the range of 630 - 690 mg/fc.
Table 37. Metabolic Activities of 34 Strains of Soil Bacteria Belonging to
Pseudomonas Group Towards Derivatives of Berizoic Acid
(Kameda et al., 1957)
Behzoic Acid
Derivative Tested
Number of Strains
Which Showed
Visible Growth
Number of Strains
Which Failed
To Grow
Strains Not
Tested
_p_-Nitrobenzoic acid
m-Nitrobenzoic acid
o-Nitrobenzoic acid
3
0
0
31
33
33
0
1
1
145
-------
to act as sole source of carbon and nitrogen for a species of Nocardia
(referred to as Nocardia V) and found that only p-nitrobenzoic acid promoted
full growth; the o- and m-isomer gave much smaller growth yield in the same
period.
The results of the laboratory biodegradation
experiments indicate that some removal of a- and p_-isomer of mono nitro-
benzoic acids would probably occur in the environment. The m-isomer has
consistently been shown to be persistent in pure culture, mixed culture, and
enrichment culture tests, and in view of this information it appears unlikely
that the m-isomer would degrade to a measurable extent in the natural environ-
ment.
Routes of Degradation
Nitrobenzoic acid has been reported to degrade
by different pathways depending upon microorganisms. The major route of
breakdown of nitrobenzoic acids in Nocardia sp. was suggested to be via oxi-
dation by way of hydroxylated intermediates. From the studies on the effect
of known metabolic inhibitors on the oxidation of nitrobenzoic acid and
through application of simultaneous adaptation methods, Cartwright and Cain
(1959 a and b) have suggested the scheme for oxidative metabolism of nitro-
benzoates depicted in Figure 16.
Although aryl amines are formed in the culture
fluid under certain conditions, they have been shown not to lie on the
direct oxidation pathway of nitrobenzoic acid in Nocardia sp. This is also
supported from the experiments of Villanueva (1960), who found that Nocardia V.,
which grows on ^-nitrobenzoic acid, fails to grow when provided with p_-amino
146
-------
para-Isomer
meta-Isomer
A-Nitrocatechol
OH
'Protocate-
chuic acid
C02H
CO
C02H
CH
H00-
-^•CH2 '*—> CH2
6-Carboxymu-
conic acid
2°
-------
benzole acid as the sole source of carbon and nitrogen. During the metabolism
of nitrobenzoic acid by Nocardia sp., the nitrogen is liberated as nitrite in
the case of the m-isomer, and in the form of ammonia with the £- and £-isomer.
A strain of Pseudomonas fluorescens capable of
utilizing £-nitrobenzoic acid as a sole source of organic carbon and nitrogen
for aerobic growth, has been found to metabolize nitrobenzoic acid via a dif-
ferent pathway. In this organism, £-aminobenzoic acid is a direct inter-
mediate in the breakdown of p-nitrobenzoic acid (Durham, 1958). Based on the
experimental result, the following sequence was suggested for p-nitrobenzoate
transformation by P_. fluorescens.
COOH
COOH
COOH
COOH
-NH3
p_-Nitrobenzoic
acid
£-Aminobenzoic £-Hydroxybenzoic
acid acid
Protocatechuic acid
Figure 17. Metabolism of £-Nitrobenzoate by Pseudomonas sp.
(Durham, 1958)
148
-------
Further metabolism of protocatechuic acid was
suggested to be via B-oxoadipic acid as reported by Stanier et al. (1950) and
shown in Figure 16. Symons et^ ad. (1961) have reported a similar pathway of
£-nitrobenzoate degradation by activated sludge.
The o-isomer of nitrobenzoic acid, on the other
hand, has been shown to be metabolized in Flavobacterium sp. via intermediates
nitrosobenzoic acid and hydroxyl amino benzoic acid without the involvement of
o-aminobenzoic acid (Ke et al., 1959).
COOH
COOH
cellular
materials
A
COOH
Figure 18. Metabolism of £-Nitrobenzoate by Pseudomonas sp. (Ke £t^ £l., 1959)
o-Nitrobenzoic acid metabolism by activated sludge was also shown to
proceed via a similar pathway (Symons et^ al_., 1961). Symons and his co-
workers further reported that the intermediates which followed included o-
hydroxylaminobenzoic acid and possibly catechol, protocatechoic acid and succinic
acid. The results of Ke et^ aJ. (1959), however, suggest that protocatechuic
acid does not occupy an intermediary position in the metabolism of the £-ispmer.
149
-------
As indicated above, the routes of degradation
of nitrobenzoic acid by pure cultures of microorganisms appear to have been
fairly well investigated. However, no studies have been reported in the
literature which deal with the route of breakdown under environmental con-
ditions. It is uncertain if the degradation of nitrobenzoic acid in the
environment will be catalyzed predominantly by Nocardia sp., Pseudomonas sp.
or by some other species of microorganisms which has not yet been enriched,
or by a mixture of these microorganisms. Furthermore, it is also uncertain
if the pathway of degradation by an organism will be similar under laboratory
and field conditions. In the absence of this information, the route of deg-
radation of c>-, m-, and p_-nitrobenzoic acid under environmental conditions
remains obscure.
(b) Di- and Trinitro-substituted Benzoic Acids
The studies reported to date reveal that di-
and trinitro-substituted benzoic acids are not attacked by microorganisms.
Symons &t_ a±. (1961) noted that neither 3,5-dinitro-Na-benzoate nor 2,4,6-
trinitro-Na-benzoate could be degraded by activated sludge adapted to p_-
nitrobenzoate. Degradation could also not be initiated by introducing an
easily metabolizable carbon source (e.g., mononitrobenzoic acids or by addi-
tion of fresh sludge to supply new microorganisms and nutrients). The studies
undertaken by Chambers et al. (1963) further support the inability of micro-
organisms to degrade di- and trinitro-substituted benzoic acids. These
authors tested the ability of a culture highly adapted to degrade phenol (phenol
was presumed to have the same basic configuration as nitrobehzoates, and thus be
able to induce requisite enzymes) to attack various nitrobenzoic acids. The
150
-------
experimental findings indicated little or no activity of the phenol-adapted
cells with 3,5-9 3,4-, or 2,5-dinitrobenzoic acid, or with 2,4,6-trinitro-
benzoic acid, which suggests that these compounds are difficult to degrade.
It must be pointed out, however, that nonbiodegradability of nitrobenzoates
in this study may be attributed to the inability of phenol to induce appro-
priate enzymes for the degradation of di- and trinitro-substituted benzoic
acids. Cain (1958), on the other hand, has noted that one of the dinitro-
substituted compounds - 2,4-dinitrobenzoic acid - could serve as the sole
carbon source to the species of Nocardia enriched and grown on p_- or o_-
nitroberizoates. However, since only a small amount of growth was observed
after the test period of seven days, the biodegradable nature of the dinitro-
substituted benzoic acid is somewhat inconclusive.
(c) Halogen Analogues of Nitrobenzoic Acids
A number of studies dealing with biodegradation
of halogen analogues of nitrobenzoic acids have been reported. The utili-
zation of halogen-substituted nitrobenzoates by p-nitrobenzoate-grown cells
of Nocardia erythropolis was examined by Smith et^ al. (1968). The authors
found that 2-fluoro-, 2-chloro-, or 2-bromo-4-nitrobenzoate did not support
growth of this organism, nor did they increase the growth yield when added
to the cultures utilizing fumarate as carbon source. The halogenated nitro-
benzoic acid, when present together with £-nitrobenzoic acid, inhibited the
induction of the. £-nitrobenzoate-oxidation system, and caused inhibition of
growth. The halogen analogues of £-nitrobenzoates were, however, oxidized
by washed £-nitrobenzoate-grown cells of 1>J. erythropolis (Cain et
al., 1968). The ease of oxidation decreased in the order £-nitrobenzoate>
151
-------
2-fluoro-4-nitrobenzoate>2-chloroderivative>2-bromo and 2-iodo derivatives
(Figure 19).
For detailed metabolic study, Cain and his co-
workers used only one of the halogen analogues: 2-fluoro-4-nitrobenzoate.
The results indicated that 2-fluoro-4-nitrobenzoate was oxidized to fluoro-
acetate; the pathway of degradation appeared similar to that reported for
£-nitrobenzoate (Cartwright and Cain, 1959 a, see Figure 16), except that
corresponding fluoro-intermediates were formed. Several fluorine containing
metabolic intermediates were detected by the authors, and 2-fluoroproto-
catechuate was identified as one of them. No fluoride ions were released
into the incubation medium. The nitrogen of the nitro group in 2-fluoro-4-
nitrobenzoate was recovered entirely as ammonia. Incubation of p_-nitroben-
zoate grown cells with 3-fluoro-4-nitrobenzoate and 3-methyl-4-nitrobenzoate
resulted in the formation of £-dihydroxy compounds: 5-fluoroprotocatechuate and
5-methylprotocatechuate, respectively. Further breakdown of these compounds
was not reported.
(iii) Nitrophenols and Related Compounds
Nitrophenols are generally toxic and have a pro-
nounced inhibitory effect upon the processes of assimilation in cell metabolism.
For example, the selective action of dinitrophenol on cell respiration is well
known (Simon and Blackman, 1953). These properties of nitrophenols, coupled
with the fact that a number of nitrophenols are commercialized as pest con-
trol agents or are used for synthesis of other more selective pesticidal
agents, have inspired both the basic scientist as well as those concerned with
the pollution potential of nitrophenols from their commercial applications,
to investigate the biological transformation of these compounds. The fate of
152
-------
240-1
40 60
TIME I mint
60
100
Fig. 19. Oxidation of halogenonitrobenzoates by p-nitro-
benznate-grown N^. erythrnpolis. o, p-Nitrobenzoate; A,
2-fluoro-4-nitrobenzoate; «, 2-chloro-4-nitrobenzoate; A,
2-bromo-A-nitrobehzoate; ?, 2-iodo-A-nitrobenzoate; ,
3-fluoro-4-nitrobenzoate. Oxygen uptakes were corrected
for endogenous respiration (185 yl/hr.). (Cain et al.,
1968)
153
-------
nitrophenols and the routes of their degradation have thus been reasonably
well investigated. The experimental details of the studies dealing with the
decomposition of nitrophenols with pure cultures or with natural communities
of microorganisms are summarized in Table 38.
(a) £-, m-, and £-Nitrophenols
Microbial Degradation
As can be noted from the table, in studying the
environmental fate of nitrophenols, efforts of the majority of researchers have
been directed towards isolation of the microorganism(s) which can modify these
chemicals either by using them as sources of energy, carbon, or nitrogen, or
by cometabolism (concomitant metabolism of a non-growth substrate). Simpson
and Evans (1953) isolated two organisms (Pseudomonas sp.), which used o-
nitrophenol (referred to as strain SO) and £-nitrophenol (referred to as
strain SP) as the sole source of carbon. The organisms were isolated from
the filter beds of a biological treatment plant. The nitro group was oxi-
datively eliminated as nitrite in this process. An organism which appeared to
be a strain of either Flavobacterium or a closely related bacterium, capable
of using £-nitrophenol as carbon and energy source, has been isolated from soil
by Raymond and Alexander (1971). The organism required some component of
soil extract for its multiplication in the nitrophenol medium. In addition,
the medium employed for studying degradation of nitrophenol also contained
small quantities of yeast extract. Cells grown on _p_-nitrophenol were able
to cometabolize the m-isomer to nitrohydroquinone but were not able to use
it as a carbon source for growth.
In a number of pure culture studies dealing
with the degradation of nitrophenols, researchers have used organisms which
154
-------
Table 38. Experimental Conditions Used by Various Investigators in Studying the Fate of Nitrophenols
and Related Compounds
Nitrophenols
01
Reference
Alexander and
Lustigman, 1966
Cain, 1958
Chambers et al. ,
1963
Guillaume et al. ,
1963
Gundersen and
Jensen, 1956
Jensen and Lautrup-
Larsen, 1967
Madhosingh, 1961
Raymond and
Chemicals Tested Concn. Used
o-, m-, £-Nitrophenol 8-15 yg/ml
o-, m-, £-Nitrophenols 0.025%
o_-, m-, £-Nitrophenol; 60-100 mg/X,
2,4-, 2,6-dinitro-
phenol; 2,4,6-, trini-
trophenol; 2-chloro-4-
nitrophenol; 4-chloro-
2-nitrophenol; 2,6-
dichloro-4-nitrophenol
m- and £-Nitrophenol 50 yg/ml
o-, m-, £-Nitrophenol; 0.01-0.05%
2,4-, 2,5-, and 2,6-
dinitrophenol; 2,4,6-
trinitrophenol
2,4-Dinitrophenol; 2,4, 0.2-0.5 mM
6-trinitrophenol ; o-,
m-, £-nitrophenol; 2-
methyl-4-nitrophenol ;
dinitro(sec)butylphenol
2,4-DinitrophenoL 0.001-0.006%
-4
£-, m-Nitrophenol 5.5 x 10 M
Criteria for
Source of . Duration of Test Chemical
Microorganisms the Test Alteration
Niagara silt loam
Nocardia sp. enriched
on £-nitrobenzoic acid
Microorganisms from soil,
compost, or mud from a
catalytic cracking plant
waste lagoon, adapted to
phenol
Mycobacteria sp.
Arthrobacter simplex
isolated from soil
Arthrobacter and
Pseudomonas-like
organisms isolated
from soil
Wood decaying fungus
tolerant to DNP: £.
oxysporum and C. micaceus
Flavobacterium sp .
64 Days Loss of UV
absorption
7 Days Growth
180-230 min. Oxygen uptake
— Formation of the
product-4-nitro-
pyrocatechol
Up to Growth, nitrite
10 days release, loss of
color
20 Days for Growth, formation
growth; 24 of nitrite, loss
hours for of color
nitrite
'30 Days Reduction of the
nitro groups
6-72 Hours Growth, nitrite
Alexander, 1971
isolated from soil
formation
-------
Table 38. Experimental Conditions Used by Various Investigators in Studying the Fate of Nitrophenols
and Related Compounds (Cont'd)
ui
Nitrophenols (cont.)
Reference
Chemicals. Tested
Concn. Used
Source of
Microorganisms
Duration of
the Test
Criteria for
Test Chemical
Alteration
Simpson and Evans,
1953
Villanueva, 1960
Villeret, 1965
Nitrocresols
Chambers et al.,
1963
Gunderson and
Jensen, 1956
Jensen and
Lautrup-Larsen,
1967
Tewfik and Evans,
1966
f>-, £-Nitrophenol; 100 ppra
2,4-dinitrophenol
p_-, m~> £-Nitrophenols; 0.025%
2,4-dinitrophenols;
2-amino-4-nitrophenol;
picric acid
°~» 5-> £-Nitrophenols 10~5-10~2 M
4,6-Dinitro-p_-cresol;.
2,4,6-trinitro-m-
cresol
60-100 mg/H
4,6-Dinitro-o-cresol 0.02%
4,6-Dinitro-o-cresol 0.2-0.5 mM
4,6-Dinitro-o-cresol 0.01% (w/v)
Pseudomonas sp. isolated
from the filter beds of
a biological detoxifica-
tion plant
Nocardia V
Chlorella vulgaris
Growth
16 Days Growth
Growth with nitro
group as the
nitrogen source
Microorganisms from soil, 210 Minutes Oxygen uptake
compost or mud from a
catalytic cracking plant
waste lagoon, adapted to
phenol
Arthrobacter Simplex
isolated from soil
Arthrobacter and Pseudo-
monas -like organisms
isolated from soil
Pseudomonas sp.
isolated from garden soil
Up to 10
days
Growth, nitrite
release, loss of
color
20 Days for Growth, release
growth; 24 of nitrite, loss
hours for of color
N0_~ release
Growth, release
of nitrite ion
-------
Table 38. Experimental Conditions Used by Various Investigators in Studying the Fate of Nitrophenols
and Related Compounds (Cont'd)
Nitroresorclnols
Reference
Chemicals Tested
Concn. Used
Source of
Microorganisms
Duration of
the Test
Criteria for
Test Chemical
Alteration
Brebion et al. ,
1967 (Abstract)
Chambers et al.,
1963
2,4-Dinitroresorcinol;
2,4,6-trinitro
resorcinol
2,4,6-Trinitro-
resorcinol
Approx. 200 ppm
60 mg/fc
Bacterial cultures taken
from earth, water, and
mud, especially from areas
already polluted
Elimination of
the parent
compound
Microorganisms from soil, 210 Minutes Oxygen uptake
compost, or mud from a
catalytic cracking plant
waste lagoon, adapted to
phenol
en
—i
-------
were enriched on chemical compounds resembling nitrophenols. For example,
Cain (1958) tested the ability of the Nocardia sp. isolated by en-
richment on £-nitrobenzoic acid to utilize £-, m-, and p_-nitrophenols. The
authors noted no growth of the organism on any of the nitrophenol isomers.
The microorganisms which were enriched from soil with herbicide 4,6-dinitro-
o-cresol were capable of decomposing p_-nitrophenol with release of nitrite;
other nitrophenol isomers were not attacked (Gundersen and Jensen, 1956;
Jensen and Lautrup-Larsen, 1967) (Table 39). Chambers et^ al^ (1963) noted
Table 39. Degradation of Mononitrophenols by DNOC-Grown Bacteria
(Gundersen and Jensen, 1956; Jensen and Lautrup-Larsen,
1967)
Nitrophenols Attacked
Organism Enriched on
Herbicide 4,6-Dinitro-
ortho-cresol o-Nitrophenol m-Nitrophenol p_-Nitrophenol
Arthrobacter Simplex +
Arthrobacter X. ~
Pseudomonads +
(from acid loam field soil)
that a culture adapted to degrade phenol was capable of oxidizing o-, m-,
and p_-nitrophenol at a slow rate; o_- and m-isomers were oxidized at relatively
higher rates than the p_-isomer. The culture was also able to oxidize a number
of chloro-substituted nitrophenols at slow rates. The compounds attacked were
2-chloro-4-nitrophenol, 4-chloro-2-nitrophenol and 2,6-dichloro-4-nitrophenol,
the rate of oxidation being the highest with 4-chloro-2-nitrophenol. The
158
-------
assessment of biodegradation from the oxygen uptake data is susceptible to
many criticisms. For example, by their uncoupling action, nitrophenols may
stimulate the endogenous oxygen uptake rates which may be confused with in-
creased oxygen uptake due to the breakdown of nitrophenol. One of the well-
known uncouplers of oxidative phosphorylation is 2,4-dinitrophenol; however,
a similar action may also be exerted by mononitro- and certain trinitrophenols.
Several researchers have investigated the
biodegradability of nitrophenols using the microorganisms which were selected
on the basis of metabolic versatility. Guillaume et^ al^. (1963) studied the
oxidation of _p_- and m-nitrophenol by certain Mycobacteria. The experimental
findings revealed that both the isomers could be attacked and that product of
oxidation was identified as 4-nitropyrocatechol. Since some species of
the genus Nocardia were reported to utilize aromatic compounds (Sergey*js Manual
of Determinative Bacteriology, 1948), Villanueva (1960) tested the ability of
a Nocardia sp. (referred to as Nocardia V) to utilize nitrophenols for growth.
The results of this study are, however, inconclusive perhaps because the esti-
mation of growth was based on the visual examination of the culture. Villeret
(1965) reported the utilization of the nitro group of m-, p_-, and ^-nitrophenols
as a source of nitrogen during growth of freshwater algae Chlorella vulgaris.
The nj-isomer supported better growth than the p_~isomer, with the £-isomer
giving minimal growth.
The studies described above, in which pure cultures
of microorganisms obtained from a variety of sources have been used, appear to
suggest that mononitrophenols are susceptible to microbial attack. The p_-
and £-isomer are attacked by a greater number of microorganisms than the
159
-------
m-isomer. Whether mononitrophenols are degraded in the natural environ-
ment, and whether the order of biodegradability is the same as derived
from the laboratory pure culture studies, is debatable. The main difficulty
encountered in extrapolation of the pure culture data is that the concentra-
tion of the test chemical employed for enrichment of an organism and for
obtaining a reasonable amount of cell growth at the expense of the chemical
is far removed from the concentrations generally encountered in nature.
Whether the appropriate enzyme(s) can be induced and/or growth can occur at low
environmentally significant concentrations is not clearly understood.
There are only a few published studies regarding
the breakdown of nitrophenolic compounds by. natural communities of micro-
organisms. Brebion et^ £l_. (1967) examined the ability of the microorganisms
taken from soil, water, or mud, and grown in a porous mineral bed to attack
p_-nitrophenol. The cells were cultivated on a mineral nutrient solution in
which nitrophenols were added as the sole source of carbon. The experimental
findings revealed no significant removal of the compound under these conditions.
The fate of £-, m-, and £-nitrophenols by natural communities of microorganisms
in soil has been studied by Alexander and Lustigman (1966). These authors
found that the ^-isomer was far more resistant to degradation (persisted for
>64 days) than the m- and j>-isomer (m, 4 days; p_, 16 days). This is unlike
the observations made with pure cultures of microorganisms where the £-isomer
was found to be attacked easily. The degradation of the test compounds in this
study was determined by following the loss of ultraviolet absorbancy when the
benzene ring is cleaved by microorganisms present in the soil. The assay
technique makes use of a small soil inoculum to minimize interference, and
160
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thus, in turn, limits the concentration of microorganisms and soil nutrients
in the assay system. Furthermore, the test conditions used in this study
simulate a soil suspension rather than the soil. In view of these short-
comings, extrapolation of the laboratory data to assess environmental fate
of nitrophenols is difficult.
Routes of Degradation
Very little is known about the metabolic sequence
of the breakdown of a-, m-, or p_-nitrophenol. The available information has
been derived predominantly from the pure culture work. Simpson and Evans
(1953) using Pseudomonas sp. which utilize ^-nitrophenol as its sole source
of organic carbon, obtained evidence which supported the view that degradation
of ^-nitrophenol proceeded via an oxidative elimination of the nitro group
and resulted in the formation of catechol. The catechol thus formed was
suggested to be utilized by well-known pathways involving cis-muconic and
3-ketoadipic acid (Evans et^ a!L., 1951). Similarly, in the metabolism of p_-
nitrophenol, the corresponding dihydroxy intermediate formed was shown to be
£-dihydroxybenzene (quinol). Raymond and Alexander (1971) reported a somewhat
different pathway for nitrophenol metabolism in a Flavobacterium sp. These
authors obtained evidence which suggested that an initial step in the reaction
with nitrophenol is its hydroxylation. The metabolite generated from p_-
nitrophenol was identified as 4-nitrocatechol; the metabolite underwent
further degradation as evidenced by the considerable amount of oxygen con-
sumption and growth of the bacterium on p_-nitrophenol. The actual metabolites
were, however, not directly identified in this study. The product of cometab-
olism of the m-isomer was shown to be nitrohydroquinone; the metabolite was not
161
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degraded further by the Flavobacterium sp. A similar mechanism of oxidation
for nitrophenol has been reported in certain Mycobacteria (Guillaume et^ al.,
1963).
(b) Di- and Trinitrophenols
The most extensively investigated compounds in
this group are 2,4-dinitrophenol (2,4-DNP) and 2,4,6-trinitrophenol (picric
acid). As early as 1953, Simpson and Evans (1953), in a brief communication,
reported isolation of an organism from soil which formed nitrite from 2,4-
dinitrophenol. A number of microorganisms enriched from soil on dinitro-
^-cresol were shown to metabolize the nitro group of 2,4-dinitrophenol and
2,4,6-trinitrophenol with the formation of nitrite; 2,5- and 2,6-dinitro-
substituted phenols were not attacked and only negligible amounts of nitrite
appeared with these compounds (Jensen and Lautrup-Larsen, 1967; Gundersen and
Jensen, 1956). The picture is somewhat obscure as far as the carbon nutrition
from nitrophenolic compounds is concerned. It is unclear from the results
if the microorganisms cause ring cleavage and/or derive any carbon from the
compound. One of the organisms in this study was identified as Arthrobacter
simplex; other organisms were provisionally referred to as Arthrobacter x. and
Pseudomonas. The percentages of organic nitrogen converted to nitrite-nitrogen
from different compounds were different. Whereas nearly all the trinitrophenol
nitrogen was converted to nitrite, only 50% could be detected from 2,4-dinitro-
phenol. The fate of the remaining nitrogen in the case of 2i4-dinitrophenol
is obscure, except that a small portion was found to be assimilated by the
organism (Jensen and Lautrup-Larsen, 1967). The metabolic attack on 2,4,6-
trinitrophenol was accompanied by formation of an unidentified soluble rust
162
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brown pigment (structure unknown). A similar pigment was reported to be
formed in the cultures of Arthrobacter simplex incubated with 2,5-dinitro-
and 2,6-dinitrophenol.
The experimental findings of Chambers et a^l.
(1963) indicate that a phenol-adapted culture was able to slowly oxidize 2,4-
and 2,6-dinitrophenol and 2,4,6-trinitrophenol. The oxidative activity was
slowest with the 2,6-dinitro isomer. The results of oxygen uptake studies with
nitrophenolic compounds must be interpreted with caution as discussed earlier.
The possibility cannot be excluded that the increased oxygen uptake observed by
the authors was due to the enhancement of endogenous oxygen uptake by nitrophenols
due to their uncoupling effect.
A number of studies have indicated that enzyme
systems in bacteria and fungi can reduce dinitrophenolic compounds to the cor-
responding arylamines in an attempt to detoxify the compounds. For instance,
the aerobic bacterium Azotobacter chroococcum, anaerobic bacterium Clostridium
butyrjcum, and the fungus Fusarium (a DNP-tolerant fungus) may all reduce 2,4-
DNP according to Radler (1955), Lehmber (1956), and Madhosingh (1961). The
reduction of the nitro group in the fungus was postulated to take place in
stages, involving the intermediate formation of the nitroso and hydroxylamino
groups as shown in Figure 20.
OH OH
,NO
N00 N=0
Dinitrophenol 4-Nitroso-2- 4-Hydroxylamino- 4-Amino-2-
nitrophenol 2-nitrophenol nitrophenol
Figure 20. Sequence of the Reduction of Nitro Group In Fusarium sp.
(Madhosingh, 1961)
163
-------
The reduction of the nitro group could occur either in position 2 or 4, thus
giving rise to 2-amino-4-nitrophenol and 4-amino-2-nitrophenol, respectively.
Both of these products have been identified by Madhosingh (1961) in the DNP-
treated media which had maintained the growth of the fungus. No other in-
formation regarding the pathway of degradation or detoxification of dinitro-
and trinitrophenolic compounds is available in the literature.
From the experimental evidence presented above,
it can be concluded that the nitro compounds 2,4-dinitrophenol and 2,4,6-
trinitrophenol are susceptible to partial degradation by certain microorganisms.
It may be speculated that nitrophenols will be subjected to microbial attack
in the environment by the microorganisms adapted to phenol or the herbicide
dinitro-o-cresol. Both phenol and dinitro-o-cresol appear to be widespread
contaminants and thus the microorganisms adapted to them will also be expected
to be widespread. Since 2,5- and 2,6-dinitro-substituted phenols have not
beeri shown to be altered by DNOC- or phenol-adapted culture, it appears
likely that these isomers may persist in the environment for extended periods
of time.
(c) Nitrocresols
A number of nitrocresols are well-known her-
bicides, and their fate in soil has received much attention. DNOC (4,6-
dinitro-^-cresol) usually disappears from the soil within a few weeks to two
months (Petersen and Hammarlund, 1953; Bruinsma, 1960; Jensen, 1966). The
elimination of DNOC at least in part was attributed to the effect of micro-
organisms (Jensen and Lautrup-Larsen, 1967). Jensen and coworkers (Gundersen
and Jensen, 1956; Jensen and Lautrup-Larsen, 1967) isolated an Arthrobacter
164
-------
and a Pseudomonas that grew on DNOC with the release of nitrite. The
Pseudomonas sp. was able to attack DNOC within a wide range of pH (4.4 - 8.8).
The authors reported that certain strains of Nocardia isolated from 2,6-dinitro-
phenol-treated soil could also metabolize DNOC. A Pseudomonas capable of
metabolizing DNOC in the presence of hydrogen donors (e.g., glutamate, lactate,
yeast extract, etc.) has been isolated from soil by Tewfik and Evans (1966).
To Some extent dinitro- and trinitro-substituted cresols were oxidized by a
culture which had been adapted to degrade phenol (Chambers et_ al^., 1963). The
compounds investigated in this study were 4,6-dinitro-£-cresol and 2,4,6-
trinitro-m-cresol. The oxidative activity was found to be relatively greater
with dinitro- than with trinitrocresol.
The degradation pathway of dinitro-^-cresol in
pure cultures of microorganisms has been investigated by Tewfik and Evans
(1966). The findings indicated that in Pseudomonas sp., degradation proceeded
via formation of an aminocresol; in contrast, Arthrobacter simplex was found
to employ initially a reaction involving a hydroxylated intermediate (Figure 21).
The evidence indicated that the pathway of degradation after the inital step
was similar in both organisms.
(d) Nitroresorcinol
Chambers ejt al_. (1963) investigated the sus-
ceptibility of 2,4,6-trinitroresorcinol to microbial attack using a culture
which had been adapted to degrade phenol. The criterion for degradation was
the oxidation of the molecule by microbial action. The experimental findings
indicated that negligible amounts of oxygen were consumed with trinitroresorcinol
as the substrate, suggesting the recalcitrant nature of the molecule. It
165
-------
OH
Pseudoraonas
NO,
N02
4,6-dinitro-fi-cresol
4-amino-6-nitro-a-cresol
I Arthrobacter
I
4-methyl-6-nitro-catechol
i
I
l
I
M'
4-methyl-6-amino-catechol
I
I
2,3,5-tr ihydroxytoluene
Ring Cleavage
Figure 21. Metabolism of 4,6-Dinitro-o-Cresol by Soil Microorganisms
(Tewfik and Evans, 1966)
166
-------
is also likely, however, that adaptation of the microorganisms to phenol failed
to induce appropriate enzymes for the degradation of trinitroresorcinol. The
inability of microorganisms to attack 2,4,6-trinitroresorcinol was also in-
dicated from the studies of Brebion et^ _al_. (1967). These investigators in-
cubated the microorganisms (from soil, water, or mud especially from areas
already polluted) with a nutrient solution to which nitro-substituted resorcinols
were added as the sole carbon source. The incubation was carried out in a
porous mineral bed. The experimental findings revealed no significant removal
of 2,4,6-trinitroresorcinol and about 36% removal of 2,4-dinitroresorcinol.
The results of the studies of both Chambers et al. (1963) and Brebion et al.
(1967) point to the recalcitrant nature of the 2,4,6-nitro-substituted
resorcinol. On the other hand, the 2,4-nitro-substituted compound may be de-
graded to a small extent.
(iv) Nitrotoluenes
There has been a considerable amount of concern over
the level of trinitrotoluene pollution generated by military munitions pro-
duction. In order to minimize water pollution, a number of researchers have
investigated the biodegradability of trinitrotoluene and other explosives and
the feasibility of using biological processes for treatment of the waste water
resulting from their manufacturing and loading in munitions products. The
studies can be classified into three major categories, depending upon the choice
and source of biological agent and environmental conditions used in degradation:
(a) Degradation by pure cultures of microorganisms.
(b) Degradation by natural communities of microorganisms.
(c) Degradation under sewage treatment plant conditions.
167
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All the studies dealing with the environmental fate of nitrotoluenes are
summarized in Table 40.
(a) Degradation by Pure Cultures of Microorganisms
As early as 1951, Russian worker Rogovskaya
noted that certain microorganisms can utilize TNT better as a source of nitrogen
than as a source of carbon. Enzinger (1970) isolated several Pseudomonas-like
microorganisms from the mixed liquor suspended solids of a TNT laboratory
scale biooxidation unit and from contaminations found in prepared TNT agar;
the organisms could be acclimated to TNT and grown successfully on trypticase
soy broth in the presence of high concentrations of TNT (29-100 ppm) (one of
the organisms grew without prior acclimation). Acclimation was achieved by
subculturing into media containing successively higher TNT concentration. TNT
concentration during growth could be reduced to as low as 1.25 ppm (from the
starting concentration of 100 ppm) within a 5 day period; the reduction in
the concentration of TNT was linked to the appearance of two unidentified
products in the medium. No unaltered TNT was detected in the cells. In
addition to testing the pure cultures of microorganisms isolated as described
above, the author also investigated the ability of Zoogloea ramigera 115 to
degrade TNT. The organism is relatively abundant in sewage treatment facilities
and has been isolated by Friedman and Dugan (1968). The cells of Zoogloea
were found to be relatively more sensitive than Pseudomonas to TNT. It is
unclear from the paper, however, if Zoogloea was able to remove TNT after an
acclimation period.
Three Pseudomonas-like organisms capable of
metabolizing TNT have been isolated by Won et al. (1974) from mud and water
168
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Table 40. Salient Features of the Biodegradation Studies with Nitrotoluenes
VO
Reference
Villanueva,
Chambers et
1960
al.,
Test Compound
p_-Nitro toluene
2,4-Dinitrotoluene ;
Concn. Used
0.025% (w/v)
LOO mg/Jl
Source of Microorganisms
Nocardia v
Microorganisms
in soil.
Duration of.
the Test
16 Days
3 Hours
Criteria for Test
Chemical Alteration
Growth
Oxygen uptake
1963
Osmon and Klaus-
meier, 1973
Won et al. ,
1974
Enzinger, 1970
2,4,6-trinitro-
toluene
Trinitrotoluene,
ammonium picrate
2,4,6-Trinitro-
toluene
2,4,6-Trinltro-
toluene
100 mg/Jl
12-35 x
10~5 M
100 ppm
compost, or mud from a
catalytic cracking plant
waste lagoon, adapted to
phenol
Sewage effluent, TNT loading 4r6 Days
facility effluent, soil,
pond, and aquarium water,
pure culture of Pseudomonas
aeruginosa
Mud and water sample
obtained from U.S. Naval
Ammunition Depot at
MacAlester, Okla.
Mixed liquor suspended
solids from laboratory scale
biooxidation units; pure
cultures of Zooglea ramigera
115
Oxygen uptake:
4 hours; growth:
70 hours
120 Hours
Assay of the
parent compound
Growth, oxygen
consumption
Measurement of
the parent
compound
Nay, 1972 ; Nay
et al. , 1974
Trinitrotoluene 2.5-40% TNT
manufacturing waste waste con-
water; 2,4,6-TNT taining
2 mg/fc TNT
added to the
samples;
2,4,6-TNT
12-64 mg/J.
Munitions plant domestic 8-18 Days
waste
BOD test using
manotnetric apparatus ;
TNT analysis
-------
samples collected from a naval base. At the naval base, TNT had been used in
large amounts for long periods of time, thus enhancing the possibility of
selection of TNT degrading microflora. The conversion of TNT was found to
vary with the isolates. An isolate (tentatively named as isolate Y) was able
to metabolize TNT most effectively. At 24 hours, the TNT concentration was
reduced from 100 yg/m£ to less than 1 yg/mfc in yeast extract enriched medium;
the rates were relatively slower in glucose supplemented cultures. Thin layer
chromatographic analysis of the incubation mixture revealed that TNT was
metabolized to yield a variety of reduced TNT metabolites and azoxy toluenes
(see Figure 22, p. 177); extensive degradation of TNT did not take place
since no ring breakdown products were identified. During the enrichment of
the microorganisms capable of using TNT, Osmon and Klausmeier (1973) noted the
predominance of pseudomonas among the isolated TNT transforming cultures.
Consequently, they examined the ability of a pure culture of Pseudomonas
aeruginosa (ATCC 13388) to degrade TNT. The organism was found to be able to
transform TNT only when provided with extraneous nutrients. For instance, in
the presence of 1-2% glucose, TNT concentration was decreased from 100 ppm to
30 ppm in 5 days based on the residual TNT analysis. It was concluded from
these observations that degradation occurred via a co-metabolic process (con-
comitant metabolism of a non-growth substrate). The authors noted the accumu-
lation of small quantities of a number of unidentified TNT metabolites in the
medium. The metabolites were reported to be transient in nature and they
eventually disappeared. The disappearance of TNT as well of certain TNT
metabolites to some extent may be due to their accumulation in the cell. Since
the authors did not extract the cells, the extent of removal by this mechanism
is unclear.
170
-------
The pure culture studies described here provide
evidence that TNT can undergo minor modification by the action of certain
microorganisms; however, any degradation of the compounds is questionable.
Furthermore, the concentration of TNT employed in these studies is more repre-
sentative of the concentration of TNT in the waste water resulting from the TNT-
manufacturing process, and thus the results are more applicable to determining
the feasibility of biological treatment for disposing of TNT, rather than for
assessing its environmental fate.
Very little information is available concerning
microbial decomposition of mono- and dinitro-substituted toluenes. Douros and
Reid (1956) briefly examined the biodegradability of 2,4-dinitrotoluene using
the cultures of soil microorganisms _P. aeruginosa and ]?. putida which had been
adapted to degrade the herbicide 3-(p_-chlorophenyl)-l,l-dimethylurea. Both
strains yielded fair amounts of growth with 2,4-dinitrotoluene as the sole
organic compound, which suggests that the compound was attacked by the micro-
organisms. Villanueva (1960) reported failure of a strain of Nocardia (Nocardia
V) to use p-nitrotoluene as the sole source of carbon for growth. Nocardia V
was chosen for these studies on the basis of the earlier reports regarding
catabolic versatility of the genus Nocardia (Moore, 1949; Cain, 1958).
(b) Degradation by Natural Communities of Microorganisms
Osmon and Klausmeier (1973), while attempting
to develop a biological disposal method for waste from an explosive factory,
studied the ability of inocula from a variety of natural sources to degrade TNT.
The following sources of inoculum were tested: effluent from sewage treatment
plants; waste water from an ordnance loading facility using TNT; a soil suspension
171
-------
and pond water; and water from a laboratory aquarium. Transformation was
studied in a mineral salts medium containing 100 mg/fc TNT both alone and in
the presence of 1% yeast extract. The microflora from all the sources tested
catalyzed a complete and rapid removal of TNT (based on residual TNT analysis)
from the yeast extract-enriched medium but not from the medium containing
mineral salts and TNT only. The majority of the organisms which showed posi-
tive degradative ability on TNT were noted to be Pseudomonas-like. The authors
also tested the ability of raw sewage and sewage sludge digester supernatant
to degrade TNT. The results showed that while raw sewage is ineffective, sludge
liquor caused a 64% reduction in the concentration of TNT. Since only the
disappearance of TNT was followed in all the above studies, it is not clear
if TNT underwent minor modification or was extensively degraded, or to what
extent the removal was due to adsorption on the particulate matter.
Chambers et^ al. (1963), in their biodegradation
studies with nitro-substituted toluenes, used a culture consisting of several
species (Pseudomonas predominating) which had been adapted to degrade phenol.
Oxygen consumption at the expense of the test substrate was measured using
respirometric technique. The ratio of the test oxygen uptake rate to the endo-
genous rate was found to be nearly 2.5 - 2.6 for 2,4-dinitrotoluene and 2,4,6-
trinitrotoluene, implying that these compounds were biodegradable to some degree.
(c) Degradation Under Sewage Treatment Plant Conditions
A considerable amount of work has been reported
concerning biological treatability of trinitrotoluene, and of the waste waters
resulting from TNT manufacturing operations and from munitions loading plants.
Consideration of the fate of these chemicals under waste water treatment plant
172
-------
systems is important because their fate in these systems can be a determining
factor in whether the chemicals become environmental pollutants or degrade to
innocuous materials.
The early investigations designed primarily to
determine the effect of TNT waste on domestic sewage treatment (since non-
acclimated samples of activated sludge were used) led researchers (Ruchhoft
et^ a!L. , 1943, 1945 a, b) to conclude that TNT waste should, not be treated by
either activated sludge or trickling filter units because inhibition of the
BOD removal efficiency was observed. The results of the BOD test on TNT-
waste revealed that the waste was not biodegradable (upon incubation for up
to 129 days, the BOD value was only 11 mg/£ as compared to the COD value of
673 mg/1).
Soviet scientists under the direction of Madera
et^ al. (1959) investigated the biological oxidation of TNT by activated sludge
during digestion of the sludge. They incubated a-TNT (ranging from 5-50 mg/fc)
with various concentrations of raw and digested activated sludge, and found that
the majority of the TNT disappeared fairly rapidly. The metabolism of TNT
was reported to have gone to completion via pathways similar to those reported
for TNT breakdown in animals (Channon £t aJL., 1944; see Section III-B). A
two-stage model waste water purifier consisting of an aerator (1st stage),
which was inoculated with Azotobacter agilis (reasons for using this organism
are unclear), and a 2nd stage overflow basin which was inoculated with con-
ventional activated sludge, was used by Bringmann and Kuehn (1971) to study the
biological decomposition of a synthetic waste water which contained nitro-
toluenes and nitrobenzenes (118 - 146 mg/fc). It was revealed in this study
173
-------
that dinitro- and trinitrotoluenes (2,4,6-trinitrotoluene, 2,4-dinitrotoluene,
2,6-dinitrotoluene) were removed to the extent of 95-97% after the 2nd stage;
the mononitrotoluenes were practically completely removed. Only in the case
of di- and trinitro-substituted compounds were small quantities of the reduced
metabolites found to be present in the overflow from the aeration stage. Since
only a moderate volume of oxygen was consumed, it appeared probable that some
of the material was removed by absorption.
The results of the BOD tests performed by Nay
e^t al. (1974) on pure cx-TNT and TNT waste water resulting from the counter
current-continuous flow TNT manufacturing process revealed that TNT was oxi-
dizable at slow rates. The authors noted that mixing was very important to
aid in keeping TNT in solution and in contact with the seed microorganisms.
Furthermore, the TNT:microorganisms ratio had to be below the toxic level.
The oxygen consumption was extremely concentration dependent and decreased
considerably as the TNT loading increased. Nay (1972) performed another series
of BOD tests in which the BOD water was supplemented with glucose; the increase
in BOD was considered to be due to TNT consumption. The results of these studies
also confirmed the oxidizability of TNT (Table 41). The authors' efforts to
relate the BOD to the TNT removed were unsuccessful (Table 42). As can be
seen, for approximately the same amount of TNT removed, the BOD values varied
considerably. The authors suggested that the dilution factor was of significant
importance in controlling their experiment. A likely explanation may be that
higher TNT concentrations are toxic to certain TNT oxidizing microorganisms, and
thus TNT may undergo only incomplete oxidation.
174
-------
Table 41. Results of the Biodegradability Test on TNT and TNT Waste in
Combination with Ammunition Plant Domestic Waste and Glucose.*
(Nay, 1972)
A. TNT Waste
B. a-TNT
Concn .
of TNT
(ng/D
18.5
it
n
12.4
16.3
20.2
Waste
Neutralization
Procedure
Slowly with lime
Slowly with soda ash
Rapidly with soda
Slowly with lime
Slowly with soda ash
Rapidly with soda ash
16 Day BOD**
Attributed to TNT
(mg/A)
71
52
43
Less than control
6
59
TNT Removal
(based on TNT
Analysis) (%)
42.7
43.2
45.4
0.0
65.6
32.7
* Glucose equivalent to 5 mg COD, and domestic waste 50 ml (average
composition/50 ml: C, 5.5 mg; N, 1.18 mg; P, 0.175 mg) per total test volume
of 157 ml.
** Seed: Settled mixed liquor suspended solids from the bench scale continuous
flow pilot plant that had been acclimated to Redford Army Ammunition Plant waste.
Table 42. Effect of TNT Concentration on the Biodegradability of TNT-Waste.
(Nay, 1972)
TNT Concn. in
Diluted Waste
(mg/A)
1
3
8
16
5 Day BOD**
(ing/A)
450
190
48
36
TNT Removed Based
.on TNT Analysis
mg/£ %
0.9
0.8
0.7
1.0
90.0 "
26.6
8.8
6.3
* Seed: Settled secondary influent of the Redford Army Ammunition plant
trickling filter treatment plant.
** BOD test data cannot be expressed in terms of % COD or TOG since the COD
value exerted per mg/Jl TNT oxidized varied considerably from sample to
sample, and with the initial concentration of TNT in the sample.
175
-------
Following the BOD testing, Nay ejt al. (1974)
evaluated the biological treatability of TNT waste in static tube and continuous
flow runs. In the treatability experiments, TNT waste was diluted with the
Army Ammunition plants domestic waste. The experimental findings from the
static tube runs indicated that biological treatment can oxidize TNT from the
waste at slow rates. The food to microorganisms ratio near 0.4 was found to
be more amenable to biological treatment. The authors noted that biosorption
or bioprecipitation of the TNT waste was much faster than the rate of oxidation
of the waste. The biological treatment was ineffective in removing the color
of the waste. In the continuous flow treatability runSj the activated sludge
acclimated to TNT-waste for 10 days was used as inoculum. The treatability runs
were made using 5 different concentrations of TNT (5-25 mg/Jl); for each con-
centration tested, three different detention times were investigated to determine
the effect of different organic loading rates. The average TNT removal effi-
ciency for all 15 runs was nearly 65%, and the removal efficiencies showed a
tendency to decrease with a decrease in detention time and increase in TNT
concentration. From these findings, the authors concluded that TNT waste water
can be biologically treated when combined with domestic waste. Although some
TNT loss is evident from these studies, it should be emphasized, however, that
a portion of TNT loss was due to biosorption on the activated sludge micro-
organisms and not due to molecular transformation.
In summary, the reports concerning biodegrad-
ability of nitrotoluenes are conflicting. There is no doubt that certain
microorganisms possess the ability to alter trinitrotoluene and related com-
pounds. No organisms have, however, been shown to use TNT as sole source of
176
-------
carbon and/or nitrogen. Degradation appears to occur by a cometabolic process.
The majority of the organisms capable of degrading TNT were noted to be
Pseudomonas-like. The experimental findings regarding biological treatability
of the waste waters resulting from TNT manufacturing operations revealed that
TNT-waste water can be biologically treated when combined with domestic waste.
(d) Routes of Degradation of Nitrotoluenes
The biochemical mechanism and pathway of TNT
degradation by microorganisms are not well established. Won et_ a\^. (1974).
reported identification of 2,2',6,6'-tetranitro-4-azoxytoluene, its isomer
2,2',4,4'-tetranitro-6-azoxytoluene, 4,6-dinitro-2-aminotoluene, 2,6-dinitro-
4-hydroxylaminotoluene, nitrodiaminotoluene and trace quantities of the 2-
amino- and the 4-amino-compounds in the cultures of Pseudomonas-like organisms
(referred to as isolate Y) upon incubation with TNT. Based on their experimental
findings, the authors proposed the following pathway for TNT metabolism (Figure 22),
diamino
compound
2,6-dinitro-4-hydroxyl- 4-amino compound
aminotoluene
V"^ ""O
NHOH
2,4,6-trinitrotoluene
diamino
compound
2,4-dinitro-6-hydroxylaminotoluene 6-amino compound
Figure 22. Proposed Pathway for TNT Metabolism
(Won et al., 1974)
177
-------
The azoxy compound may not be the product of direct TNT metabolism as proposed
by Channon et^ al_. (1944) in the mammalian systems.
(v) Nitroanilines
A number of pure culture and mixed culture studies
dealing with the decomposition of nitro-substituted anilines have been pub-
lished. Salient features of these studies are presented in Table 43. The
isolation of microorganisms active in decomposing £-nitroaniline, from a
biofilm and the activated sludge of a laboratory purification installation,
has been reported by Udod et al. (1972). The microorganisms belonged to the
genera Pseudomonas and Bacillus, and had the ability to utilize £-nitroaniline
(up to a concentration of 250 ppm) as the sole source of carbon and nitrogen.
The ^-isomer was degraded to the extent of 85-90% at a concentration of 100 mg/£,
An increase in the concentration of nitroaniline resulted in a lag in the de-
composition of nitroaniline. The mixed culture of microorganisms prepared
by mixing the isolated microorganisms was successful in degrading £-nitro-
aniline even in saturated solutions (approximate concentration 2 g/fc).
The intermediate products of £-nitroaniline decompo-
sition were identified as £-phenylenediamine and £-aminophenol. The pathway
for their formation is shown in Figure 23 (Udod et_ al., 1972).
Figure 23. Intermediate Products of _p_-Nitroaniline Decomposition
(Udod et al,., 1972)
178
-------
Table A3. Summary of the Degradation Studies with Nitroanilines
Reference
Malaney, 1960
Test Chemical
o-, m~, and _p_-
Concn. Used
500 mg/Jl
Source of
Microorganisms
Aniline-acclimated
Duration
of
the Test
8 Days
Criteria
for Test
Chemical
Alteration
Oxygen uptak
Chambers
al., 1963
Nitroaniline
m- and £-Nitro-
aniline
100 mg/£
Udod et al., £-Nitroaniline
1972
100-500 mg/fc
activated sludge
Microorganisms in 210 Min.
soil compost or mud
from a catalytic
cracking plant waste
lagoon, adapted to
degrade phenol
Microorganisms iso- 12-24
lated from a biofilm Hours
and the activated
sludge of a laboratory
purification
installation
by Warburg
Method
Oxygen uptake
by Warburg
Method
Growth, loss
of the parent
compound
Villeret,
1965
Alexander
and Lustig-
man (1966)
jn-, £-»
Nitroaniline
m-, £-, and £-
Nitroaniline
0.69-.38.0
mg/X,
5-10 mg/£
Fresh water alga
Chlorella vulgaris
Soil
64 Days
Growth with
nitroaryl
compounds as
the nitrogen
source
Loss of U.V.
absorbancy
179
-------
The degradation products beyond p-aminophenol were not characterized in this
study. However, since the cells were able to utilize £-nitroaniline for growth,
it appeared that £-aminophenol underwent further degradation.
A number of investigators have studied the biodegrad-
ability of nitroanilines using a culture which has been adapted to degrade a
compound similar in chemical structure to nitroaniline. Malany (1960) reported
slow oxidation of £-, m-, and £-nitroaniline by the activated sludge which
had been acclimated to aniline as sole source of carbon and energy. The sus-
ceptibility to oxidation decreased in the order m-, £-, o-. Chambers et al.
(1963) reported rapid oxidation of m-nitroaniline by a culture which had been
adapted to degrade phenol. The £-isomer was also attacked by these microorganisms,
however, at a considerably slower rate.
The utilization of the nitro and amino groups of
nitroanilines as the source of nitrogen for growth by fresh water algae
Chlorella vulgaris was reported by Villeret (1965). The author found that the
meta-isomer supported better growth than the para-isomer, with ortho-isomer
giving minimum growth. No effort was made in this study to identify the
transformation products.
The information presented here tends to suggest that
nitroanilines are susceptible to rapid degradation by microorganisms. The £-
isomer has been reported to be utilized as the sole source of carbon and
nitrogen by certain microorganisms belonging to the genera Pseudomonas and
Bacillus. The microorganisms capable of utilizing £- or m-isomer as the sole
source of carbon and nitrogen have not been reported. This may be interpreted
to mean that the £-isomer is relatively more biodegradable. On the
180
-------
other hand, the oxygen consumption studies with various isomers of nitro-
aniline as substrate suggest decreasing susceptibility to degradation in
the order m-, p-, £-. The information available concerning the biodegrada-
bility of the various isomers of nitroaniline is conflicting and does not
permit definite conclusions to be drawn regarding the order of their sus-
ceptibility to microbial attack.
(vi) Summary of the Biodegradation Studies With
Nitroaromatics
A large number of nitroaromatic compounds have been
tested for biodegradation. The major groups of compounds tested are nitro-
anilines, chloronitro- and nitrobenzenes, nitrobenzoic acids, nitrophenols,
and nitrotoluenes. A variety of sources of seeds have been used by researchers
in these studies. Some of the major sources are sewage, soil, river water, and
pure cultures. However, very rarely in the biodegradation studies have re-
searchers attempted to simulate other media than water. Although chemicals
are first released into one specific medium, they often move from one medium
to another. For example, nitrotoluenes usually pass through sewage treatment
plants; however, if they are not degraded and/or removed there, they will con-
taminate the natural waters. It then becomes important to examine
the persistence of nitrotoluenes in river water. The absence of information \
concerning the persistence of nitroaromatic compounds in a variety of media
imposes a limit on the conclusions that can be drawn regarding the environ-
mental fate of these chemicals.
The available information on the biodegradability of
t
nitroaromatics is summarized in Table 44. Since all the compounds have not
181
-------
Table 44. Biodegradability of Nitroaromatic Compounds under Varying Test
Conditions.
Relative
Compound Seed Test. Media Biodegradability
Nitroanilines
m-Nitroaniline
o-Nitroaniline
£-Nitroaniline
Chloronitro- and Nitrobenzenes
o-Chloronitrobenzene
£-Chloronitrobenzene
m-Dinitrobenzene
o-Dinitrobenzene
£-Dinitrobenzene
2 ,4-Dinitrochlorobenzene
Nitrobenzene
1,3, 5-Trinitrobenzene
Nitrobenzoic Acids
2-Bromo-4-nitrobenzoic acid
2-Chloro-4-nitrobenzoic acid
3,4-Dinitrobenzoic acid
2 ,4-Dinitrobenzoic acid
2,5-Dinitrobenzoic acid
3,5-Dinitrobenzoic acid
2-Fluoro-4-nitrobenzoic acid
3-Fluoro-4-nitrobenzoic acid
3-Hydroxy-4-nitrobenzoic acid
2-Hydroxy-4-nitrobenzoic acid
2-Iodo-4-nitrobenzoic acid
3-Methyl-4-nitrobenzoic acid
m-Nitrobenzoic acid
o-Nitrobenzoic acid
£-Nitrobenzoic acid
2,4,6-Trinitrobenzoic acid
Nitrocresols
4, 6-Dinitro-o-cresol
2,4, 6-Trinitro-m-cresol
Nitrophenols
2-Chloro-4-nitrophenol
4-Chloro-2-nitrophenol
2 , 6-Dichloro-4-nitrophenol
AS, S .
AS
AS, S, EP
S, RW, SW
S, EP
S, P, AS
S, P
S, P
S, EP
S, P, AS, EP
P, S, AS
EP
EP
S, AS
EP
S
AS
EP
EP
EP
EP
EP
EP
S, AS, P, EP
S, RW, AS, P, EP
S, RW, AS, P, EP
S, AS
EP
S
S
S
S
A
A
A
A .
A
A, SW
A
A
A
A, SW
A, SW
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
+ +
+ +
+ + +
-
-
+
+
+
-
±
±
+
+
-
+ + +
-'
-
+
+
-
-
+
+
±
+ + +
+ + +
-
+ + +
+ +
±
±.
±
182
-------
Table 44. Biodegradabllity of Hitroaroroatic Compounds Under Varying Test
Conditions (Cont'd.)
Compound
Seed
Relative
Test Media Biodegradability
Nitrophenols (cont.)
2,4-Dinitrophenol S, EP
2,6-Dinitrophenol S, EP
2,5-Dinitrophenol EP
Dinitro-(sec)butyl phenol EP
2-Methyl-4-nitrophenol EP
m-Nitrbphenol ' S, EP
o-Nitrophenol S, EP
£-Nitrophenol S, EP
2,4,6-Trinitrophenol S, EP
Nitroresorcinols
2,4-Dinitroresorcinol S, RW
2j4,6~Trinitroresorcinol S, RW
Seed
SW
AS
RW
S
P
EP
Sewage seed
Activated sludge
River water
Soil
Stocked pure culture
Enriched pure culture
A
A
A
A
A
A
A
A
A
A
A
Nitro toluenes
2 , 4-Dinitrotoluene
2~Nitrotoluene
2,4, 6-Trinitrotoluene
S, EP
P
S, P, SW, EP
A
A
SW, A
+ +
±
+
Relative Biodegradability
Susceptible (extensive degradation, ring cleavage) + + +
Moderate (partial degradation, slow degradation) + +
Minor molecular alteration +
Resistant
Uncertain +
Test Media
S Soil
A Aqueous
SW Sewage treatment
183
-------
been tested under similar conditions, the biodegradability information pro-
vided in the table is of qualitative significance only. One must also keep in
triihd that the order of biodegradability shown in the table is under the con-
ditions of the laboratory test; whether the relative biodegradability will be
similar under environmental conditions is questionable.
2. Environmental Transport
Little experimental data are available concerning the extent
and mechanism of transport of nitroaromatics in the environment. In the absence
of this information, the environmental transport consideration of the nitro-
aromatics has been based primarily on the physical and chemical properties of
these compounds.
a. Volatility
(i) Volatilization From Water
Organic chemicals are gradually lost to the atmosphere
from aqueous solutions by codistilling with water. The volatility of a com-
pound is dependent on its vapor pressure (which varies with temperature), water
solubility, and adsorption properties (Kenaga, 1972). The amount volatilized
increases with concentration until the maximum water solubility is reached.
Mackay and Wolkoff (1973) have derived equations to predict the rate of evapor-
ation of compounds from aqueous solutions using the water solubility and vapor
pressure of the compound. In predicting the residence times, the authors
assume that the water column undergoes continuous mixing and that the compound
is present in the solution and not adsorbed, complexed, etc. Using this approach
the half lives for certain nitroaromatic compounds have been calculated and are
presented in Table 45. Unfortunately, the solubility and vapor pressure data
1*4:
-------
Table 45. Rate of Evaporation of Nitroaromatics from Bodies of Water (Calculated
According to MacKay and Wolkoff, 1973).
Compound
Solubility*
Vapor Pressure*
(mm Hg)
M.W.
Calculated Half Life**
at Ambient Temp.
(approximate values)
Nitroanilines
2-Nitroaniline
3-Nitroaniline
4-Nitroaniline
Nitrobenzenes
Mononitrobenzerie
Nitrophenols
1000
20°
138
138
138
123
<8.8 Days
£-Nitrophenol
Nitrotoluenes
£-Nitro toluene
o-Nitrotoluene
m-Nitrotoluene
38°
3200
' _ «
15°
4030o
65230o
50020o
2,4,6-Trinitrotoluene 130
Other Compounds for
DDT
Benzene
Comparison
12 x 10~3
1780
I1*9'30
±53-7°
-[50°
^50- 2o
0.053850
1 x 10~7
95.2250
139
137
137
137
227
354.5
78
<25 Days
<44.6 Minutes
<12.3 Hours
<9.45 Hours
-
317 Days
37.3 Minutes
* Data from Matsuguma, 1967a; Nay, 1972. The superscripts after the solubility
and vapor pressure refer to the temperature of measurement.
** At less than saturation concentration in a square meter of water.
185
-------
are not available for the same temperature. For Table 45, half lives have been
calculated based on the values of the two parameters at the available temper-
ature, and from the anticipated variation of solubility and vapor pressure with
temperature.
The experimental data on the loss of ot-TNT from
solution due to aeration has been obtained by Nay (1972). He conducted an
air stripping experiment on both raw and neutralized waste samples. Incu-
bations were performed in the absence of activated sludge to exclude the losses
due to biological degradation. The results of this study revealed that only
a small portion of the TNT concentration (about 8-10%) was vaporized during
the experimental period of 18 days. The low evaporation rate of a-TNT into
the atmosphere is supported by the fact that TNT has an extremely low vapor
pressure (Table 45).
(ii) Volatilization From Soil and Other Surfaces
Volatilization of chemicals from soil and other
surfaces is dependent upon the vapor pressure of the chemical as modified by
the adsorptive interactions with the surface (Spencer and Cliath, 1975). The
extent of reduction in vapor pressure is dependent upon the nature of the
chemical, its concentration, water content of the soil, and soil properties
(see Spencer et al., 1973). It has been reported that chemicals in soil
water are more easily lost than those that are adsorbed on soil particles
(Huang, 1970).
Experimental data regarding the loss of pentachloro-
nitrobenzene (PCNB, a fungicide) from soil has been obtained by Wang and
Broadbent (1972). The authors found that the fungicide was lost from three
186
-------
California soils (differing in their properties) mainly through volatili-
zation. The calculated half times for PCNB in three soils were in the range
of 4.7 - 9.7 months. Higher organic matter was associated with slower
PCNB loss. Considering the solubility increasing effect of the loss of
chlorines from the molecule, it can be predicted that the lower substituted
chloronitrobenzenes will be lost more easily than pentachloronitrobenzene.
b. Leaching and Downward Movement of Nitroaromatics
The leaching potential of a chemical from soil depends
on the extent to which it is adsorbed and its water solubility (see Browman
and Chesters, 1975). The behavior of pentachloronitrobenzene in soil has
been experimentally determined by Ko and Lockwood (1968). The authors re-
ported that about 75% of the PCNB was still retained in natural soil (50%
by soil, and 25% by soil organic matter and microorganisms) after three ex-
tractions (each for two days) with excess water. From these observations, it
can be concluded that PCNB is unlikely to be lost by leaching. Con-
sidering that the loss of chlorine from the molecule tends to decrease its
hydrophobicity and subsequently its retention by soil, it can be predicted
that the lower halonitrobenzenes will be relatively more susceptible than
PCNB to losses by leaching.
The factors governing the movement of a chemical to the
ground water are the nature of the compound, its concentration and solubility
in water, the composition and pH of the soil, the amount of rainfall and the
height of the water table (see Browman and Chesters, 1975). The pertinent
information needed to evaluate the ground water movement of nitroaromatics
could not be found in the literature. The fairly high water solubility of
187
-------
many nitroaromatics suggests that these compounds will migrate through the soil
a"nd eventually contaminate ground water. The sorption of certain nitro com-
pounds to clay and organic matter will tend to reduce their ground water move-
ment. For example, Yariv and coworkers (Saltzman and Yariv, 1975, Yariv et al.,
1966) have reported that nitrobenzene and nitrophenol can enter the inter layer
space of the cation-saturated montmorillonite and form hydrogen bonds with water
molecules in the hydration shell of the highly polarizing exchangeable cation.
c. Mobility in Water
Generally, the monitoring data provide a good indication
of the mobility of a chemical in the aquatic environment. Unfortunately,
very little monitoring information is available for nitroaromatic compounds.
One of the chemicals, £-chloronitrobenzene has been found to travel long
distances in surface waters; the compound was followed down the Mississippi
River from St. Louis to the Gulf of Mexico (about 900 miles) at a concentra-
tion that could be simply explained by dilution (Kramer, 1965). This obser-
vation suggests only minimum adsorption to river sediment and a fairly high
mobility of ^-chloronitrobenzene in the aqueous environment.
Information regarding the mobility of other nitroaromatic
compounds is not available. However, it may be possible to predict their
mobility by considering the factors which affect the mobility of chemicals in
the aqueous environment. Some of the important considerations in this respect
are: adsorption of chemicals on hydrosoil or other particulate matter (those
adsorbed will not be easily lost), water solubility, losses due to evapor-
ation or degradation, etc. The nitro-substituted aromatic compounds dealt
with in this report are fairly water soluble, and will most likely be .
188
-------
contained in water. Subsequently, they will be expected to be freely mobile.
However, a number of these chemicals may not persist long enough to be trans-
ported long distances. For example, mononitro-substituted benzoic acids
(except the m-isomers) have been shown to be easily attacked by microorganisms.
Nitrotoluenes are hard to degrade, but since they codistill fairly rapidly
from water (except for a-trinitrotoluene), some .losses may occur prior to their
transport by water. Unlike these compounds, jo-nitrophenol has a long cal-
culated half life in water and is extremely water soluble; it can be predicted
that this compound will travel to a certain extent in the aqueous environment
at undiminished concentrations (except reduction by dilution or degradation).
3. Bioaccumulation
The nitroaromatic compounds may be present in water in very
low concentrations. However, organisms of the lower aquatic food chain may
be able to increase the concentration by accumulation of these compounds from
their surrounding environment by various processes including absorption, ad-
sorption, ingestion (bioaccumulation). In the absence of experimental data
regarding the bioaccumulation potential of nitroaromatics, the assessment
of their bioaccumulation potential can be estimated from their physical and
chemical properties. Accumulation of any molecule occurs when the molecule
is selectively concentrated in biological material and is accumulated
faster than it is eliminated. Most nitroaromatic compounds (except for p_-
nitrotoluene, ct-TNT, and perhaps certain halonitrobenzenes) are fairly water
soluble, and it is unlikely that they will be taken up to a significant ex-
tent by aquatic organisms. Furthermore, in the case of nitrobenzoic acid, the
available information suggests that these compounds will be rapidly
189
-------
attacked by microorganisms and therefore are not likely to be around to be
taken up by food-chain organisms.
The octanol-water partition coefficient has been used in many
instances to assess the bioconcentration potential of chemicals. Neely et^
al. (1974) have noted a linear relationship between octanol-water partition
coefficients of several chemicals and bioconcentration factors (ratio of the
concentration between the organism and the exposure water) in trout muscle.
The water-octanol partition coefficients of a number of nitroaromatics are
available from the literature (Leo et^ al^., 1971). Using the equation of the
straight line of best fit derived by Neely and coworkers (1974), the biocon-
centration factors for nitroaromatics have been calculated (Table 46). As
can be seen, nitroaromatic compounds in general do not have high bioconcentration
potential. Of the compounds listed in the table, chloro-substituted nitro-
benzenes and nitrotoluenes may perhaps be of some concern. However, it should
be pointed out that the calculated values do not take into account the loss
of the compounds from the organism due to metabolism and other mechanisms, so
that the calculated value for a chemical will generally be higher than that
determined experimentally as noted by Neely £t aJL (1974). Thus, it appears
unlikely that nitrotoluenes or lower isomers of chloronitrobenzenes will bio-
accumulate to a significant extent.
Further support for relatively low bioconcentration potential of
the lower isomers of chloro-substituted nitrobenzenes comes from the data of
Ko and Lockwood (1968). These investigators were able to show little accumu-
lation and concentration above ambient levels of pentachloronitrobenzene
in the mycelia of fungi. Incubation (48 hours) in soil containing 42 yg of PCNB
190
-------
Table 46. Bioconcentration Factor of Nitroaromatics in Trout Muscle.
(Calculated according to Neely et al., 1974)
Compound
Log, Octanol-Water
Partition Coefficient*
Calculated Bioconcentration
Factor (Ratio of the Concen-
tration between the Organism
and the Exposure Water)
Nitrobenzenes
Nitrobenzene
A-Chloro-1-nitrobenzene
3-Chloro-l-nitrobenzene
2-Chloro-l-nitrobenzene
m-Dinitrobenzene
o-Dinitrobenzene
£-binitrobenzene
Nitrophenols
m-Nitrophenol
o-Nitrophenol
£-Nitrophenol
2,4-Dinitrophenol
2 , 5-Dinitrophenol
2 , 6-Dinitropheriol
3 , 5-Dinitrophenol
Nitroanilines
m-Nitroaniline
o-Nitroaniline
_p_-Nitroaniline
Nitrobenzoic Acids
m-Nitrobenzoic acid
o-Nitrobenzoic acid
_p_-Nitrobenzoic acid
Nitrotoluenes
m-Nitro toluene
£-Nitro toluene
£-Nitrotoluene
Other Compounds for Comparison
End r in
Benzene
1.85
2.41
2.41
2.24
1.49
1.58
1.46
2.0
1.79
1.91
1.51
1.75
1.25
2.32
1.46
1.79
1.19
1.66
1.31
1.85
2.45
2.30
2.37
5.6
2.13
13.37
26.92
26.92
21.78
8.531
9.550
8.222
16.13
12.42
14.46
8.399
11.81
7.603
24.04
8.222
12.42
5.862
-
10.54
6.823
13.37
28.25
23.45
25.59
2953 + 10
19.0
* Data from Leo et al., 1971
191
-------
per gram of moist soil led to the accumulation of PCNB in mycelia about 7 fold.
Since reducing the number of chlorines on a molecule tends to reduce its hydro-
pfabbicity, which in turn is presumed to reduce its bioaccumulation potential,
it can be predicted that less chlorine-substituted chloronitrobenzenes will be
bioconcentrated to a lesser extent, if at all, than PCNB.
4. Biomagnification
Biomagnification refers to concentration of a compound through
the consumption of lower organisms by higher organisms with net increase in
tissue concentration (Isensee ^t_ a^L. , 1973). The only reported study dealing
with the biomagnification of nitroaromatic compounds is that of Metcalf and
Lu (1973). The authors studied the ecological magnification of nitrobenzene
in a model aquatic ecosystem which permitted passage of the chemical through
C ' " " ' ~ —•—
the aquatic fauna and flora. The findings revealed low overall biomagnifi-
cation of nitrobenzene in fish (biological magnification* = 20; for comparison
"* ' — - - — .
biological magnification for DDT under similar conditions = 16,950). Most of
*C~"""""""""
the radioactivity present in the fish was in the form of £-nitrophenol and its
conjugate. Knowing the biomagnification potential of nitrobenzene may enable
one to predict the behavior of halogenated nitrobenzenes in the food chain.
Since substitution of chlorine on nitrobenzene will presumably increase its
hydrophobicity, it appears reasonable to conclude that halonitrobenzenes will
have a greater biomagnification potential.
For other nitroaromatic compounds, their water solubility may
also be helpful in predicting their biomagnification potential. Metcalf and Lu
(1973) have reported a straight line relationship between ecological magni-
fication of several chemicals in their model aquatic ecosystem, and the water
* concentrations in organism/concentration in water
192
-------
solubility. Using this relationship, the ecological magnification values for
several nitroaromatics have been calculated (Table 47).
In summary, the solubility characteristics of nitroaromatic
compounds, in general, indicate that they will not biomagnify or bloaccumulate
to a significant extent in the food chain. However, it is likely that certain
haldnitroaromatics may biomagnify since chlorine substitution will tend to make
them more hydrophobic.
Table 47. Ecological Magnification in the Model Aquatic Ecosystem for
Nitroaromatics (Calculated According to Metcalf and Lu, 1973).
Compound
o-Nitrophenol
_p_-Nitrophenol
o-Nitrotoluene
m-Nitrotoluene ..
Nitrobenzene li?8)r>o
4-Chloronitrobenzene
2-Chloronitrobenzene
3-Chloronitrobenzene
3 , 4-Dichloronitrobenzene
2 , 5-Dichloronitrobenzene
2 , 3-Dichloronitrobenzene
2,4, 5-Trichloronitrobenzene
2,3, 4-Trichloronitrobenzene
2,3,4, 6-Tetrachloronitrobenzene
2,3,4 , 5-Tetrachloronitrobenzene
2,3,5, 6-Tetrachloronitrobenzene
2,3,4,5, 6-Pentachloronitrobenzene
DDT (for comparison)
Solubility
at 20°C
(mg/A)
38°
3.2 x0106
40.03QO
652.0
500.0300 /Q ^
c^TTsj^x lov
T7137 x 10-
X
2.80 x 10,
X
1.73 x 10,
6.29 x 10
4.8 x 10
3.25 x 10
1.3 x 102
1.15 x 10
29.0
28.0
8.0
1.5
Calculated Ecological
Magnification in Fish
at Ambient Temperature
(approximate values)
<2.7
>43
<7.5
<8.8
78.9
224 (J •£
227 .«
i
307
579
686
876
1555
1682
3987
4074
8933
25520
16950
t-TKidCuJf
Data from Matsuguma, 1967a, and Eckert, 1962.
A =
0,3
(,
193
-------
B. Biology
The biological activities of foreign organic compounds are
determined in large part by their metabolic fate. Metabolic pathways
have evolved in which foreign compounds are chemically transformed by oxi-
dation, reduction, hydrolysis, and conjugation reactions into metabolites
which are inactive, or which are more water-soluble and therefore more
readily excreted. These metabolic pathways have been described as detoxi-
cation mechanisms, since foreign compounds seem to be converted to less toxic
or inert products. However, in some cases, biologically active metabolites
are produced.
Most biotransformations of foreign organic compounds are catalyzed
by a series of enzymes present in any of several tissues and organs. By far,
the greatest number of the enzymes of importance in foreign compound metabolism
are in the liver; however, other sites include the kidney, the skin, the
intestine, the lung, or the placenta.
While the transformations of most foreign compounds are accomplished
by enzyme-catalyzed reactions, other factors influence their metabolism. These
factors include the structure of the foreign compound, the route of absorption,
storage in fat, binding to plasma proteins, localization in tissues, and sen-
sitivity of target molecules. Metabolism may also be affected by diet and
by the genetic makeup of the species.
1. Absorption and Elimination
Although exposure to most industrial poisons occurs by absorption
through the respiratory tract, nitroaromatic derivatives are also readily ab-
sorbed through the skin. A large number of occupational and accidental poisoning
194
-------
cases have directly resulted from dermal exposure to nitroaromatic chemicals
(see Section III-C). Subsequent to skin absorption, localization and storage
of these compounds in the body fat often occurs. The stored chemical can
be readily mobilized by ingestion of alcohol or exposure to sunlight (Rejsek,
1947), resulting in an episode of serious intoxication.
The relationship between toxicity and mode of absorption for
the nitroaromatics is largely dependent upon their molecular structure, lipid
solubility, and degree of ionization. As a group, the nitroaromatic compounds
are readily soluble in organic solvents and therefore can usually penetrate
the intact skin with ease. Passive diffusion across the skin and other
lipoidal membranes is greatest for lipid-soluble neutral molecules (i.e., un-
dissociated compounds having a high lipid/water partition coefficient).
Absorption and rate of passive diffusion decrease with increasing ionization,
as illustrated in Table 48. The absorption of chemicals that can dissociate
i
will be favored by a change in pH that increases the proportion of the un-
dissociated form.
Table 48. Intestinal Absorption in Rats From Solutions of Various pH Values
- The percent absorbed is expressed as the mean + the range. The
figure in parentheses indicates the number of animals. -
(From Hogben et al., 1959)
1
Drug
Bases
Aniline . i
Aihinopyrine
jj-Toluidihe
Quinine . >
Acids
5-Nitrosalicylic .... ..
Salicylic
Acetylsalicylic
Benzole
jj-Hydroxypropiophenone .
pKa
4.6
5.0
5.3
8.4
2.3
3.0
3.5
4.2
7.8
Per Cent Absorbed
pH of the intestinal solution
3.6-4.3
40 ± 7 (9)
21 ± 1 (2)
30 ± 3 (3)
9 ± 3 (3)
40 ± 0 (2)
64 ± 4 (4)
41 ± 3 (2)
62 ± 4 (2)
61 ± 5 (3)
4.7-5.0
48 ± 5 (5)
35 ± 1 (2)
42 ± 3 (2)
11 ± 2 (2)
27 ± 2 (2)
35 ± 4 (2)
27 ± 1 (2)
36 ± 3 (4)
52 + 2 (2)
7.2-7.1
58 ± 5 (4)
48 ± 2 (2)
65 ± 4 (3)
41 ± 1 (2)
<2 (2)
30 ± 4 (2)
35 ± 4 (3)
67 ± 6 (5)
3.0-7.8
61 i 9 (.1.0)
52 L 2 (2)
64 ± 4 (2)
54 1 5 (5)
<2 (2)
10 ± 3 (6)
5 ± 1 (2)
60 e. 5 (2)
195
-------
Nitroaromatic chemicals which act as weak acids or weak bases
will often be incompletely ionized at physiological pH. The degree of ioni-
zation will depend on both the physiological pH and the pKa value for the
particular chemical. The absorption of several nitroaromatic compounds and
other organic electrolytes from rat small intestine was shown to be related
to their pKa values as illustrated in Tables 49 and 50. Acidic compounds
having pKa values greater than 3 and basic compounds with pKa values less
than 8 were rapidly absorbed.
Table 49. Absorption of Organic Acids From the Rat Small Intestine
- The percent absorbed is expressed as the mean + the
range followed by the number of experiments in parentheses
(From Schanker et^ al., 1958) -
Acid
5-Sulfosalicylic
Phenol red
Bromphenol blue
.a-Nitrobenzoic
5-Nitrosalicylic
Tromexan
Salicylic
jn-Nitrobenzoic
Acetylsalicylic
Benzoic
Phenylbutazone
Acetic
Thiopental
Barbital
jj-Hydroxypropiophenone
Phenol
pKa
(strong)
(strong)
(strong)
2.2
2.3
2.9
3.0
3.4
3.5
4.2
4.4
4.7
7.6
7.8
7.8
9.9
Per Cent Absorbed
Actual
<2 (2)
<2 (4)
<2 (2)
5 ± 2 (2)
9 ± 2 (3)
35 ± 7 (3)
60*
53 ± 0 (2)
20 ± 4 (6)
51 ± 5 (2)
65 ± 7 (3)
42 ± 1 (3)
55 ± 6 (3)
30 ± 4 (2)
61 ± 9 (5)
51+8 (3)
Relative
to aniline
<2
<2
<2
5
9
37
—
50
21
54
54
40
67
25
61
60
* Standard deviation, ± 10 per cent for 30 experiments.
196
-------
Table 50. Absorption of Organic Bases From the Rat Small Intestine
- The percent absorbed is expressed as the mean + the
range followed by the number of experiments in paren-
theses (from Schanker, jet al, 1958) -
Base
Acetanilide
Theophylline
£-Nitroaniline
Antipyrine
m-Nitroaniline
Aniline
Aminopyrine
jv-Toluidine
Quinine
Ephedrine
Tolazoline
Mecamylamine
Darstine
Procaine amide
1
ethobromide
Te t r ae thy lammonium
Tensilon
pKa
0.3
0.7
1.0
1.4
2.5
4.6
5.0
5.3
8.4
9.6
10.3
11.2
(strong)
(strong)
(strong)
(strong)
Per Cent Absorbed
Actual
42 ± 5 (2)
29 ± 1 (3)
68 ± 7 (2)
32 ± 6 (3)
77 ± 2 (2)
54*
33 ± 4 (4)
59 ± 3 (3)
15+2 (6)
7 ± 3 (2)
6 ± 1 (2)
<2 (2)
<2 (2)
<2 (2)
<2 (2)
<2 (2)
Relative
to salicylic
acid
43
30
61
30
63
—
27
56
15
6
5
<2
<2
<2
<2
<2
* Standard deviation, ± 10 percent for 43 experiments.
The nitroaromatic compounds with a relatively high vapor
pressure, particularly the nitrobenzenes, are quickly absorbed upon in-
halation. The pulmonary epithelium, unlike most other body membranes, is
easily penetrated by both lipid-soluble molecules and large lipid-insoluble
molecules and ions (Enna and Schanker, 1969). This factor is often of greater
practical importance in determining the hazards of environmental exposure
than a simple comparison of toxicity data obtained by oral or parenteral ad-
ministration of various substances.
197
-------
a. Nitrophenol Derivatives
Several investigators have undertaken to study the ab-
sorption and elimination of nitroaromatic compounds by measuring their levels
in the blood after administration by various routes. For the most part, these
studies have involved the derivatives of dinitrophenol which are important as
agricultural chemicals and, therefore, pose the greatest risk of exposure.
Parker _et^ ^1. (1951) investigated the fate of 4,6-dinitro-
^j-cresol (DNOC) in rabbits, cats, rats, and dogs after subcutaneous injection.
Figure 24 shows the exponential drop in blood serum levels of DNOC after a
single injection. It can be seen that a species variation exists in the rate
of fall.
100-1
3 4
Days after injection
"I
7
Figure 24.
Changes in Serum Level of DNOC After a Single Subcutaneous Injection
of 10 mg DNOC/kg (Parker et al., 1951)
198
-------
The administration of a series of daily subcutaneous injections in the rabbit
did not change the rate of DNOC elimination from the blood, nor did it produce
a cumulative rise in the blood level (Figure 25).
100-i
8
o
o
z
0
O)
40
20-
Figure 25.
DAYS
Effect of Repeated Subcutaneous Injections (10 mg DNOC/kg)
on Serum Level of DNOC in Rabbit. Samples of Serum Analyzed
Immediately After Injection and Then at One, Three, Five,
and Twenty-four Hours (From Parker et^ al. , 1951)
Similarly, in the rat and dog, blood levels of DNOC still fell to the same
level after repeated daily injections, even though the rate of DNOC elimination
by these species is slower than in the rabbit, as shown in Figure 24 above. In
addition, Parker and his coworkers demonstrated that the rate of DNOC elimin-
ation from the blood was not altered by pre-treating the animal with a series
of daily injections (Figure 26).
199
-------
R
Q
o>
70-|
60-
50-1
40-
30-
20-
10-
= Injection of 10 mg. DNOC/Kilo
0 1 2 34 5 6 7 89
DAYS
10 11 12
Figure 26. Blood Levels of DNOC in the Dog After Repeated Subcutaneous
Injections
- The upper curve shows the effect of five daily subcutaneous
injections on the DNOC concentration in serum of dog (serum
samples taken 24 hours after the previous injection). After
the fifth day, no further injections were given.
The lower curve shows the rate of fall of DNOC in serum
following a single injection 24 hours before the first
sample was taken. (From Parker'_et_ al_., 1951)
Overall, there is clearly no evidence from these studies to indicate any likeli-
hood for significant DNOC accumulation in the blood of laboratory animals.
Further observations on the absorption and elimination of DNOC
were made possible through a number of animal studies conducted by King and Harvey
(1953a, 1953b). Levels of DNOC in the blood were determined after its absorption
200
-------
through the gut, the lungs, and the skin of rabbits and rats. When given by
stomach tube to rats, DNOC reached maximum blood levels within seven hours
(Table 51) i Large quantities of unchanged DNOC did not remain in the gut.
One hour after dosing, 20% of the dose could be recovered from the gut, and
after two hours, only about 10% remained unabsorbed.
Table 51. Absorption of DNOC Following Single Dose of 30 mg/kg Given by
Stomach Tube to Albino Rats (King and Harvey, 1953 a)
; (Rats bled and then killed immediately by ether; entire gut removed;
contents well washed out; tissues and contents homogenized before
analysis.)
Dose
(mg.) '
4.14 :
6.00
4.62
5.01 '
4.65
4.62 ;
4.89
3.60 '
4.86
3.90
3.45
3.66 !
1
Time
after
dose
(hr.)
1
2 .
4
7
24
48
Blood
(Ug./g.)
34.0
30.0
60.5
45.5
36.5
29.3
57.2
52.0
22.5
25.0
3.0
2.8
Stomach
110.0
292.0
55.0
61.0
22.0
26.0
33.0
33.0
5.0
6.0
1.0
1.0
DNOC (Mg.)
Small
Intestine
13.0
10.0
16.3
13.0
13.0
12.0
13.0
13.0
11.0
11.0
4.0
3.0
Large
intestine
11.0
5.0
20.0
8.0
7.0
6.0
26.0
12.0
Lost
17.0
12.0
14.0
Contents of
alimentary
canal
660.0
800.0
450,0
540.0
92.0
124.0
114.0
18.0
8.0
6.0
7.0
7.0
Total DNOC
0°g.)
0.79
0.11
0.54
0.62
0.13
0.17
0.18
0.07
>0.02
0.04
0.02
0.02
I of
dose
19.2
18.5
11.7
12.4
2.9
3.6
3.8
2.1
>0.5
1.0
0.7
0.7
Intraperitoneal injection versus administration by stomach tube of identical
DNOC doses demonstrated that higher maximum blood levels could generally be
.
attained by the parenteral route (Table 52). However, the time to reach max-
imum levels following the two treatments does not seem to be consistently
shorter by either route, as shown in Table 52. On this basis, one can predict
that DNOC is readily absorbed across the gut wall but may be partially degraded
by intestinal contents, resulting in lower blood levels than can be achieved
by injection of the same dose.
201
-------
Table 52. Blood DNOC Levels in Animals Following Single Doses
(From King and Harvey, 1953 a)
(ST, administration by stomach tube; IP, intraperitoneal injection.
All animials survived dosage except two rats, 20 and 100 rag/kg.
Each experiment was on a single animal except for the 30 mg/kg
(two rats) and 40 mg/kg (twelve rats); for the latter group mean
+ S.E. is given).
Dose
(rag./
1
5
10
20
30
40
50
100
1
5
10
20
5
10
20
Admin.
Oral
ST
ST
ST
ST
ST
ST
ST
IP
IP
IP
IP
Or,)!
S'l
ST
f
' 1
2.
8.
48.
46.
34.
30.
-
-
-
4.
8.
28.
101.
5
8
0
0
0
0
-
-
-
7
8
4
0
,_
.-
"
2
—
—
—
60.5
45.5
100.3+
1.98
—
—
.
—
3
13
16
17
58
85
7
14
3
.3
.0
.0
.0
—
—
—
—
.0
.0
.1
.1
4
—
...
—
36.5
29.3
100.5+
2.85
60.0
88.0
—
_..
40.5
—
8.1
24.0
24.0
97
.0
—
—
"
—
6.4
44.0
31.0
5
3.8
9.3
26.0
48.0
—
~
—
—
—
—
5.0
15.7
33.7
63.2
—
-..
"
BNOC (wg./g.)
Time 'after dose (hr.)
6 ]_ &
Rats
2.9
„
—
—
57.2
52.0
97.4+
1.53 .
70.0 — 92.0
—
4.9
23.8
34 . 7
64.6
Rabbits
10.0 — 20.0
20.0 ~ 18.0
31.0 — 8.2
11 12 24
3.7 '
12.0
25.0
—
22.5
25.0
—
— — .
80.0 19.0
—
. —
12.2
30.6
43.5
4.6 7.4
.S.S 4.0
18.8 2.5
2_7 '
—
—
—
—
—
—
—
—
~
—
—
9.8
15.1
25.8
—
—
Evidence was obtained by King and Harvey indicating that
accumulation of DNOC can occur to a limited extent in animals after repeated
doses, either by stomach tube or by intraperitoneal injection. Blood level
determinations for DNOC were made in rats and rabbits given the compound on a
daily basis. Their results showed that a significantly higher DNOC blood level
was achieved in some groups of rats after two daily doses, as compared to th/it
resulting from a single dose. Increases in blood DNOC levels between days two
and three, however, were not usually significant. The rabbit, on the other hand,
did not appear to accumulate DNOC in any fashion from these experiments.
202
-------
The results of inhalation studies demonstrated that absorption
of DNOC can occur readily through the lungs and, in fact, prolonged inhal-
ation can lead to death (Figure 27).
foDied
Figure 27. Effect of DNOC Aerosol on Rats; Blood DNOC Values After Exposure
at 25° to Concentration of 0.1 mg/cu m
t • >, Four albino rats (not previously dosed
0 0, Six hooded rats previously given sixteen daily doses of
5 mg/kg DNOC by intraperitoneal injection; dots and circles
show values for individual rats; lines show mean value
(King and Harvey, 1953 a)
(Reprinted from the Biochemical Journal with permission from
the Biochemical Society.)
Absorption via the lungs was considered by the authors to be particularly
dangerous to health, since small particles containing DNOC can be carried
directly to the lung surface. Thus, DNOC would then be rapidly absorbed into
the circulation without passing first through the liver. This route of absorp-
tion is far more efficient than exposures occurring through the gut or across
the skin.
Determining the absorption of DNOC through the skin was complicated
by several variables. Although increased environmental temperature produced
a marked rise in the metabolic rate and increased mortality in dermally-treated
rabbits, blood levels of DNOC were not apparently affected (Figure 28).
203
-------
Skin application
DNOC
12nd
Environmental temperature Values at
I A 25-28° lA+825-280 I 24 hr. |48hr.
837-40°
1 2 3 4 5 6 7 8 9 10 11 12
Time(hr.)
-r
8
Figure 28. Skin Absorption of DNOC by Twelve Chinchilla Rabbits
2% aqueous (w/v) DNOC (as Na salt) to give 2 mg/sq cm
over 50 sq cm. Group A, 0 0 (broken line mean value);
Group B, • • (continuous line mean value). Slopes
from 8.0 to 12.0 hr. A: -2.05, +2.68, -1.50, -1.48, -1.65,
-3.08; B: -2.42, -3.49, -2.35, -1.19, -2.26, -2.70.
d(XA - X ) = 1.79, S.E. (d)=0.96 not significant. (From
King and Harvey, 1953 a) (Reprinted from the Biochemical
Journal with permission from the Biochemical Society.)
Clearly, however, DNOC was absorbed through the skin and, in fact, significant
quantities remained in the blood (2.4 to 7.9 yg/g) even 48 hours after appli-
cation of the treatment dose. This would suggest a possible storage or accum-
ulation of DNOC in the skin which can result in the sustained release of small
quantities into the blood for prolonged periods.
In a study employing human volunteers, Harvey et^ a!L. (1951) admin-
istered DNOC to five men in order to study its accumulation potential. The
authors pointed out that, while DNOC and other dinitrophenol derivatives are not
cumulative poisons in animals, occupational studies have indicated that DNOC
may be accumulated in man (Bidstrup and Payne, 1951; see Section III-C).
204
-------
The results of administering repeated oral doses of 75 mg of DNOC
are presented in Figure 29.
, A only {only
All subjects + only g
nrn n i i
24-,
22-
20-
. 18-
§ 16'
« 14~
-? 12-
o
§ 10-
o 8-
6-
4-
2-
0
KEY:
A -A
o -B
A -C
• -D
+ -E
J 75 mg. DNOC by mouth
S
[ Skin application 2% aq. ONOC
2
WEEKS
Figure 29. Comparison of 24-Hour Blood Levels (Harvey et al., 1951)
These experiments showed that DNOC remained in the blood at 1 to 1.5 yg/g
for up to forty days after administration of the last oral dose. In addition,
absorption through the skin was demonstrated, as well as the fact that exercise
caused an increase in the concentration of DNOC in the blood. The latter ob-
servation may be indicative of binding to the albumin fraction of the blood.
Levels in the blood of 15 to 20 yg DNOC per gram of blood were associated with
symptoms of poisoning and corresponded to an absorption on the order of 1 mg
per kg body weight of DNOC for three to five days. These results suggest that
exposure to relatively low levels of DNOC, when continuing over a period of
several days, may pose a serious threat to health.
In further studies on the retention of DNOC by man and animals,
King and Harvey (1953 b) considered the problem of DNOC storage in the body
and the capacity for man, rats, and rabbits to eliminate DNOC. A plot of the
205
-------
regression lines expressing the decay in blood DNOC levels for man, rat, and
rabbit, calculated from the data in Table 53, is presented in Figure 30. These
results show an exponential decay in all cases, but also demonstrate that
man is relatively inefficient in eliminating DNOC.
Table 53. Decaying Blood DNOC Values in Man and in Animals (King and Harvey, 1953 b)
Species,
number and sex
Man (1)
Rat (F) (4)
Rat (F) (5)
Rabbit (F) (6)
Rabbit (F) (6)
Dally dose Time after
of D1OC dosing
(mg/kR) . .. (hr) .___
9 x 20 6.0
24.0
48.0
72.0
120.0
144.0
1 x 30 3.5
24.0
46.5
72.0
77.0
95.0
9 x 25 4.5
7.5
10.5
24.0
48.0
1 x 30 6.0
9.0
12.0
25.0
31.0
49.0
Blood DNOC
d'B/fO
Hnan.'S.E. Slope (b)
See Pollard 4 -0.002
Fllbee, 1951
72.2 1 10.0
50.7 1 7.5
17 1 • 33 -0.0105
7.1 1 1.5
2.0 l 0.5
105.0 i 10.0
64.6 1 4.8
32.5 i 4.0 - ....
19.3 '- 2.7 -u.uili
13.9 ; 1.7
11.2 t 2.1
54.7 • 6.6
44.4 • 5.3
31.2 .•• 3.1
6. 1 '. 1.07
0.7 • 0.2
49.5 .<-. 3.4
46.8 J 2.9
77 '," 13 -0.0454
4.2 • 1.0
0.8 t 0.3
Halt- rime.
(hr)
153.6
26.8
28.5
6. 7
6.6
100
024
12 16 24
Time (days)
Figure 30. Decay Curves of Blood DNOC of Man, Rat, and Rabbit (King and Harvey,
1953 b) '
(Continuous lines represent curves resulting from many doses; broken
lines from a single dose.)
(Reprinted from the Biochemical Journal with permission from the
Biochemical Society.) .
206
-------
Twenty-four hours after the administration of a single 75 mg dose
of DNOC to human volunteers, only about 40 percent of the compound could be
accounted for (Table 54).
Table 54. Distribution of DNOC in Man Following Single Dose of 75 mg DNOC
(King and Harvey, 1953 b)
Accountable DNOC 24
Volunteer
subject
A
B
C
D
E
Blood
volume
(ml.)
5200
5400
5800
5600
6000
In blood
(mg.)
28.7
30.3
35.0
26.4
26.7
^S\*-.
(% intake)^
38.2
40.4
46.6
35.2
35.5
Hr. After Dose
^
In urine
^
(mg.)
0.8
0.6
1.3
0.6.
1.5
•N^
(% intake)^
1.1
0.80
1.7
0.80
2.00
DNOC which
cannot be
j f
(% intake)
60.7
58.8
51.7
64.0
62.5
This observation suggested that excretion of DNOC may be occurring very slowly,
or that storage of DNOC in the body may have been taking place. However, it is
not possible to determine from these results whether the non-accounted for fraction
was present in the form of unchanged DNOC or as unidentifiable (and therefore
non-accounted for) metabolites.
Further studies on the elimination of nitrophenolic compounds by
various animals were conducted by Lawford £t al. (1954). Determinations were
made of the absolute slope values of the elimination rates from blood of four
nitrophenol derivatives given to various animals. Table 55 details the rates
of elimination following either oral or parenteral administration of p_-nitro-
phenol, 2,4-dinitrophenol, DNOC, and 2,4-dinitro-ct-naphthol.
207
-------
Table 55. Absolute Rates of Elimination
(Lawford et al. , 1954)
(a)
of Four Nitre-Compounds
Animal
Mouse
Rabbit
Gutnea-
Plg
Rat
Method of
dosage
Oral
Intra-
peritoneal
Oral
Intra-
perltoneal
Oral
Intra-
peritoneal
Oral
Intra-
perltoneal
Substance
mol. wt.
solubility
In water
B./100 ml.
S (a)
T
b
S
T
b
S
T
b
S
T
b
S
T
b
5
T
b
S
T
b
S
T
b
o-nltrophenol
139
1.6 (25'C)
30
30
- 0.90 + 0.06
24
24
- 1.24 + 0.12
4
64
- 0.43 + 0.036
5
45
- 0.78 + 0.006
(b)
(b)
4
32
- 0.190 + 0.012
5
25
- 0.80 + 0.06
2,4-dtnitrophenol
184
0.56 (18'C)
36
36
- 0.098 + 0.033
24
24
- 0.21 + 0.014
6
24
- 0.010 + 0.02
6
24
- 0.22 + 0.0009
16
16
- 0.12 + 0.017
16
16
- 0.135 + 0.017
6
24
- 0.062 + 0.009
6
24 •
- 0.122 4- O.OC08
4 ,6-dinItro-o-cresol
198
0.024 (19'C)
44
44
- 0.036 + 0.004
28
28
- 0.04 + 0.002
6
30
- 0.045 + 0.001
3
15
- 0.077 + 0.0109
16
16
- 0.032 + 0.001
20
20
- 0.021 + 0.003
- 0.01
- 0.02
2,4-dinltro-
-------
100-1
1
£
J3
«*•
O
01
•o
c
3
a
o
r
e
'E
"o
A — Dinitro—a—naphtho!
B — Dinitrophenol
C — Dinitro—o—cresol
D — p—Nitrophenol
X X 20 mg/kg p—nitrophenol oral
B B 20 mg/kg p—nitrophenol intraperitoneal
&-•—•-& 15 mg/kg 2 : 4—dinitrophenol oral
A A 15 mg/kg 2 : 4—dinitrophenol intraperitoneal
o........o 15 mg/kg dinitro—a—naphthol oral
•- -• 15 mg/kg dinitro—a—naphthol intraperitoneal
7 V 20 mg/kg dinitro—o—cresol oral
¥-•—.-V 20 mg/kg dinitro—o—cresol intraperitoneal
10-
RAT, - 0.01
one monkey
• one monkey
' one monkey
one monkey
one monkey
0
1 1 .
20
i 1 i
40
HOURS
i
60
i i
80
Figure 31. Elimination of Nitro Compounds By the Monkey (From Lawford et al., 1954)
-------
The comparisons presented in Table 56 depict elimination rates
expressed as ratios of the absolute slope values, with either the rat or DNOC =
1. These results indicate that in the four species of animals employed in this
study, the ability to eliminate the chemicals decreased in the order mouse >
rabbit > guinea pig > rat.
Table 56. Comparison of Rates of Elimination (Lawf ord e_t al. , 1954)
(A) By Animal Species: Rat = 1
Substance
j>-nitrophenol
2,4-dinltro-
phenol
4,6-dinitro-
S-cresol
2,4-dinitro-
a-naphthol
Animal
Mouse
Rabbit
Guinea-pig
Rat
Oral
Intra-
peritoneal
Oral
Intra-
peritoneal
Oral
Intra-
peritoneal
Oral
Intra-
perltoneal
Oral
Intra-
peritoneal
Oral
Intra-
peritoneal
Oral
Intra-
peritoneal
Oral
Intra-
peritoneal
Mouse
4.9 >
1.5 >
1.5 <
1.7 <
3.6 <
2.0 <
(N.A.)
6.0 >
(B) By
_p_-nitrophenol
25.0
31.0
9.5
10.0
(N.A.)
(N.A.)
19
40
Rabbit
2.3
1.0
1.6
1.8
4.5
3.8
4.0
4.1
Compounds :
Guinea-pig
> (N.A.) >
(N.A.)
< 2.0 >
> 1.1 >
> 3.2 >
> 1.0
> 3.4 >
> 1.9 >
dinitro-pj-cresol = 1
..... , . 2,4-dlnltro-
2,4-dinitrophenol a:napj,thol
> 2.7
5.2
> .22
> 3.0
3.9
6.5
> 6.2
> 6.1
> (N.A.) >
> 3.0 >
> 1.3 >
> 1.3 >
> 1.6 >
> 2.0 >
Rat
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
4,6-dinitro-
fl-cresol
1.0
1.0
1.0
1.0
1.0
1.0
> 1.5 > 1.0
> 1.1 > 1.0
The nitrophenol derivatives, 2-sec-butyl-4,6-dinitrophenol
(dinoseb) and 2,4-dinitro-6-octylphenyl crotonate (binapacryl), have been
measured in the blood of animals following oral and dermal applications
(Bough eit al. , 1965). Table 57 presents the results of oral treatment of
guinea pigs and the levels of free and total dinoseb in the blood.
210
-------
Table 57. Blood Levels in Guinea Pigs After Oral Doses of Binapacryl and
Dinoseb (Bough e£ al., 1965)
Binapacryl
(400 mg/kg)
Parameter
Number of animals
Deaths in 7 hours
Time of survival (hours)
Blood concentration of dlnoseb (mg/100 ml)
Before dosing
Free dinoseb:
Total dlnoseb:
At death
Free dinoseb:
Total dinoseb:
Method 2
Method 1
Method 1
Method 2
Method 1
Method 1
0
0
0
7
7
7
Group 1
Croup 2
11
7
3.0 onward
.0
.1
.0
.6
.7
.6
(+0.0)
(± 0.1)
(+0.0)
(+ 0.4)
(± 0.3)
(+ 0.3)
11
5
5
7
5C
5
4
3 .
5.2 onward
0.0 (+
0.1 (+
0.0 (+
8.2 (+
7.8 (+
7.7 (+
0.0) It
0.0) 4
0.0) 4
0.8) 3
0.4) 3
0.4) 3
Dinoseb
(40 mg/kg)
Group 3
8
8
1.7 - 3.0
0.0 (+ 0.0) 8 0
0
0
8.6 (+ 0.6) 8 7
8
8
Group 4
2
2
2.0 and 2.9
.1
.1
.0
.8
.0
.4
(±
<±
(+
(f
(i
(+
0.0) 2
0.0) 2
0.0) 2
O.S) 2
0.5) 2
0.3) 2
a. This applies only to those animals which died within 7 hours.
b. For blood concentrations the values given are means for groups of animals followed by the standard error in parentheses and the number of animals
c. Estimations made only for five of the seven animals which died.
These results tend to indicate that binapacryl is converted to dinoseb in
the body. Both free and total dinoseb levels are about the same at death
in all treatment groups, and no evidence of binapacryl was found in the blood
of guinea pigs even at large doses.
211
-------
The outcome of dermal absorption studies presents a different
picture, however, as indicated in Table 58.
Table 58. Blood Levels in Rabbits After Dermal Absorption on Binapacryl and
Dinoseb (Bough et al., 1965)
Parameter
Number of animals
Deaths
Time of survival
(hours)
Blood concentration
of free diuoseb
(mg/100 ml) :
Before dosing
Free dinoseb:
Method 2
Method 1
• Total dinoseb:
Method 1
1 hour
Free dinoseb:
Method 2
2 hours
Free dinoseb:
Method 2
4 hours
Free dinoseb:
Method 2
8 hours
Free dinoseb:
Method 2
16 hours
Free dinoseb:
riethoa 2
24 houro
Free dinoseb:
riethod 2
Method 1
Total dinoseb:
Method 1
At Jeath
Fret- dlnoseh:
Method 2
Binaparcryl dinoseb
750 mg/kg, 750 mg/kg, 10 mg/kg, 20 rag/kg, 40 mg/kg,.
Group 1 Group 2 Group 3 Group 4 Group 5
4 4 44 4 •
00 04 4
>24 >24 >24 3.5- 5.5 2.5-3
0.0(10.0)4 0.0(±0.0)4 0.0(±0.0)4 0.0(±0.0)4 0.0(±0.0)4
0.1(10.0)4
0.0(10.0)4
0.0(10.0)4
0.1(10.0)4 —
0.1(10.0)4
0.2(10.0)4 — 3.3(10.3)4
0.2(10.1)4
0.2(10.1)4 0.9(10.2)4
0.4(10.1)4
0.0(10 0)4 - —
— — - — 4.5(10.4)4 S. 3(10. 4)4
For blood concentrations, the values given are means for groups of animals followed by the standard error in
parentheses and the number of animals.
While rabbits given 20 or 40 mg/kg of dinoseb all died within 5.5 hours, no
deaths resulted from applications of binapacryl at concentrations up to 750 mg/kg.
Furthermore, blood levels of DNBP in the binapacryl-treated rabbits were not
significantly greater than zero, and binapacryl could not be detected in the
blood. Therefore, it must be concluded that binapacryl is very poorly ab-
sorbed through the skin. This observation is consistent with the results of
dermal toxicity studies employing other alkyldinitrophenols with large ring-
subs tituents. For example, 2-cyclohexyl-4,6-dinitrophenol possesses extremely
212
-------
low dermal toxicity (Spencer et al. , 1948; see Section III-D) which is probably
due to poor absorption through the skin. By comparison, an oral dose of
2-cyclohexyl-4,6-dinitrophenol at 180 mg/kg produced 100% mortality in rats,
whereas a topical dose of 1.0 g/kg produced no mortality in the guinea pig.
Dinoseb , on the other hand, is rapidly absorbed following dermal application,
as can be seen in Figure 32. Even though the time of death varied among
the individual rabbits, the blood levels of dinoseb at death are in a very
narrow range.
8
84
O I 23456
TIME [HRI AFTER APPLICATION
Figure 32. Concentration of DNBP in Blood of Individual Rabbits at Various
Times After DNBP (50 mg/kg) Had Been Applied to the Skin
(Bough £t al., 1965)
(The final point for each animal represents the blood concentration
at death.)
CReprinted with permission from the Academic Press, Inc.)
An example of the excretion pattern of a sterically-hindered
nitrophenol is provided by a study on the absorption and metabolism of 2,6-
di-tert-butyl-4-nitrophenol (BNP) in the rat (Holder e£_al., 1971). The re-
ported results indicated that BNP is slowly absorbed from the gut following
14
oral administration of C-BNP. An average of 28.1 ±9.8 percent of the
213
-------
dose was excreted as unchanged BNP in the feces. Maximum fecal radioactivity
excretion occurred 48 hours after dosing and had ceased after 72 hours. An
14
oral dose of 1.0 mg C-BNP in the rat led to the recovery of 33 and 20 per
cent of the radioactivity in the urine and feces, respectively, after five
days. Pretreatment of the rats with neomycin to kill gut microflora, however,
changed these percentages to 23 and 34, respectively. These data would indicate
that the gastrointestinal bacteria facilitate the absorption of BNP, as evi-
denced by the increased fecal radioactivity excretion after antibiotic pre-
treatment. This conclusion is supported by results obtained from parenteral
14
administration of C-BNP to rats. Comparison of the oral and parenteral
studies indicated that the excretion of radioactivity in the urine of rats is
higher after intraperitoneal injection and suggests that direct absorption of
unchanged BNP from the gut is difficult.
b. Nitrobenzene
The absorption of nitrobenzene vapor through the lungs was
measured in humans during studies conducted by Salmowa et_ _al. (1963). Seven
men were exposed to nitrobenzene in air at concentrations ranging from 5 to
30 yg/& for periods up to six hours. The results indicated that nitrobenzene"
had been absorbed by the lungs in amounts ranging from 8.4 to 67.6 mg. Figure
33 depicts the time-course of retention of nitrobenzene in the lungs, which
averaged 80%, and varied from 87% in the first hour to 73% in the sixth hour.
214
-------
IOO
I 90-
.j
t 80-
9 7O
c
3
oi>r<
Figure 33. The Percentage Retention of Nitrobenzene Vapor in the Lungs in
the Course of a Six-Hour Exposure (Mean Values)
(From Salmowa £t al. , 1963)
(Reprinted from the British Journal of Industrial Medicine with
permission from the British Medical Association.)
The kinetics of nitrobenzene elimination were investigated in this study by
relating the amount and rate of urinary excretion of £-nitrophenol, one of
the metabolites of nitrobenzene, to the amount of nitrobenzene absorbed.
These relationships are presented in Figures 34 and 35.
2
0.
SO 4O SO
Nitrobenzene mq
to 7O SO
Figure 34. The Concentration of £-Nitrophenol in Urine Collected
Two to Three Hours After the End of Exposure, as a
Function of the Absorbed Dose of Nitrobenzene
(From Salmowa et_ al. , 1963)
(Reprinted from the British Journal of Industrial
Medicine with permission from the British Medical
Association.)
215
-------
zoo •
I 50-
100-
z
4.
so-
10
20 30
-------
2. Transport and Distribution
Transport and distribution of nitroaromatic chemicals in the
body have not been studied extensively. Data from case histories of occupa-
tional poisonings have demonstrated that localization of nitrobenzene deri-
vatives in lipids can commonly occur (see Section III-C). It is well known
that highly lipid-soluble compounds such as dinitrobenzene and organochlorine
pesticides are often localized in adipose tissue due to partition between
intracellular lipids and body water.
One can predict that distribution and localization of nitro-
aromatics in the brain may commonly occur. Exposure to these chemicals often
produces pronounced CNS effects, and the uncoupling of oxidative phosphorylation
can be demonstrated in brain cell mitochondria of poisoned mice (Ilivicky and
Casida, 1969; see Section III-D). It has been pointed out by Schanker (1964)
that rapid penetration of drugs into the central nervous system is best
i
accomplished by a substance with a low degree of ionization at blood pH and a
high degree of lipid solubility for the undissociated form. These compounds
will cross the blood-brain and blood-cerebrospinal fluid barriers at a rate which
is related to the lipid/water partition coefficient of the undissociated molecules.
On the basis of this information, the passage of many nitroaromatic compounds into
the brain can be predicted. These compounds would primarily include those-
of the nitrobenzene series and their halogen-substituted derivatives. Increasing
substitution with nitro, carboxyl, sulfonyl and amino groups would tend to
hinder penetration.
Evidence has been presented which demonstrates that p_-nitro-
aniline binds to hemoglobin and localizes in the erythrocytes. Schanker et al.
(1961) measured the binding of £-nitroaniline to red cells after incubation
217
-------
with the chemical, followed by rupture of the cells, dilution, and ultrafiltra-
tion of the resulting suspensions. These results and the results from incubating
£-nitroaniline with varying concentrations of hemoglobin are presented in
Figure 36. The binding for undiluted cells was extrapolated to be 77% and
agreed closely with theoretical calculations. The extent of binding of £-
nitroaniline to hemoglobin was extrapolated from the data to be 72% and,
therefore, would account for nearly all of the observed binding of £-nitro-
aniline to red cells.
I
I
it
?
b
*
BROKEN RED C
HEMOGLOBIN
.191
.171
J5I
J3I
.III
.091
.071
.031
2 145
RECIPROCAL OF FRACTION BCCSO
.031
Figure 36.
Binding of £-Nitroaniline to Suspensions of Broken Red Cells and
to Hemoglobin (Schanker jet al. , 1961)
(Reprinted from the Journal £f Pharmacology (1961), with
permission from the Williams & Wilkins Co.)
Numerous studies on the hematologic effects of exposure to
nitroaromatic compounds (see Section III-B-4) would tend to indicate that many
nitroaromatic substances are capable of entering the red blood cell and
combining directly with hemoglobin. For example, acute poisoning by nitro-
benzene will typically produce a chocolate-brown discoloration of the blood
and a sharp decrease in hemoglobin content as a result of severe hemolysis.
218
-------
Nakagawa and his associates (1971) examined the distribution
of 2,4-dinitrophenyl groups in guinea pig skin after the topical application of
2,4-dinitrochlorobenzene (DNCB). They found that DNCB penetrated through
the epidermis into the derrais and combined with skin components within a
few minutes after application to the intact skin surface. After 24 hours,
only about five percent of the applied DNCB remained in the skin, while the
rest had been removed via regional lymphatics and veins to other non-cutaneous
tissues or excreted in the urine. Immunofluorescent techniques were used to
observe the presence of DNCB in the cytoplasm of epidermal cells, and thereby
suggested a possible selective association with cytoplasmic components. This
cytoplasmic complex has been postulated to participate in the development of
allergic contact sensitivity reactions which commonly occur upon exposure to
DNCB or 2,4-dinitrofluorobenzene (Parker and Turk, 1970) (see Section III-B-4-b).
Tissue distribution of DNOC (4,6-dinitro-o-cresol) in the rat
following subcutaneous injection has been investigated by Parker et^ al. (1951).
Their results showed that a single dose of 10 mg/kg DNOC produced very high
levels in the serum (100 vg/g at 30 minutes) but no particular accumulation in other
tissues. Large amounts of DNOC were detected in the lungs and heart but were postulated
219
-------
to be due to the high blood-content of those organs. The authors calculated
that within 30 minutes of the injection, 83% of the DNOC which could be accounted
for was present in the blood. After six hours, 0.37 mg of the 1.5 mg dose of
DNOC could be accounted for, of which 72% was in the blood.
An examination of the DNOC content of the liver and kidneys was
made in rats receiving either a single injection or a series of 40 daily injec-
tions of the compound. The results, presented in Table 59, clearly indicated
that DNOC had not accumulated in the tissues, nor had the apparent rate of
DNOC metabolism apparently been changed by repeated treatment.
Table 59. Comparison of DNOC Concentration in Serum, Kidney, and Liver of
Rats After Single and 40 Successive Daily Injections Each of
20 mg DNOC per kg (Parker et^ al. , 1951)
a.
b.
Treatment
One injection
40 daily injections
No.
of
Rats
19
9
Concentration of DNOC 24 Hours after Last
Injection (yg/g wet weight)
Liver
8 ± 0.7yg*
7 ± 0.3yg
Kidney
7 ± 0.2yg
7 ± 0.3pg
Serum
45 ± 1.6yg
38 ± l.Oyg
Results expressed as means and standard errors.
220
-------
3. Metabolism and Excretion
a. Nitrobenzene Derivatives
A number of studies have been conducted in an attempt to
identify quantitatively the transformation products and to delineate the path-
ways involved in the mammalian metabolism of nitrobenzene and related nitro-
benzene derivatives. Using nitrobenzene randomly labelled with ll+C, Parke (1956)
accounted for some 85-90% of a single dose of nitrobenzene administered orally
to rabbits. Approximately 70% of the dose was eliminated from the body in the
expired air, urine, and feces during 4-5 days after dosing. The remainder of
the nitrobenzene was retained in the body of the rabbit and slowly excreted in
the urine. The metabolic fate of a single oral dose of 14C-nitrobenzene in the
rabbit is shown in Table 60.
Table 60; Metabolic Fate of a Single Oral Dose of 14C-Nitrobenzene in the
Rabbit During 4-5 Days After Dosing (Parke, 1956)
Metabolite
Respiratory C02
Nitrobenzene
Aniline
£-Nitrophenol
m-Nitrophenol
p-Nitrophenol
o-Aminophenol
m-Aminophenol
p-Aminophenol
4-Ni trocatechol
Nitroquinol
p-Nitrophenylmercapturic acid
"(total urinary radioactivity)
Metabolized nitrobenzene in feces
Metabolized nitrobenzene in tissues
Total nitrobenzene accounted for
Percentage of Dose (Average)
1 A
0.6* > 2 in expired
0.4t| air •>
0.1
9
9
3
4
31
0.7
0.1
0.3 J
(58)
^v
\ 60 total
^ 58 in urine
J
9A
15-20
85-90%
* 0.5% in the expired air and < 0.1% in the urine.
t 0.3% in the urine and < 0.1% in the expired air.
A 6% of the dose was present in the feces as p-aminophenol
221
-------
Of the 70% of the dose eliminated from the body, about 58% was excreted in
the urine. The major urinary metabolite, jD-aminophenol, accounted for 31%
of the dose.
These quantitative findings are in good agreement with
those obtained by Robinson et^ ji!L. (1951 a) in a previous study on the metabolism
of nitrobenzene in the rabbit using the unlabelled chemical. From their re-
sults, the authors have proposed the scheme shown in Figure 37, depicting
possible pathways of nitrobenzene metabolism.
C6H5N02
jj-nitrophenol
o-amino
ihenol
C6H5NH2
aniline
nitrobenzene
1
C6H5NO
nitrosobenzene
I
C6H5NHOH
phenylhydroxylamine
m-N02C6HltOH
m-nitrophenol
N02C6H3(OH)2
-nitrocatechol
p-nitrophenol
m-NH2C6H4OH
m-aminophenol
Figure 37.
j>-aminophenol
Suggested Pathways in the Metabolism of Nitrobenzene (Robinson
1951 a) (Major metabolic paths are shown by heavy arrows.)
^t al. ,
222
-------
Analyses of urine samples revealed that the m- and £-nitro-
phehols were produced in roughly equal quantities, wheras the amount of p_-amino-
phenol produced was very much greater than the m-aminophenol. From this,
it was suggested that the major portion of the £-aminophenol was not derived
from _p_-nitrophenol. Since jg-aminophenol occurred in much greater amounts than
o-aminophenol, it was postulated that p_-aminophenol was not primarily derived
from aniline. Therefore, since a hydroxylamine has been established as an
intermediate in the biological reduction of nitro compounds (Channon et al. ,
194A), it was concluded that the major portion of the £-aminophenol may have
arisen from nitrobenzene via phenylhydroxylamine.
The metabolism of m-dinitro[ll+C]benzene in the rabbit
at oral doses of 50-100 mg/kg body weight has been studied (Parke, 1961). In
two days , some 65-93% of the dose was excreted in the urine and 1-5% in the
feces. The remainder of the radioactivity was presumed to be retained in
the tissues and slowly excreted in the urine. The metabolites of m-dinitro-
f14C]benzene excreted in the urine are listed in Table 61.
The reduction products, _m-nitroaniline and m-phenylenedia-
tnine, were among the major metabolites, together accounting for 35% of the
dose. Some 1% of the dose was excreted as m-nitrophenylhydroxylamine and
artifacts derived therefrom (e.g., 3,3'-dinitroazoxybenzene and m-nitrosoni-
trobenzene). Traces of unchanged m-dinitrobenzene were excreted (0.7%).
The major phenolic metabolite of m-dinitrobenzene was
2,4-diamindphenol which accounted for 31% of the dose. 2-Amino-4-nitrophenol,
also a principal metabolite, was excreted as 14% of the dose. 2,4-Dinitrophenol
was found to be present in trace amounts (0.1%). Approximately 30% of the dose
is excreted as glucuronide conjugates and 6% as ethereal sulphates after ad-
ministering non-radioactive m-dinitrobenzene.
223
-------
Table 61. Metabolites of m-Dinitro[ ll+C]benzene Excreted in Urine by Rabbits (from Parke, 1961)
(m-Dinitrobenzene was administered orally as aqueous suspensions. Urines were collected
for two days.)
Dose of m-dinitrobenzene (mg/kg)
Dose of 14C (pc/animal) ...
in-Dinitrobenzene
m-Nitrophenylhydroxylamine
m-Nitrosonitrobenzene
N> 3»3'-Dinitroazoxybenzene
** m-Nitroaniline (total)
m-Phenylenediamine (total)
2, 4-Dinitrophenol (total)
2-Amino-4-nitrophenol (total)
4-Amino-2-nitrophenol (total)
2,4-Diaminophenol (total)
Total metabolites
Total radioactivity in urine
Total radioactivity in f eces
1*
100
5
'<0.1
28
<0.2
0.1
12.5
1.4
19
61
65
2
70
4
0.3
— -
35
<0.2
0.1
15
1.6
37
89
89
0.3
3
70
5
Perc
10
23
<0.1
12
2.1
38
85
82
1.0
4
60
4
entage of
2.4
0.5
0.3
18
25
16
2.6
28
93
93
5
60
4
dose
0.7
0.2
0.2
71
5.2
6
50
2
0.4
1.1
0.1
0.3
1.9
(0.6)t
•
89
7
50
3
0.7
0.75
0.25
t
2.4
(l.O)t
75
Average
0.7 N
0.8
0.25
0.3
r 35
I i
0.1
14
2.0
(0.8)t
31
83
81
*Animal died on 3rd day.
t Estimated after enzymic hydrolysis.
-------
One possible sequence in the metabolism of m-dinitrobenzene
has been presented by Williams (1959) and is shown in Figure 38.
N02
N02
N02
N02 / \^NH2
m-Nitroaniline 2-Amino-4-Nitrophenol 4-Amino-2-Nitrophenol
m-Dinitrobenzene
m-Phenylenediamine
NH2
2,4-Diaminophenol
Figure 38. Metabolism of m-Dinitrobenzene (from Williams, 1959)
A study of the metabolic fate of £-, m-, and £-chloro-
nitrqbenzene in the rabbit has shown that reduction and hydroxylation are the
major metabolic processes acting on these compounds (Bray j^t _al. , 1956). The
main urinary excretion products are phenols conjugated with glucuronic and
sulphuric acids. Chloroanilines, chloronitrophenols, and aminochlorophenols
were found to be metabolites of all the chloronitrobenzenes. A summary of the
results obtained from quantitative analysis of urine from rabbits dosed with
0,1 g/kg £-chloronitrobenzene, and 0.2 g/kg m- and ^-chloronitrobenzenes is
shown in Table 62.
225
-------
Table 62. Excretion of Metabolites of £, m-, and £-Chloronitrobenzenes by the
Rabbit (from Bray £t al., 1956)
- Results are expressed as percentages of the dose, given as means with
ranges in parentheses; superscript figures indicate the number of experi-
ments. Consecutive urine samples were analysed until excretion of. metabo-
lites ceased. The unabsorbed material found in feces was completely
reduced to the chloroaniline except in the case of the para-isomer, when
it consisted of approximately 1 part of £-chloronitrobenzene and 2 parts
of _p_-chloroaniline. The values for chloroanalines were obtained from
steam distillates of the pooled urines of six rabbits.
Chloronitrobenzene
administered
ortho-
meta-
>ara-
Unabsorbed
Ether glucuronide
Ethereal sulphate
Mercapturic acid
Chloroaniline, free
Chloroaniline, conjugated
Total accounted for
0.3 (0.1, 0.5)2
42 (26-56) 3
24 (18-31) 3
7 (0-18)3
9
0
82
0.6 (Q.5,0.7)2
33 (17-58)9
18 (4-30)6
1 (0-1) 5
11
0
64
-
2.8 (2.4,3.2)2
19 (9-27)4
21 (15-37)4
7 (2-11) 6*
3 (0-19)9**
9
4
63
* Colorimetric method. This method was more sensitive than the Stekol
method and indicated that small amounts (1% of dose) were excreted on
the fourth day after dosage.
** Values by modified Stekol method.
Small amounts of unconjugated phenolic metabolites were not included
in the values given for the total percentage of the dose accounted
for.
226
-------
Possible pathways leading to the formation of the phenolic
metabolites of the chloronitrobenzenes have been suggested by Bray et al. (1956)
(Figure 39). As shown in Figure 39, reduction could precede or follow hydroxyla-
tion.
Cl
Cl
Figure 39. Phenolic Metabolites Excreted (Free or Conjugated) in the Urine by
the Rabbit After Dosage with jo-, m- and p-Ghloronitrobenzene and
_pj- , m- and p_-Chloroaniline (from Bray et al. , 1956)
(Broken arrows point to metabolites excreted only in very small
amounts. Although only a small amount of 4-chloro-3-nitrophenol
was excreted, it is likely that a much greater amount was formed
and reduced to 3-amino-4-chlorophenol before it was excreted.)
227
-------
A study of the metabolism of orally administered 2,3,5,6-
and 2,3,4,5-tetrachloronitrobenzene in the rabbit demonstrated that these com-
pounds were not readily adsorbed and that some reduction of the nitro group
occurred in the intestine (Bray et_ jl. , 1953). Excretion of 2 ,3 ,5 ,6-tetra-
chloronitrobenzene in the feces was complete within three days after dosing
and, over a dosage range of 0.1-3.0 g, 59-78% was unabsorbed. Due to the
toxicity of 2,3,4,5-tetrachloronitrobenzene, dosage levels administered to the
rabbits were usually not greater than 0.3 g/kg. During 48 hours after dosage,
27-36% of the 2,3,4,5-compound was excreted in the feces as a mixture of un-
changed 2,3,4,5-tetrachloronitrobenzene and tetrachloroaniline. Of this
27-36%, 20% was reported to be in the reduced form.
Urinary metabolites of the tetrachloronitrobenzenes were
identified as tetrachloroaniline, tetrachloroaminophenol and glucuronide,
ethereal sulfate, and mercapturic acid conjugates. A comparison of quanti-
tative results obtained with 2,3,5,6- and 2,3,4,5-tetrachloronitrobenzene and
2,3,5,6-tetrachloroaniline is shown in Table 63.
Table 63. Excretion of Metabolites of 2,3,5,6- and 2,3,4,5-Tetrachloronitro-
benzenes and of 2,3,5,6-Tetrachloroaniline by the Rabbit (Bray e± al. ,
1953)
(Results are expressed as the average percentage of the absorbed
dose. Absorbed dose = actual dose administered less material
found in feces after 48 hours.)
2,3,4,5-
2,3,5,6-Tetrachloro- 2,3,5,6-Tetra- Tetrachloro-
nitrobenzene chloroaniline nitrobenzene
Absorbed dose (g) 0.58
Metabolite:
Tetrachloroaniline
Tetrachloroaminophenol
Glucuronide
Ethereal sulphate
Mercapturic acid
Total accounted for 90
0.82
0.47
23
5
31
3
28
18
—
73
12
0
16
—
61
9
0
103
86
228
-------
The fact that the administration of tetrachloroaniline gave no detectable
mercapturic acid suggested that the mercapturic acid conjugate was formed only
from the nitro compound or an intermediate in its reduction. The following
scheme summarizes the experimental results obtained by Bray j_t al. (1953)
using a dose of 1.5 g of 2,3,5,6-tetrachloronitrobenzene:
Cl
Cl
Excreted
free
Excreted
free
NHCOCHs
Figure 40. Metabolism of 2,3,5,6-Tetrachloronitrobenzene (Bray jit al. , 1953)
The metabolism of 2,3,4,5-tetrachloronitrobenzene differed
considerably from that of the 2,3,5,6-isomer. A greater amount of the 2,3,4,5-
compound was absorbed, the extent of hydroxylation was greater, and no mer-
capturic acid formation was detected. Using a dose of 0.7 g of 2,3,4,5-tetra-
chlbronitrobenzene, Bray and coworkers (1953) observed the results presented
in Figure 41.
229
-------
Cl
Unabs orbed
(one-fifth reduced)
Cl
Figure 41. Metabolism of 2,3,4,5-Tetrachloronitrobenzene (from Bray ejt al. , 1953)
Since nitro compounds were not detected as metabolites of
either isomer and all identified metabolites were anilines, it was suggested
that reduction of the tetrachloronitrobenzenes precedes hydroxylation.
Studies on the metabolism and excretion of nitrobenzene
derivatives have also included an investigation of pentachloronitrobenzene
(PCNB) (Kuchar jat aL. , 1969). Feeding studies were conducted using beagle
dogs and rats which were treated with various levels of PCNB in the diet for
up to two years. Table 64 presents the analysis of tissues from three dogs
fed PCNB at levels of 5 and 1080 ppm in the diet over a two year period.
Chromatographic examination of hexane extracts of the tissues confirmed the
formation of four major metabolites: pentachlorobenzene (PCB) , hexachloro-
benzene (HCB), pentachloroaniline (PCA), and methylpentachlorophenyl sulfide.
230
-------
As indicated in Table 64, accumulation of PCB and HCB,
either as metabolites or chlorinated impurities in the treatment compound, had
occurred in the fat. Additional studies in rats were performed whereby PCNB
was fed in the diet for seven months at levels of 50 and 500 ppm. Some groups
were put on a control diet for two months after the end of PCNB treatment before
being sacrificed. An analysis of the fat from these animals is presented in
Table 65; the results indicate storage of HCB in the fat which persists after
the cessation of treatment.
A further analysis of the urine was made to determine the
extent of metabolic conjugation of PCA with glucuronic and sulfuric acid.
Hydrolysis of the urine with sulfuric acid resulted in an increase in PCA
content, and thereby confirmed the role of the conjugation process (Table 66).
b. Nitrotoluene Derivatives
The oxidation of the mononitrotoluenes was studied at an
early date (1874) by Jaffe in an investigation of the fate of _p_- and £-nitro-
toluene in dogs. The methyl group of o^nitrotoluene was found to be oxidized
to yield ^-nitrobenzyl alcohol and o-nitrobenzoic acid. The alcohol, excreted
as a glucuronide, accounted for 25% of the dose; the p_-nitrobenzoic acid, 10%.
The metabolism of p-nitrotoluene resulted in the formation of £-nitrobenzoic
acid and £-nitrohippuric acid (Williams, 1959).
Gillette (1959) studied an enzyme system in rabbit liver
that catalyzed the oxidation of £-nitrotoluene to £-nitrobenzoic acid (PNBA).
He found that neither the soluble nor the microsomal fractions of rabbit liver
homogenates alone could transform £-nitrotoluene to PNBA. It was shown that
£-nttrotoluene was first metabolized to £-nitrobenzyl alcohol by a TPNH-dependent
231
-------
Table 64. PCNB Studies on Twenty-Four Month Male Beagle Dog Tissues (from Kuchar et al. , 1969)
N>
Muscle
5 ppm PCNB
1080 ppm PCNB
Kidney
5 ppm PCNB
1080 ppm PCNB
Fat
5 ppm PCNB
1080 ppm PCNB
Liver
5 ppm PCNB
1080 ppm PCNB
Urineb
5 ppm PCNB
1080 ppm PCNB
Feces
5 ppm PCNB
1080 ppm PCNB
PCNB
ND
ND
ND
ND
ND
ND
ND
ND
ND
<0.004
0.059
14.1
Data in ppm
PCB
< 0.003
0.234
0.012
0.214
0.093
5.15
0.007
0.387
ND
ND
0.007
0.422
HCB
0.016
7.28
0.035
6.41
0.452
194
0.039
5.92
ND
<0.001
0.009
1.37
PCA
ND
ND
ND
ND
0.010
0.643
0.057
0.037
<0.002
0.092
0.188
16.7
MPSa
ND
0.227
ND
1.08
0.030
2.50
0.039
0.322
<0.001
<0.001
0.134
3.64
a. Methyl pentachlorophenyl sulfide.
b. Twenty-four hour samples before scarifice.
ND - None Detected
-------
Table 65. PCNB Studies on Rat Fat (from Kuchar et al. , 1969) (Data in ppm)
PCNB Level
in Food
ppm
50
500
50
500
Male
PCNB
ND
ND
Male
ND
ND
Rats Fed PCNB
and
PCB
0.019
0.30A
Rats Fed PCNB
Diet for
ND
ND
Seven Months, Sacrificed,
Fat Analyzed
HCB PCA
10.8 0.019
117 1.11
MPS3
0.46
4.74
Seven Months, Then on Control
Two Months, Fat
3.67 ND
22.3 ND
Analyzed
ND
ND
a. Methyl pentachlorophenyl sulfide.
ND - None Detected.
Table 66. Acid Hydrolysis vs. Direct Solvent Extraction (from Kuchar et al.,
1969) (Data in ppm)
Urine
Male Dog 56
(1080 ppm PCNB)
Male Dog 59
(1080 ppm PCNB)
Liver
Male Dog 56
(1080 ppm PCNB)
Male Dog 59
1 (1080 ppm PCNB)
H+
0.097
0.079
1.14
2.92
0.111
0.092
0.195
0.220
PCA
Direct Solvent
<0.005
<0.004
0.354
0.164
0.028
0.034
0.044
233
-------
enzyme system located in the microsomal fraction of liver. A second enzyme,
localized in the soluble fraction of liver, metabolized jv-nitrobenzyl alcohol
to PNBA by a two-step DPN-dependent process. First, p_-nitrobenzyl alcohol was
oxidized to £-nitrobenzaldehyde by an alcohol dehydrogenase; then followed by
the oxidation of £-nitrobenzaldehyde to PNBA by an aldehyde dehydrogenase.
This scheme would appear as follows:
TPNH +0
£-nitrotoluene >- £-nitrobenzyl alcohol
microsomes
DPN
p_-nitrobenzyl alcohol >• £-nitrobenzaldehyde
alcohol
dehydrogenase
DPN
£-nitrobenzaldehyde + PNBA
aldehyde
dehydrogenase
The oxidation of p_-nitrotoluene by several insect species
has revealed the formation of jj-nitrobenzoic acid as the only metabolite de-
tected (Chakraborty and Smith, 1964). Since alkyl side chains are structural
features of several insecticides, the study of the metabolism of such alkyl
groups is of interest. As Chakraborty and Smith have suggested, different
rates of oxidation of insecticides having aliphatic side chains could be a
factor in differing rates of detoxication.
234
-------
The metabolism of 2,4,6-trinitrotoluene (a-TNT) was studied
by Channon e_t^ _al. (1944) in an attempt to learn the fate of trinitrotoluene
in the body. The isolation of 2,6-dinitro-4-hydroxylaminotoluene, 2,6-dinitro-
4-aminotoluene, and 2,4-dinitro-6-aminotoluene from the urine of rabbits receiving
2,4,6-trinitrotoluene demonstrated the existence of the reduction mechanism:
CH3C6H2(N02)3 * CH3C6H2(N02)2NHOH >• CH3C6H2(N02) 2NH2
The 2,2',6,6'-tetranitro-4,4'-azoxytoluene, which had been previously obtained
from the urine of rabbits (Dale, 1921) and from workers in TNT factories (Moore,
1917; Webster, 1921), was shown not to be a metabolic product. The azoxytoluene
appears to be an artifact present in freshly voided urine. Thus, there was
no evidence for the in vivo formation of the azoxy compound.
About 30% of the trinitrotoluene administered was excreted
as aromatic amino compounds and 47% in combination with glucuronic acid.
In an investigation of the chemical nature of the red
pigment present in urine after exposure to TNT, the authors administered pos-
sible intermediate metabolic substances to rats and observed the color of the
urine excreted. Of the compounds used, only a-TNT and 2,4,6-trinitrobenzyl
alcohol caused the urine to be red. Thus, the red pigment in trinitrotoluene
urine may be due to the presence of 2,4,6-trinitrobenzyl alcohol or one of its
derivatives. : This finding indicates the possible existence of the oxidation
mechanism:
CH3C6H2(N02)3 »• HOH2CC6H2(N02)3
Doses of up to 150 mg/kg of a-TNT were eliminated within 24 hours; elimination
of larger doses required up to 48 hours. The excretion of unchanged a-TNT was
not observed.
235
-------
c. Nitroaniline Derivatives
The metabolism of several derivatives of 4-nitroaniline in
the rat has been studied by Mate £t _al. (1967). Treatment of rats with [14C]2,6-
dichloro-4-nitroaniline by oral and intraperitoneal routes produced an excretion
pattern for 14C as outlined in Table 67.
Isolation of radioactive metabolites by reverse isotope
dilution of the 24-hour urine revealed that 70.4 +3.9 percent of the urinary
radioactivity was in the form of 4-amino-3,5-dichlorophenol. An additional
2.4 + 0.3 percent of the activity was present as 4-amino-2,6-dichloroaniline.
There was no evidence of any parent 2,6-dichloro-4-nitroaniline being present
in the urine. Another in vivo^ study using 14C-labelled 2,6-dibromo-4-nitro-
aniline produced a similar pattern of metabolic reduction. After intraperitoneal
injection of 5 mg of starting material, 81 +_ 3 percent of the dose was excreted
in the 24-hour urine, of which 80 percent was 4-amino-3,5-dibromophenol. None
of the parent compound was present.
When rats were treated with unsubstituted [ll* C]4-nitro-
aniline, about 80 percent of the dose was excreted in the 24-hour urine regardless
of the route of administration (Table 68).
Analysis of the acid-hydrolyzed urine by reverse isotope
dilution demonstrated that 14.1 + 2.0 percent of the activity was 4-nitroaniline,
26.1+ 6.8 percent was 4-phenylenediamine, and 43.1+ 2.5 percent was 2-amino-
5-nitrophenol. The unhydrolyzed urine contained only two percent 4-nitroaniline
in the unconjugated form.
The mechanism of formation of aminophenols from halogenated
nitroanilines was postulated to begin with N-hydroxylation, as illustrated in
236
-------
Table 67. Excretion of~14C by Rats Dosed with [14C]2,6-Dichloro-4-nitroaniline (from Mate et al.
1967)
to
Route of
Intraperitoneal
Oral
Intraper itoneal
Oral
Percentage of dose excreted in
Urine Feces Bile
Dose
(rag/rat) 24 hr 48 hr 72 hr 72 hr 6 hr 12 hr
10 69.7 ± 4.5 11.9 ± 5.2 1.0 ± 0.5 1.5 ± 0.5
10 77.1 ± 3.8 13.75 ± 6.1 0.5 ± 0.3 1.0 ± 0.4
S • 1 0 + 0 9
-------
Table 68. Excretion of ll*C by Rats Dosed with [1'tC]4-Nitroaniline (from Mate et al. , 1967)
ro
OJ
00
Route of
administration
Intr aper itoneal
Oral
Dose
(mg/rat)
5
5
Percentage of dose excreted in
Urine Feces
24 hr 48 hr 72 hr 48 hr
76.5 ± 1.4 2.8 ± 1.4 1.6 ± 0.6 0.4 ± 0.2
83.0 ± 1.7 1.7 ± 0.4 1.0 ± 0.8 0.6 ± 0.3
Results are expressed as the mean ± SE. A group of six rats was used in each experiment.
-------
Figure 42. This is followed by nucleophilic attack by water, and then proton
rearrangement leading to quinonimine formation which is then reduced to the
athinophenol.
Figure 42. Proposed Mechanism for the Displacement of Nitro by Hydroxyl in the
Metabolism of 2,6-Dihalo-4-Nitroanilines (Mate et al., 1967)
(Reprinted with permission from C. Mate, A.J. Ryan, and S.E. Wright,
"Metabolism of Some 4-Nitroaniline Derivatives in the Rat" (1976),
Food Cosmet. Toxicol., Pergamon Press Ltd.)
The replacement of the nitro group by the hydroxyl group
appeared to be limited to the halogenated compounds, however. In the case of
4-nitroaniline, it was suggested that there is no hindrance to nucleophilic
attack at the ortho position. Therefore, rearrangement of the hydroxylamine
would preferentially lead to 2-amino-5-nitrophenol, as was reported in these
experiments.
239
-------
d. Nitrophenol Derivatives
A study of the metabolism of the o_-, m-, and p_-nitrophenols
itl the rabbit has shown that, with doses of 0.2-0.3 g/kg, conjugation in vivo
with glucuronic and sulfuric acids was almost complete (Robinson et_ _al. , 1951 b).
With all three of the mononitrophenols, the major conjugation product was nitro-
phenyl glucuronide. Reduction of the nitrophenols occurred to a small extent,
the reduction of the £-isomer being slightly greater than that of the m- and
o-isomers. That the nitrophenols are oxidized in vivo to dihydric nitrophenols
has been demonstrated by paper chromatography; however, the oxidation products
comprise less than 0.5% of the dose. A summary of the metabolism of the mono-
nitrophenols is shown in Table 69.
Table 69. Summary of the Metabolism of Mononitrophenols (from Robinson jrt jd.,
1951 b)
Percentage of Dose Excreted as
s
/
'
Nitro Amino Ethereal
Compounds Compounds Glucuronides Sulphates
Nitrophenol N A (N + A)* G E (G + E)*
Ortho 82 3t 85 71 11 82
Meta 74 10 84 78 19 98
Para 87 14 101 65 16 81
* (N + A) should be roughly equal to (G .+ E), since the amounts of free
phenols excreted were very small.
t This figure is low since only o-aminophenol combined with glucuronic
acid was estimated. To allow for <>-aminophenol combined with sulfuric
acid, this figure could probably be doubled.
240
-------
Parker (1952) studied the enzymatic reduction of dinitro-
phenol by rat liver homogenates in order to ascertain the metabolic products.
Of the possible amino derivatives, A-amino-2-nitrophenol was found to be the
major metabolite, comprising 90% of the total metabolites formed, and 2-amino-
4-nitrophenol accounted for less than 10% of the amines produced. Definite
.identification of the second reduction product, 2,4-diaminophenol, was not made.
In an investigation of the rate of destruction of dinitrophenol and the rate of
formation of amino compounds, loss of amine occurred after three hours, while
destruction of dinitrophenol continued. When the two aminonitrophenols were
incubated with liver homogenates, the 2-amino-4-nitrophenol was slowly destroyed,
while 4-amino-2-nitrophenol was destroyed more rapidly. In an in vivo study
of rats poisoned by 2,4-dinitrophenol, the urinary excretion products were
unchanged 2,4-dinitrophenol and 2-amino-4-nitrophenol. There was no evidence
of 4-amino-2-nit:rophenol. Since the 4-amino isomer was destroyed more rapidly
in vitro than the 2-amino compound, the authors suggested that 2-amino-4-
nitrophenol might be expected to appear as the main reduction product of dinitro-
phenol in the urine.
Eiseman et al. (1972) examined the in vitro metabolism of
ll*C-2,4-dinitrophenol by rat liver homogenates. Of the 81+4% of the dinitro-
phenol metabolized during a 30-minute incubation period, 75+4% was metabolized
to 2-amino-4-nitrophenol, 23+2% to 4-amino-2-nitrophenol, and 1% to 2,4-diamino-
phenol. These amounts differ significantly from Parker's results, in which
Parker reported 4-amino-2-nitrophenol to be the major metabolite. Eiseman
et_ al. (1972) stated that a contributing factor in the difference in results
could be the lower stability of the 4-amino isomer and its possible oxidation
to a non-extractable product.
241
-------
Although the quantitative aspects of the metabolism of
2,4-dinitrophenol remain to be elucidated, the outline of its metabolic fate as
reported by Williams (1959) is as follows:
NH,
NH,
A recent investigation on the disposition of 4,6-dinitro-
2-sec-butylphenol (dinoseb) in female mice was conducted by Gibson and Rao (1973).
It had previously been established that dinoseb is teratogenic in mice (Gibson,
1973; see Section III-D-5). When administered by oral and intraperitoneal routes,
ll*C-dinoseb was excreted in the urine, feces, and bile as outlined in Table 70.
Three hours after administration to pregnant mice, the
liver and kidney were found to contain about 50 percent unchanged dinoseb and
50 percent as unidentified metabolites. In the embryo, 85 percent of the radio-
activity present three hours after the oral administration of dinoseb was in the
242
-------
Table 70. Excretion of [luC]Dinoseb by Female Mice Following Oral or IP Administration (from Gibson
and Rao, 1973) .
Tune after
administration
(hr)
0.5.
1
2
4
8
16
32
64
Mean cumulative excretion (% of administered radioactivity) in
Bile Urine Feces
Treatment Oral Ip Oral Ip Oral Ip
Dose (mg/kg). 32 17.7 32 17.7 32 17.7
0.1 ± 0 0.2 ± 0.1
0.4 ± 0.1 0.6 ± 0.1 0.8 ± 0.2 1.4 + 2.0 -
0.6 ± 0.1 1.4 ± 0.4 1.9 ± 0.4 3.9 ±0.1
0.9 ± 0.4 3.9 ± 0.6 3.2 ± 0.4 7.0 ± 0.1
1.4 ± 0.6 9.6 ± 1.4* 6.8 ± 1.4 13.4 ± 1.3 0.5 ±0 3.3 ± 0.9
14.4 ± 2.0 22.1 ± 2.1 4.3 ± 1.1 11.1 ± 1.1*
23.2 ± 3.5' 26.3 ± 1.9 9.7 ± 3.7 28.7 ± 4.8*
26.3 ± 3.3 28.2 ± 2.5 30.4 ± 7.5 40.8 ± 6.5
Values are means for groups of three mice ± SEM. Those marked with asterisks differ significantly from the corresponding
value for orally treated animals: *P < 0.05.
-------
form of the unchanged compound. After intraperitoneal injection, however, only
57 percent of the radioactivity in the embryo was present as unchanged dinoseb.
This difference may explain the apparent lack of teratogenic and embryotoxic
effects of dinoseb when it is administered by the oral route, as opposed to its
definite toxic effects when given intraperitoneally (Gibson, 1973). Such obser-
vations illustrate the importance of conducting studies in toxicologic metabolism
prior to making judgements concerning the potential hazards of environmental
chemicals.
e. Metabolic Reduction of Nitroaromatic Compounds
A comprehensive investigation of the in vitro metabolism
of numerous nitroaromatic compounds was conducted by Fouts and Brodie (1957).
They described a nitro reductase system present in the liver and kidney of
rabbits which can reduce various nitroaromatic compounds to the corresponding
amines. The system was localized in both the soluble fraction and the micro-
somes of the liver cell. This system was completely inactivated in the presence
of oxygen. Both TPNH and DPNH could act as hydrogen donors for the system,
which also included a flavoprotein whose prosthetic group may be replaced by
any of the three flavins, riboflavin-5-phosphate, flavin adenine dinucleotide,
or riboflavin. The wide variety of nitroaromatic compounds which could be
reduced by these enzymes is illustrated in Table 71. The nitrophenols are
notable exceptions to the rapid reducing action of the system described.
The formation of arylhydroxylamines as intermediates in
the metabolic reduction of nitro compounds is of great significance in deter-
mining their threat to human health. Sternson (1975) has pointed out that the
carcinogenicity of nitroaromatic compounds may depend upon their metabolic
244
-------
Table 71. Reduction of Various Nitro Compounds by Liver Homogenates (from
Fouts and Brodie, 1957)
Supernatant fraction (900 x g) equivalent to 1 gm liver, 4 micromoles
of substrate, 0.6 micromole of TPN, and 100 micromoles of nicotinamide were
incubated for two hours at 37°C with nitrogen as the gas phase.
Substrate
Chloramphenicol
m-Diriitrobenzene
£-Nitrotoluene
£-Nitrobenzoic acid
Nitrobenzene
m-Nitrobenzoic acid
2 ,4-Dinitrophenol
£-Nitrophenol
m-Nitrophenbl
£j-Nitrobenzyl alcohol
m-Nitroacetophenone
m-Nitrobenzaldehyde
p-Nitrobenzaldehyde
Reduced Product
Formed
(ymoles)
3.07
1.83
1.60
1.50
1.15
0.61
0.10
0.20
0.00
—
—
—
--
Relative Optical
Density (540 my) of
Reduced Product
0.650
1.150
0.230
1.335
0.335
0.550
0.075
0.150
0.010
0.840
0.620
0.530
0.530
245
-------
activation to the corresponding hydroxylamine (see Section III-D-6). He studied the
reductive metabolism by the action of hepatic nitro reductases of a series of
nitroaromatic compounds in rabbit liver microsomal suspensions. The detection
of hydroxylamine intermediates formed by metabolic reduction in biological
systems had previously been very difficult due to their high reactivity and
lability. A sensitive electrochemical detection method was employed, however,
based on the anodic oxidation of hydroxylamines at carbon paste electrodes. By
using this technique, hydroxylamines were demonstrated in liver microsome in-
cubations containing either 1-nitronaphthalene, nitrobenzene, nitrofluorene,
or p-nitrocresol. These reactions were carried out under anaerobic conditions
because the presence of oxygen destroyed nitro reductase activity, which indicated
that the process was mediated by cytochrome P-450.
In an investigation of the enzymatic reduction of 2-nitro-
naphthalene and similar nitroaromatic compounds by rat liver in vitro, 2-naph-
thylamine was slowly produced as 2-nitronaphthalene disappeared (Poirier and
Weisburger, 1974). Similarly, the reduction of 1-nitronaphthalene led to the
formation of the corresponding arylamine. In both cases, there was no evidence
of hydroxylamine accumulation. Previous studies on the in vitro enzymatic re-
duction of £-nitrobenzoic acid (Kato et^ ai., 1969), nitrobenzene (Uehleke, 1963),
and 4-nitrobiphenyl (Uehleke and Nestel, 1967) reported the formation of hydroxy-
lamine intermediates. Poirier and Weisburger (1974) attributed their lack of
detection of arylhydroxylamines to a low activity of nitroreductase and the
fact that the arylhydroxylamines, when formed, are rapidly reduced to amines by
liver extracts.
246
-------
f. Metabolism by Gastro-Intestinal Microorganisms
The reduction of nitro groups by intestinal microorganisms
has been known for some time (Glazko et^ _al. , 1949; Zachariah and Juchau, 1974)
and can often determine the form in which a nitroaromatic chemical is absorbed.
In a study on the metabolism of 2,3,4,5- and 2,3,5,6-tetrachloronitrobenzene
in rabbits (Bray et al., 1953) it was noted that these compounds had very
low water solubility and were only partially absorbed from the intestine.
In this case, bacterial reduction prior to absorption may have accounted for
most of the reduced metabolites identified. On the other hand, nitroaromatic
compounds which are quickly absorbed would avoid extensive bacterial reduction.
A study on the metabolism of the herbicide trifluralin in rats and dogs revealed
that a large portion of the dose was excreted in the feces as reduced amino
metabolites (Emmerson and Anderson, 1966). These results suggested that re-
duction had taken place in the intestine prior to absorption (Scheline, 1968).
Bacteria of the rumen are also known to reduce nitro
groups. Golab and co-workers (1969) demonstrated that trifluralin labelled
with ltfC when incubated with artificial rumen fluid, was rapidly transformed
by reduction of both nitro groups (Figure 43). Subsequent investigations
performed jn vivo on a lactating cow confirmed the results of the in vitro
assay.
An observation of the relative lack of tpxicity of the
pesticide parathion when given orally to cows was suggested to be due to
nitro reduction by rumen bacteria to the corresponding amino compound
(Cook, 1957).
247
-------
o
-------
Recent evidence has been presented which indicates that
intestinal microflora may play a major role in the metabolic reduction of
_p_-nitrobenzoic acid (PNBA) (Wheeler ej^ ai^. , 1975). The authors established
that the anaerobic flora of the rat cecum was capable of reducing PNBA to
p_-aminobenzoic acid (PABA) by incubating rat cecal contents with PNBA under
various conditions (Figure 44). Destruction of the microflora by autoclaving
or the use of cecal contents from germfree rats resulted in the loss of PNBA
reducing activity.
Noting that the majority of the flora of a rat is con-
tained in the cecum, Wheeler and coworkers (1975) compared the reduction of
PNBA in vivo using both conventional and cecectomized rats. They found that
conventional rats were able to convert more than 20 percent of PNBA to PABA,
whereas rats converted only 7 percent of the same dose when it was administered
one week after cecectomy (Table 72). There was some evidence, however, of
regaining the ability to reduce PNBA after cecectomy, as evidenced in rats
fed PNBA 14 days after surgery.
These results cannot characterize the extent to which
mammalian enzymes participate in the reduction of nitro groups, nor can they
identify a possible joint participation of the liver and other organs with
bacteria in metabolic nitro reduction. It is known, however, that induction
of microsomal enzymes by DDT or phenobarbital increases PNBA reduction in
vitro but not in vivo (Carlson and DuBois, 1970). Furthermore, the depression
of xanthine oxidase function, an enzyme which reduces p_-nitrobenzenesulfonamide
in rat liver, causes a decreased rate of PNBA reduction in vitro but does not
diminish the reduction of p_-nitrobenzenesulfonamide in vivo (Westerfeld
et al., 1956). Wheeler and his associates (1975) found that the reduction of
249
-------
£-nitrobenzenesulfonand.de when fed in the diet of rats occurred at the rate
of 5 to 6% in germfree animals, compared to 46 to 47% in conventional rats.
These results strongly suggest an important role of the microflora in the
metabolic reduction of nitro compounds in-general.
*t
to
z
0.
o
•o
0)
ee
10
-------
Table 72. The Reduction of PNBA by Conventional and Cecectomized Rats'
(Wheeler et al., 1975)
Type of Rat
Conventional
Conventional
Cecectomized
Cecectomized
No.
of
Rats
5
2
3
3
Urinary
Total
PABA
% of
20 (18-23) °
26, 25
7.0 (6-8)
13 (9-19)
Excretion
Recovery of
PNBA and its.
metabolites
dose
76 (72-85)
85 (40-110)
86 (70-97
a. PNBA (25 mg) was given orally unless otherwise noted. Urine was
collected for 24 hours and analyzed.
b. Includes PABA and its conjugates (Total PABA).
c. Numbers are the average recovery of metabolites; the range is shown
in parentheses.
d. PNBA (25 mg) given subcutaneously.
e. PNBA was fed seven days after the cecectomy operation.
f. Rats were refed PNBA 14 days after cecectomy.
251
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4. Metabolic and Pharmacologic Effects
The nitroaromatic compounds as a group exert a varied and pro-
found effect on metabolic and physiologic processes. Individual compounds in
this group can cause severe hematologic changes including methemoglobinemia,
sulfhemoglobinemia, Heinz body formation, and red cell destruction resulting
in anemia. Certain nitroaromatics are unique in their ability to "uncouple"
oxidative phosphorylation by suppressing the coupling of electron flow to
synthesis of ATP. Several nitroaromatic chemicals cause allergic contact
dermatitis, and one of these compounds, 2,4-dinitrochlorobenzene, is among
the most potent primary skin sensitizers known. The major organs of foreign
compound metabolism and detoxification, principally the liver and kidneys,
are adversely affected by excessive exposure to any of the nitroaromatic com-
pounds, and irreversible cellular damage may occur.
a. Hematologic Effects
Methemoglobinemia is probably the most notable mani-
festation at the biochemical level resulting from exposure to most of the
nitrobenzene derivatives. The production of methemoglobin, a chemical analog
of normal blood hemoglobin, results from an oxidation of the heme moiety
of the molecule from its usual Fe-H- (ferrous) state to an abnormal Fe-H-+
(ferric) state (Bodansky, 1951; Kiese, 1966; Nakajima and Kusumoto, 1963).
In contrast to normal hemoglobin, which functions as an oxygen transporter
to body tissues, methemoglobin binds oxygen so firmly that it cannot be
released to the cells which require it.
Methemoglobin is normally present in the body at low
concentrations and exists in equilibrium with normal hemoglobin. It is
252
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continually reduced by a methemoglobin reductase, diaphorase (Smith, 1969),
and is thereby prevented from reaching excessive levels. Large amounts of
methemoglobin produced in response to exposure to nitroaromatic chemicals
will overload this reducing mechanism and diminish the oxygen-carrying
capacity of the blood. This occurs not only by irreversible binding of
oxygen to methemoglobin but also by interference with the release of oxygen
from normal hemoglobin. Tissue hypoxia and cyanosis (bluish discoloration
of the skin and mucous membranes) become evident when the oxygenated hemo-
globin level falls below the critical cellular demand for oxygen.
The nitroaromatic compounds themselves are not generally
regarded as direct methemoglobin producers, but rather their corresponding
reduced metabolites are considered to be the active proximate agents (Linch,
1974). The hydroxylamine analogs of nitrobenzene, p-nitrotoluene, and _p_-
\
nitroacetophenone were administered to mice and found to be active methemo-
globin-inducing agents (Smith et^ al^., 1967). These results, summarized in
Table 73, demonstrated that £-hydroxylaminoacetophenone (p-HAAP), phenyl-
hydroxylamine (PHA), and p-hydroxylaminotoluene (p-HAT) were all quite simi-
lar in their methemoglobinemia-inducing properties. The authors noted that
Table 73. Per Cent Circulating Methemoglobin at Various Times
After the Injection of Aromatic Hydroxylamines in
Female Mice* (From Smith et^ al., 1967)
Compound 10 min 20 min 40 min 60 min
p-HAAP 38.3 ± 5.8 327867! 677
PHA 42.1 ± 4.3 28.9 8.4 3.6
p-HAT 33.4 ± 5.7 18.5 5.1 . 2.'3
* All chemicals given i.p. in propylene glycol, O.lm-mole/kg.
Values are either mean ± S.D. for six animals or the simple
average for three. Ten-minute values for PHA and p-HAT are
significantly different (P<0.01).
253
-------
methemoglobinemia produced by PHA in vivo was more short-lived than that
produced by treatment of mouse red blood cells in vitro. This observation
suggested a methemoglobin-reducing process which is external to the red cell
and is functional only in the intact animal.
Metabolic reduction of the nitrophenols or aniline pro-
duces the corresponding aminophenol without necessarily forming a hydroxyl-
amine intermediate. The ability of aminophenol to produce methemoglobin
in vivo was studied in female mice (Smith et^ a]L , 1967). Table 74 shows that,
while £-aminophenol was considerably more active than £-aminophenol, it was
nevertheless about tenfold less active than PHA. This observation supports
the general assumption that the nitrophenolic compounds are not potent
methemoglobin-inducers.
Table 74. Per Cent Circulating Methemoglobin at Various Times
After the Injection of Aminophenols in Female Mice*
(From Smith et^ al., 1967)
Isomer 10 min
p-Aminophenol 7 . 2
o-Aminophenol 32.6 ± 5.2
20 min
4.9
11.9
40 min
2.3
1.4
* Compounds given i.p. in aqueous solution, 1.0 m-mole/kg.
Values shown are either mean ± S.D. for six animals or
the simple average for three.
The supposition that nitroaromatic compounds must be
reduced before they can produce methemoglobin has not been totally substan-
tiated. One recent study (Kusumoto and Nakajima, 1970) indicated that
methemoglobin could be formed in vitro by incubation of nitrobenzene with
254
-------
human and rabbit hemoglobin. As shown in Figure 45, nitrobenzene converted
a relatively small fraction (15%) of the hemoglobin to methemoglobin. This
action was considerably less potent, however, than that produced by amino-
phehol incubated under similar conditions. Evidence was obtained in this
study which demonstrated that nitrobenzene caused a structural change of
hemoglobin protein which was similar in nature but less than that caused by
aminophenol.
Figure 45* Methemoglobin Formation by Nitrobenzene In Vitro (Kusumoto and
Nakajima, 1970)
(Final concentration, hemoglobin, 0.3 mM; nitrobenzene, 5 mM)
(•— • , with nitrobenzene; 0 0, without nitrobenzene)
Most recently, Brewer and Carr (1974) have also reported
that incubation of human red blood cells with nitroaromatic compounds resulted
in the direct production of methemoglobin. This activity was found to be
related to the corresponding Hammett constant for the various nitrobenzene
derivatives, with certain disubstituted compounds being even more potent.
Another hematologic indication of exposure to nitro-
aromatic compounds is the presence of Heinz bodies in the blood, usually
255
-------
accompanied by methemoglobinemia (Hanley and Mauer, 1961). These bodies are
characterized as protein inclusion granules seen in the erythrocytes during
poisoning by aromatic amines, nitro compounds, and nitrates and nitrate esters.
The nature of this protein is not known, but it may be a denatured globulin.
Heinz bodies are readily observed on staining with vital dyes and appear as
dark particles in the bright yellowish-red erythrocytes. Their detection can
serve as a valuable aid in the diagnosis and confirmation of hematologic
poisoning. Table 75 illustrates the large number of Heinz bodies that can be
formed within a very short time from administration of a nitroaromatic sub-
stance. Many investigators have found methemoglobinemia to be a variable
Table 75. The Occurrence of Heinz Bodies in the Peripheral Blood
of Rabbits Following the Administration of a Single
Oral Dose of 3-Nitronaphthalene (BNN)
(Dosage of BNN: 0.94 to 4.7 gm/kg) (From Treon and
Cleveland, 1960)
Percentage of
Erythrocytes
Containing
Heinz Bodies
Average Range
0 0
0.75 0- 2
0.80 0- 4
8.5 7-10
33.0 32-34
87.6 63-98
90.6 75-98
88.4 85-95
81.15 75-89
7.7 6-13
7.0
7.0
8.0
Time After
Adminis tr ation
of BNN (Days)
0
0.18
0.25
0.32
1.0
1.25
2
3
4
8
9
11
15
No. of
Rabbits
Examined
7
4
5
2
2
7
7
7
6
3
1
1
1
256
-------
response at best to nitroaromatic exposure in certain animals and man. The
mouse is known to be particularly resistant to methemoglobin formation, pre-
sumably due to its very efficient methemoglobin-reducing ability (Smith et
al. , 1967). The production of Heinz bodies, however, can be a much more
sensitive indicator of intoxication and one which is usually observed in
cases of nitroaromatic poisoning.
Hasegawa and Sato (1963) investigated the formation of
methemoglobin and Heinz bodies in the blood of rabbits poisoned with £-
chloronitrobenzene. Twenty-four hours after subcutaneous administration of
500 mg/kg 'body weight, each erythrocyte of the rabbit contained one Heinz
body. After 48 hours, multiple Heinz bodies were observed both inside and
outside the red blood cells. The ratio of methemoglobin to total hemoglobin
rapidly increased upon injection of the chemical, then leveled somewhat after
seven hours, thereby indicating a biphasic formation process (Figure 46).
25
20
15
O 10
20
16
Si
o
12 f
24 48
Time After Injection ( hri)
72
Figure 46. Plots of the Amount of Methemoglobin and Oxygen Affinity of Hemoglobin
(pO , ) versus Time After Injection (Hasegawa and Sato, 1963)
2=5
257
-------
The authors cited previous studies that confirmed this process and noted that
methemoglobin produced in the initial stage was more easily reduced to normal
hemoglobin than that which was formed later. An analysis of hemoglobin iso-
lated from poisoned rabbits indicated that a functional change had taken place
which greatly increased its oxygen affinity with increasing time after in-
jection of jg-chloronitrobenzene. This change, of course, has serious physio-
logic consequences. They found that, 72 hours after treatment, the hemoglobin
of poisoned rabbits was 95 percent oxygenated, where normally one third of
the oxygen from oxyhemoglobin should have been removed. This would cause a
severe deprivation of oxygen to body tissues due to the increased strength
of its bond with circulating hemoglobin.
The appearance of sulfhemoglobin in the blood following
exposure to nitroaromatics usually parallels the production of methemoglobin,
but sulfhemoglobin is much more persistent. It is probably produced as an in-
termediate of bile pigment formation from hemoglobin. Sulfhemoglobin was
found to remain in the body for more than three months after formation, and
the length of its retention is related to the life-span of the red blood
cell (116 days). Methemoglobin, on the other hand, is usually eliminated in
two to five days.
The pharmacologic effect of several nitroaromatic der-
ivatives on blood platelet levels was found to be significant only in the case
of £-nitrophenol (Gabor et^ al_. , 1962). When 31 rats were administered p_-
nitrophenol by intraperitoneal injection at 1 mg/kg body weight, the platelet
count was observed to increase significantly (Figure 47). Even at doses of
0.1 mg/kg body weight, a similar effect was produced. The administration,
258
-------
200
Figure 47. The Platelet-Count of Rats After the Administration of Phenol
Derivatives, a 2,4-dinitrophenol; b m-aminophenol; c p_-amino-
phenol; d m-nitrophenol; e £-nitrophenol. Dose: 10 mg/100 g i.p.
(Gabor e_t al., 1962)
however, of 2,4-dinitrotoluene, nitrotoluenes, 2,4-dinitrophenol, or p_- and
m-nitrophenol did not produce a rise in platelet levels. Additional data
are not available to explain this unique phenomenon.
b. Skin Sensitization
Several halogen-substituted nitrobenzene derivatives have
been demonstrated to produce allergic contact dermatitis. Most notably, 2,4-
dinitrochlorobenzene (DNCB) is an extremely potent skin sensitizer in both
animals (Polak and Frey, 1974) and man (Catalona et^ al., 1972a, 1972b;
Malaviya ejt al., 1973; Lowney, 1971).
Contact sensitization to DNCB results from its ability to
act as a hapten when topically applied and to form covalent bonds with lysine
groups of epidermal proteins (Eisen e_t al., 1952; Eisen and Tabachnick, 1958;
259
-------
Nakagawa et al., 1971). Sensitlzatlon occurs in greater than 95 percent of
those exposed, taking place in regional lymph nodes (Kligman and Epstein, 1959).
An allergic response occurs when sensitized lymphocytes
contact the protein-bound DNCB. Circulating (humoral) antibodies do not
participate in reactions to DNCB, since the process involves only cell-
mediated immunity. Sensitization may take place 7 to 21 days after DNCB
exposure and will be manifested by a spontaneous flare reaction at the site
of application, resulting from minute concentrations of bound DNCB remaining
in the skin (Catalona et al., 1972a). Those who do not respond spontaneously
to DNCB application can be made to exhibit the allergic response by reappli-
cation of a weak solution of DNCB.
In addition to being a potent skin sensitizer, DNCB is
also an extremely strong primary skin irritant. Catalona et^ al. (1972a)
reported that doses normally used for sensitization, ranging from 1000 to
2000 yg, would cause erythema and edema within 12 hours, followed by de-
squamation and pigmentation after several days.
Allergic sensitization of the skin can also be achieved
by contact with 2,4-dinitrofluorobenzene (DNFB). DNFB acts in a similar
fashion to DNCB, combining with epidermal and dermal proteins to form an
antigenic hapten-carrier complex. Schneider (1974) described the so-called
"blastogenic response" to the application of DNFB on mice whereby lymphocytes
migrate to the regional lymph node draining the site of sensitization and
become blast cells. These blast cells rapidly proliferate for several days
and then disappear from the lymph node after four or five days. These cells
apparently enter the circulation and bone marrow to function as memory cells
260
-------
to maintain the state of contact sensitivity to future applications of
DNFB.
The monochloronitrobenzenes, specifically the ortho- and
para-isomers, are allergic skin sensitizers in rats and guinea pigs (Rusakov
et al. , 1973). Neither compound, however, is as strong an allergen as DNCB.
c. Uncoupling of Oxidative Phosphorylation
One of the most outstanding features of exposures to
nitroaromatic compounds is the relationship of the dinitrophenols to bio-
energetics. The oxidation of foodstuffs to produce energy and numerous bio-
synthetic oxidation-reduction and oxidative phosphorylation reactions are
essential to the maintenance of life. The presence of dinitrophenol compounds
in biological systems causes severe physiologic disturbance by the uncoupling
of oxidative phosphorylation.
Oxidative phosphorylation and the electron transport
sequence in aerobic organisms account for the reoxidation of reduced nico-
tinamide or flavin nucleotides and subsequently the generation of adenosine
triphosphate (ATP) by phosphorylation of adenosine diphosphate. The amount
of ATP available to the cell is critical in providing adequate energy sources
for metabolic and biosynthetic reactions. The oxidation of the nicotinamide
and flavin coenzymes is necessarily coupled to phosphorylation. Figure 48
portrays the scheme for reoxidation of the nicotinamide coenzymes. The
flavin coenzymes may also be reoxidized by the electron-transport sequence
by entering the reaction at the point of CoQ.
261
-------
Phosphorylation
ATP
SH, NAD*
S NADH
ADP + Pi
2H*
Phosphorylation
ADP + Pi
FPH2 CoQ f ^2Fe2* _
v' \\r \
||(2 cytochrome b
FP CoQH2
2 cytochrome c, j 1 2 cytochrome c ) 1 2 cytochrome a j
Phosphorylation
ATP
ATP
ADP + Pi
°
H,0
Figure 48. The Electron Transport Sequence and Probable Sites of Coupled
Oxidative Phosphorylation (This diagram shows for example that 2 molecules
of reduced cytochrome b react with 2 molecules of oxidized cytochrome
GI, and this oxidation-reduction process is coupled with the trans-
formation of ADP to ATP.) (From Feuer, 1970)
The uncoupling action of 2,4-dinitrophenol results in the
reoxidation of the reduced coenzymes without the concomitant production of
ATP (Feuer, 1970). This uncoupling effect will prevent the utilization of
foodstuffs and deprive tissues of the energy needed for muscular work and
biosynthetic reactions. Because the coenzymes are reoxidized and used to
further metabolize foodstuffs, but no ATP is generated, the organism can
take in large quantities of nutrients and yet starve, due to a lack of bio-
chemically useful energy.
Several theories have been advanced concerning the under-
lying mechanism of this uncoupling effect by dinitrophenol. Weinbach and
Garbus (1969) believed that the ability of uncoupling agents to combine with
proteins is a key to their action. Based upon their investigations, the
authors concluded that uncoupling reagents can bind to mitochondrial proteins,
as well as to soluble cellular and serum proteins. In their laboratory, it
262
-------
was demonstrated that the halo- and nitrophenols interact with mitochondria!
proteins to produce conformational changes which were thought to result in pro-
found changes in their biological activity, and hence to form the basis for
the uncoupling phenomenon.
A proposed sequence of events In the uncoupling of oxi-
dative phosphorylation would be: 1) the entrance of the undissociated reagent
into the mitochondria followed by ionization of the compound and interaction
with charged e-amino groups of the protein, 2) reorganization and conforma-
tional transition of protein structure leading to, 3) alteration in the activity
of enzymes catalyzing the coupling of phosphorylation to electron transport.
In the past, many investigators have attributed the un-
coupling action of dinitrophenol to an acceleration of the hydrolysis of oxi-
dative phosphorylation intermediates (Ernster and Lee, 1964). Studies by
Pinchot (1967), however, disagree with this theory. He suggested that phos-
phorylating particles containing a coupling enzyme formed a high energy inter-
mediate of oxidative phosphorylation. Dinitrophenol interfered with the
ability of the enzyme to bind with the electron transport particle and thereby
inhibited the oxidative phosphorylation process.
Taken together, the above studies, while not providing a
single definitive explanation for the mechanism of action of dinitrophenol,
provide strong evidence for disruption of a catalytic enzyme or enzyme com-
plex essential to the oxidative phosphorylation process.
In addition to the dinitrophenols and dinitrocresols,
several of the nitrosalicylanilides are known to be among the most effective
uncouplers of oxidative phosphorylation ever tested (Williamson and Metcalf,
263
-------
1967). An examination of the structural similarities of three potent aromatic
uncouplers, including a nitrosalicylanilide (Figure 49), reveals that strong
electron-withdrawing groups are positioned at a fixed distance from the halo-
genated aryl ring. The mode of action for these compounds in uncoupling oxi-
dative phosphorylation, as suggested by their common structural features, may
be a preferential adsorption to an active enzyme site.
Bachmann jet ajL. (1971) have reported that 2,6-dichloro-
4-nitroaniline (DCNA) is also an effective uncoupler of oxidative phosphoryl-
ation. At a concentration of 5 x 10 M with rat liver mitochondria i.n vitro,
DCNA uncoupled oxidative phosphorylation and inhibited mitochondrial electron
transport. Dinitrophenol was similarly effective at a concentration of 1 x
10 M. When administered orally to rats for four days at 1000 mg per kg body
weight per day, DCNA caused an inhibition of oxidative phosphorylation in
isolated mitochondria.
These authors have related the uncoupling potency of DCNA,
and phenolic compounds in general, to their degree of dissociation and lipid
solubility. Critical factors appear to be the ease with which a compound can
enter the mitochondria and its capacity to form a phenolic anion which can
interact with an electron-deficient enzyme of the phosphorylation reaction
chain. Alternatively, evidence has been presented (McLaughlin, 1972) to show
that the anion of dinitrophenol can bind to mitochondrial membranes and produce
a substantial negative surface potential. The significance of this effect to
the uncoupling phenomenon is not entirely clear. Additional data have been
provided (Verma e£ al., 1973) which indicate that uncouplers can change the
organization of phospholipid multibilayers. It was suggested that these changes
264
-------
Figure 49. Structural Similarities Among Various Uncouplers of Oxidative Phos-
phorylation. (A) Represents the plane of symmetry through the halogenated
aromatic rings and (B) is the corresponding plane of symmetry through the
electron withdrawing groups . (Williamson and Metcalf, 1967)
265
-------
could alter the properties of lipid-bound membrane enzymes involved in oxi-
dative phosphorylation and thereby uncouple the system.
(i) Metabolic Disruption
Metabolic effects which accompany the uncoupling of
oxidative phosphorylation are increases in body temperature, respiration rate,
oxygen consumption, glycogenolysis, and thyroxin secretion, as well as the
production of hypertension and effects on neuromuscular transmission and bile
secretion.
Hyperthermia (Jnyperpyrexia, elevated body temperature)
by the action of uncoupling chemicals results from increased heat production
rather than decreased heat loss. Gatz and Jones (1970) explained that high
phosphate bond energy normally captured in the production of ATP is lost as
heat when oxidation is uncoupled from phosphorylation. They found that the
time of onset and degree of dinitrophenol-induced hyperthermia were exponen-
tially dose-related when the compound was administered intraperitoneally to
rats (Figure 50). The authors hypothesized that a 10 percent uncoupling of
oxidative phosphorylation would increase heat production by 65 to 100 percent,
and a 20 percent uncoupling would raise heat production by 275 to 425 percent.
In studies by Hull et_ al. (1971) , 5 mg per kg body
weight of dinitrophenol injected intravenously in dogs produced little tem-
perature change. The same dose, however, administered 20 minutes after the
induction of general anesthesia by halothane produced a dramatic and lethal
rise in temperature.
Similarly, Hoch and Hogan (1973) measured the met-
abolic rate in rats treated with halothane and dinitrophenol, both alone and
266
-------
15
30 45 60 75
MINUTES
90 W5 120
Figure 50; Effect of Logarithmically Increasing Intraperitoneal Doses of
2,4-DNP on Rectal Temperature in Rats. Each point on a curve rep-
resents the mean of between 6 to 8 determinations of temperature at
the specified times. (Gatz and Jones, 1970)
(Reprinted with permission from the International Anesthesia Research
Society.)
in combination. The intraperitoneal injection of 10 mg per kg body weight of
dinitrophenol alone promptly doubled the metabolic rate and increased body
temperature by 10 percent. Gradual recovery and no mortality resulted following
this treatment. In combination with halothane, dinitrophenol evoked an addi-
tional sharp rise in temperature of about four degrees C. and a tripled
metabolic rate. Death followed within two to seven minutes of the sharp tem-
perature rise.
The above cases of hyperthermia typify the hazards
of synergistic reactions posed by substances which probably act in a similar
fashion on the body. Since dinitrophenol, and presumably halothane, can both
267
-------
act on the cellular mitochondria to disrupt oxidative phosphorylation, sub-
threshold doses of each compound when combined may produce severe physiological
disturbance through additive action at a common site.
The effect of dinitrophenol in stimulating the
t
conversion of glycogen to glucose is due, in part, to its action on the
phosphorylase enzyme system. Studies by Vercesi and Focesi (1973) and Focesi
^ aJL (1969) established that dinitrophenol could markedly increase the
level of phosphorylase b kinase in skeletal and cardiac muscle of the rat.
Phosphorylase b kinase is an enzyme which catalyzes the conversion of inactive
phosphorylase b to the activated enzyme form phosphorylase a. The relation-
ship of the phosphorylases system to glycogenolysis is explained by Segal
(1973) and illustrated in Figure 51. Glycogenolysis is normally stimulated by
ATP
Phosphorylase kinase b / phosphorylase kinase a
Epinephrine, ' /*" *%
glucagon , Phosphorylase Phosphorylase
i ' ~
« Cyclic /
Adenylate '
Protein kinase Glycogen G-I-P
\ ^ UDPG
^ Glycogen synthetase a_
cyclase
^AMP—-
Glycc£>;n synthetase b.
Figure 51. Epinephrine and Glucagon Control of Glycogen Metabolism
(Segal, 1973)
(Reprinted from Science (1973), H.L. Segal, 180, 25-32.
Copyright 1973 by the American Association for the
Advancement of Science.)
268
-------
the hormones glucagon and epinephrine via the conversion of phosphorylase b
to a through the mediation of cyclic AMP. In the case of dinitrophenol
poisoning, however, it may be possible to elevate cyclic AMP levels without
hormonal stimulation.
In skeletal muscle, dinitrophenol administered at
25 mg per kg body weight in rats produced a threefold increase in phosphorylase
a levels. Similarly, in cardiac muscle of the rat the same dinitrophenol
treatment tripled phosphorylase a content and more than doubled the level of
phosphorylase b kinase (Table 76).
Table 76. Content of Phosphorylase a and Total and of Phosphorylase b
Kinase in Hearts of Rats Poisoned With DNP 2.5 mg/100 g
(From Vercesi and Focesi, 1973)
DNP poisoned
Determination Normal Rats rats
Phosphorylase a (10 rats) 7.0+ 1.7a 25.2+ 3.1
Total phosphorylase (+AMP)
<(10 rats) 67.2 + 7.1 85.3 + 13.0
Ratio Phosphorylase a/Total
Phosphorylase X 100 10.4 + 1.1 30.7 + 6.5
Phosphorylase b kinase (10 rats) 1346 + 480 3384 + 334
a
S.E.M. The activity of phosphorylase a and total are expressed in units
according to Cori et^ _al. (1955) per gram of heart. The activity of phos-
phorylase b kinase is expressed in units of phosphorylase a formed from
phosphorylase b in 15 min according to Fischer and Krebs method (1962).
269
-------
In studying the effects of dinitrophenol on pulmonary
function in dogs, Cardus and Hoff (1963) measured oxygen consumption, venti-
lation, and respiratory frequency. They found that intravenous injection of
dinitrophenol at doses of 3, 6, or 9 mg per kg body weight invariably produced
elevations in rectal temperature within five to ten minutes after injection,
and a rapid increase in oxygen consumption (Figure 52). Increases in the
frequency and amplitude of respiratory movements were seen at the two higher
doses. In all dogs, there was an effect on the ventilation within 20 seconds
after injection of the drug. Sudden increases in respiratory frequency and
amplitude which lasted for about 10 seconds were observed, followed by re-
establishment of the control pattern. A more gradual pattern of rise in
frequency and amplitude was seen thereafter in the 6 and 9 mg per kg groups.
The authors noted that the correlation between ven-
tilation and rectal temperature was considerably greater than the correlation
between ventilation and oxygen consumption. The relation between dinitro-
phenol and increased ventilation could only be explained by an indirect
action on ventilation by a primary change in temperature or by a direct
action on some unknown receptor which elicits an independent respiratory
response.
An earlier study by Harvey (1959) measured the effect
of 4,6-dinitro-j>-cresol on oxygen consumption in guinea pigs. Similar to the
case of dinitrophenol in dogs, dinitro-o-cresol produced a marked rise in
oxygen consumption from 6 to nearly 100 percent when doses of 5 to 20 mg
per kg were administered (Figure 53).
270
-------
15 30
45 1:00 1:15 1:30 1:45
Time ( hr)
2:00
30 45 1:00 1:15 1:30 1:45 2:00
Respiratory
Frequency
Group 0
Group 3
Group 6 -
Group 9
30 45 1:00 1:15 1:30 1:45 2:00
Rectal
Temperature
Group 0
Group 3
Group 6
Group 9
30 45 1:00 1:15 1:30 1:45 2:00
Figure 52., Response to Intravenous Dinitrophenol Injection in Dogs*
(Cardus and Hoff, 1963)
271
-------
r
1100
>>
•o
o
.o
I0
5 10 15 20
DNC mg./kg.
Figure 53. Oxygen Consumption of Guinea Pigs — Average Weight of Group
1500 g, in Response to Varied Doses of DNOC Given Intraperitoneally
(Harvey, 1959)
(Measurements taken 1.0-1.25 hours after injection. Line by obser-
vation. )
(Reprinted with permission from the Pharmaceutical Society of
Great Britain.)
The nature of dinitrophenol-induced changes in
thyroid function has been the subject of several investigations. Early
studies indicated that dinitrophenol depressed the uptake of iodine into the
thyroid (Goldberg £t ajL. , 1955; Freinkel and Ingbar, 1955) and lowered the
plasma protein-bound iodine (Wolf f e£ al_. , 1950). Dinitrophenol-induced
changes in pituitary histology have also been reported (Goldberg e_t al.,
1957).
272
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In a study where human volunteers were each fed
225 mg of dinitrophenol per day for two consecutive days, a significant de-
crease in protein-bound iodine and a rise in the metabolic rate were observed
(Castor and Beierwaltes, 1956). Changes were not seen either on thyroidal
I uptake and release, or on the excretion of I in urine and feces. The
authors postulated that the mechanism of dinitrophenol action in lowering
protein-bound iodine was due to an increased utilization of the thyroid
hormone.
Later studies (DeFelice and Rupp, 1963; Reichlin, 1960)
indicated that altered thyroid function by dinitrophenol may be due to an inhi-
bition of the production or release of thyroid-stimulating hormone in the
pituitary. Maayan (1968) demonstrated that the thyroidal growth response to
thyroid-stimulating hormone was blocked by dinitrophenol and accompanied by a
131
decreased uptake of I into the thyroid.
Most recently, England et al. (1973) have established
that dinitrophenol in fact increases thyroid output and thyroid hormone ex-
cretion in the bile, while at the same time lowering the plasma protein-bound
iodine. They suggested that the mechanism of action for dinitrophenol on
thyroid hormone metabolism may be a competition for thyroid hormone binding
sites on plasma proteins leading to a fall in protein-bound iodine concentra-
tion. Consequently, newly formed thyroid hormone would be displaced from the
plasma and wasted by excretion in the bile. This wastage would serve to in-
crease demand via the pituitary feedback system for thyroid hormone. As a
result, thyroid-stimulating hormone secretion would rise, and subsequently
thyroid hormone output would be increased.
273
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(ii) Neurologic Effects
The mode of action of nitroaromatic compounds on
nervous transmission is not entirely clear at this point. It has been reported
(Takagi and Takayanagi, 1965) that picric acid, 2,4-dinitrophenol, and all
nitro-derivatives of benzoic acid induce the contraction of guinea pig small
intestine by increasing the liberation of acetylcholine from cholinergic
nerve endings. The mononitrophenol derivatives, on the other hand, had very
low activity, and, in fact, inhibited the acetylcholine liberation induced
by the active nitroaromatics. The concentrations of the compounds tested
that produce contractions of smooth muscle of guinea pig small intestine
are shown in Table 77. When applied to the rectus abdominus muscle of the
frog, £-nitrophenol induced contraction by the liberation of acetylcholine.
This finding suggested that the nerve ending of guinea pig small intestine
behaved differently than that of skeletal muscle in the frog.
Studies were designed by Beani e£ al. (1966) to
determine the effect of dinitrophenol in vitro on the neuromuscular junction
of rat and guinea pig phrenic nerve-diaphragms. Their results indicated that
dinitrophenol reduced acetylcholine release from electrically-stimulated
diaphragms. Reduction of acetylcholine output corresponded with the depletion
of tissue stores, thereby indicating a possible exhaustion of acetylcholine
available for release (Table 78).
In studying the actions of dinitrophenol on neurons
of the cerebral cortex, Godfraind et^ al_. (1970) discounted the hypothesis of
a primary metabolic action of acetylcholine. They found instead that dinitro-
phenol causes a profound decrease in electrical excitability associated with a
274
-------
Table 77. Effective Concentration of Nitrocompounds Tested on the Small
Intestine of Guinea Pig (Takagi and Takayanagi, 1965)
Compound
Effective concentration (g/ml)
NO,
NO,
OH
NO.
z ^^ 2
OH
NO,,
•NO,
5 X 10 6 — 5X105
NO,
OH NQ OH
NO,
NO,
COOH
NO,,
NO,
COOH
NO,,
COOH
3 X 10"5 — 3 X 10
C1)
5X106—5X105
ID'5 -
10~4 — 10~3
( ) Their intrinsic activity is low or zero, so they acted as an antagonist of
the agonists listed in this table.
275
-------
Table 78. Effect of 2,4-Dinitrophenol Upon Total Acetylcholine Content of
Guinea Pig Hemi-Diaphragms (From Beani e_t al., 1966)
Measurements were made at the end of the fourth period of stimu-
lation at 6/sec, lasting 10 min; temperature 33°C, dyflos 500 ug/ml
pretreatment. The % reduction of acetylcholine release during
stimulation at 6/sec is given for comparison.
*; significantly different (0.05 > P > 0.02) from the control group.
**; significantly different (P < 0.001) from the control group.
Hemi- Acetylcholine
DNP diaphragm ng/hemi- Acetylcholine
ncentration No. Expts. weight + S.D. diaphragms + S.D. stores %
(M)
16 274.0 + 29.0 122.3 + 28.0 100
3 X 10~6 10 229.0+38.0 103.1+15.6 91.8
3 X 10~5 10 255.0+27.0 87.3+24.0* 77.7
3 y 10~5 12 276.0 + 38.0 71.4 + 12.8** 63.5
Acetylcho
release
100
92.7
64.4
41.2
tendency to hyperpolarization and an increased permeability of the membrane to
K+. Similarly, Dimov et^ al. (1972) related the suppressive effect of dinitro-
phenol on cortical bioelectric activity to its penetration into the nervous
cells. They found that dinitrophenol disrupted the development of an epilep-
togenic focus in the brain of cats and suggested that a certain "critical"
level of ATP must be available for the induction of paroxysmal activity.
d. Organoleptic Properties
Studies from the Russian literature have been encountered
which reported a threshold concentration for odor, taste, and color of several
nitroaromatic compounds in reservoir waters. Makhinya (1964) reported that
the threshold concentration of o-nitrophenol was 3.83 mg/£.for odor, 8.6 mg/fc
276
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for taste, and 0.6 mg/Jl for color. Concentrations of j>-nitrophenol were
58.3, 43.4, and 0.24 mg/£ for odor, taste, and color, respectively. The
values for m-nitrophenol were given as 389, 164.5, and 26.3 mg/£, respectively.
In a later report (Makhinya, 1967) threshold limit concentrations for m-
nitrophenol were given as 350.3 mg/& for odor, 144.8 mg/£ for taste, and
26.3 mg/£ for discoloration of aqueous solutions.
In a similar study by Kosachevskaya (1967), the ortho-,
met a-, and para-nitrotoluene isomers imparted a bitter taste and an odor of
bitter almonds to water at concentrations of 0.01 to 0.2 mg/£. The meta- and
para-nitrotoluene isomers had the greatest effect upon taste (threshold value =
0.01 mg/£); the limit value for ortho-nitrotoluene was 0.05 mg/£.
C. Toxicity - Humans
There is little doubt concerning the potential for adverse human
reaction when dealing with the nitroaromatic compounds. The danger from
human exposure to these substances, even in small amounts, can range from the
production of a mild and transient episode of cyanosis to a dramatic and sudden
collapse culminating shortly in death. While it appears that a threshold
limit of exposure exists, below which no toxic effects are manifested, chronic
exposure to subacute doses can ultimately produce severe and irreversible
physiologic damage. The greatest danger of poisoning generally exists among
those persons who are involved in manufacture or who directly handle and use
these compounds on a large scale.
1 Frequently, careless work habits or insufficient protection from
chemical dusts and fumes have led to incidents of serious intoxication. A
major factor in determining the severity and permanent damage resulting from
277
-------
nitroaromatic exposure has been the failure of the person exposed to recognize
the symptoms of a toxic reaction and to act immediately in seeking medical
care. Although the signs of poisoning can be quite varied for the different
nitroaromatic compounds, the potential for serious illness is sufficient to
warrant the recommendation of special monitoring measures in several indus-
tries. It is of primary importance in the treatment of occupational poison-
ing that the patient be removed from the workplace and that all contaminated
clothing, including shoes and underwear, be removed immediately and the patient
bathed. Absorption via the skin from contaminated clothing has often been
found to result in chronic intoxication and relapses into acute poisoning.
The dangers of the nitroaromatic compounds have been commonly referred
to since about the beginning of this century, and many texts dealing with toxic
materials have devoted considerable discussion to their effect upon humans
(Hamilton and Hardy, 1974; Moeschlin, 1965; Hunter, 1969; Arena, 1974). How-
ever, despite the fact that several hundred nitroaromatic compounds are pro-
duced commercially, documented reports of poisoning and assessment of health
hazard potential have been limited to a relatively small number of these chem-
icals. Substances which have received the greatest amount of attention in the
literature, due to their toxic nature and long history of varied use, include
nitrobenzene, dinitrobenzenes, chloronitrobenzenes, nitrotoluenes, dinitrotoluenes,
trinitrotoluenes, nitroanilines, nitrophenols, dinitrophenols, dinitrocresols , and
tetryl.
The major toxic symptom from exposure to the above compounds, ex-
cepting the nitrophenol derivatives, is undoubtedly cyanosis and the production
278
-------
of methemoglobin. This response is often the only manifestation of exposure,
and in most cases is rapidly reversible without specific treatment other than
temoval from exposure. Fatalities rarely occur by hematologic disturbance.
Les3 common but more severe reactions may include liver damage, severe anemia,
bone marrow changes, renal failure, and acute dermatitis. Dinitrophenol and
dinitrocresol, on the other hand, can produce severe metabolic disturbances
and death by respiratory paralysis.
A characteristic observation among cases of occupational poisoning
is that the onset of acute symptoms often occurs several weeks or months after
the last exposure to the substance, or after many months of continuous exposure
on the job. The appearance of a toxic episode, however, can be sudden, ex-
tremely severe, and many times can end shortly in death. The ingestion of
alcoholic beverages has been related to the precipitation of many cases of
acute poisoning in persons who have handled nitrobenzene derivatives. This
phenomenon is presumably due to a sudden "washing-out" effect of ethanol on
these substances from fat deposits in the body where they are selectively
accumulated.
1. Occupational Studies
Occupational exposure to the nitroaromatic compounds has pro-
vided numerous cases of poisoning and death. Prior to the institution of
rigid industrial hygiene standards, fatalities due to nitroaromatic exposure
were common, as indicated in a summary of the early literature by Von Oettingen
(1941).
The outstanding manifestations of toxic exposure to most nitro-
aromatics are cyanosis, anemia, and the production of methemoglobin. The
279
-------
biochemical basis and mechanisms of methemoglobin formation have been dis-
cussed in Section III-B-4. A comprehensive medical surveillance program by
Linch (1972, 1974) resulted in the evaluation of 187 cyanosis cases, diagnosed
on the basis of laboratory findings, during the ten years from 1956 to 1966.
He found that acute exposure produces cyanosis and possible loss of hemoglobin,
while chronic subacute absorption may lead to reversible anemia. The relative
cyanogenic and anemiagenic potentials of many of the nitroaromatics are pre-
sented in Table 79. Increased environmental temperature, in addition to
chemical structure, was also shown to be a factor in cyanosis production
(Figure 54).
Table 79. The Chemical Cyanosis Anemia Syndrome-Hazards of the
Nitroaromatic Compounds (Linch, 1974)
Rank
1
2
3
4
5
6'
7
8
9 '
10
11
12
13
14
Cyanogenic Potential
d initrobenzene
jg-nitroaniline
j>-nitroaniline
nitroanilines
nitrobenzene
C-nitrochlorobenzene
£-nitrochlorobenzene
mixed -nit rochlorobenzene
j>-dinitrosobenzene
fl-nitrotoluene
£-nitrotoluene
mixed-nit rotoluene
d initrotoluenes
nitronaphthalene
Anemiagenic Potential
nitrobenzene
mixed-, _£-nitrochlorobenzene
mixed-nitrotoluene
d initrobenzene
nitroanilines
p-dinitrosobenzene
nitronaphthalene
1
Over-all Potential
d initrobenzene
nitrobenzene
mixed-, jj-nitrochlorobenzene
nitroanilines
mixed-nitrotoluene
£-dinitrosobenzene
nitronaphthalene
280
-------
90 n
I
60-
50-
40-
30-
20
T 1 1 i 1 i i r
4 6 8 10 12 14 16 18 20 22 24 26
Cyanosis Cases ( % of Total)
Figure 54. Effect of Temperature Upon Cyanosis Occurrence
(Linch, 1972)
An early attempt was made by Pasceri and Magos (1958) to
attach a diagnostic significance to quantitative differences in the levels of
methemoglobin, sulfhemoglobin, and Heinz bodies in the blood of chemical pro-
duction workers. Their investigation at eight industrial plants included air
contamination studies, medical examination of workers, and urine testing for
the presence of toxic chemicals and metabolites. The most common routes of
exposure to nitroaromatic chemicals were found to be (1) inhalation and (2)
skin contact resulting from hand-feeding and discharge of chemicals into
processing vessels and cleansing and repair of containers. The results of
biochemical determinations made on the blood of exposed workmen are presented
in Table 80.
281
-------
Table 80. Blood Examinations of Workmen in Aromatic Chemical Production Plants
(From Pasceri and Magos, 1958)
to
oo
to
Plant
Anlsidine
production
Phenacetine
production
Production of
•nitroaromatic
compounds
Compound
nitroanisole
anisidine
j>-nitrochloro~
benzene
nitrophenetol
phenetidine
nitrobenzene
£-nitroctiloro-
benzene
dinitrochlor-j-
benzene
nitroethyl-
benzene
period
VI. -XII.
1954
IX. -XII.
1954
VIII. -XII.
1954
"
of
Subjects
23
30
39
zation
some
cases
trans-
itory
some
cases
transitory
some
cases
transitory
Met hemoglobin
average stand, dev.
g. *
0.67
0.78
0.61
(+0.39)
(±0.37)
(+0.38)
Sulphhaemoglobin
average stand, dev.
g- *
0.32
0.20
0,27
(+0.18)
J
(+0.14)
(+0.14)
Heinz body
formation
(_V_-._ 1 »\
aDove XAJ
in percentage
of tests
43.0*
10.0*
2.8*
Individually
1-3Z, except
2 cases , in which
5.71
'Individually
1-3%
* Individually
1-3Z
-------
The authors established an average normal value for methemoglobin at 0.22 +
0.1A g% (upper limit: 0.50 g%) and for sulfhemoglobin at 0.08 + 0.05 g% (upper
limit: 0.18 g%). The upper normal limit for Heinz body formation was set at
10 per 1000 erythrocytes or one percent. By these standards, excessive ex-
posure to toxic chemicals had occurred in every plant studied. Among their
observations, it was noted that methemoglobin was short-lived in the blood,
being quickly reconverted to hemoglobin, and its presence, therefore, was In-
dicative of acute exposure of short duration. Sulfhemoglobin, on the other
hand, disappeared much more slowly from the blood and was formed in response
to subacute exposure levels below the threshold amount for subjective symptoms
of poisoning. Consequently, elevated Sulfhemoglobin levels were regarded as
good indicators of prolonged exposure to low concentrations of nitroaromatics.
Heinz body appearance in the erythrocytes generally seemed to be an early sign
of intoxication as well as low-level exposure, although their formation was
often a variable response. Increased Heinz body production paralleled the
development of toxic anemias accompanied by erythrocyte destruction, and was
claimed to precede many of the clinical signs of anemia, thereby making it a
sensitive indicator of exposure.
On the basis of their experiences in this study, the authors
established criteria for distinguishing mild, moderate, and severe exposure to
aromatic nitro and amino compounds.
Mild Exposure:
1. The contamination of air should not exceed the
permissible level.
283
-------
2. The erythrocyte count and hemoglobin level should
be normal.
3. The average methemoglobin value should not exceed
the upper limit of normal.
4. The average sulfhemoglobin level should not
exceed the upper limit of normal.
5. Heinz bodies should be absent.
6. Urinary excretion of p-aminophenol should be
less than 0.7 mg/100 ml.
Moderate Exposure:
1. Air contamination should exceed normal limits but
be less than the toxic level.
2. The erythrocyte count and hemoglobin level should
be normal.
3. The methemoglobin level should exceed the upper
limit of normal, but not be higher than 1.4-2.0 g%.
4. The sulfhemoglobin level should exceed the upper
limit of normal, but not be higher than 0.35-0.50 g%.
5. The number of Heinz bodies should exceed one percent,
Severe Exposure:
1. Air contamination should exceed the toxic limit.
2. Erythrocyte count and hemoglobin level should be
below normal, or anemia should be present.
3. Methemoglobin values should exceed 1.4-2.0 g%.
284
-------
4. Sulfhemoglobin values should exceed 0.35-0.50 g%.
5. The number of Heinz bodies should exceed one percent.
It has recently been proposed that the determination of un-
saturated iron-binding capacity (UIBC) in the blood can be a sensitive early
indicator of exposure to aromatic nitro and amino derivatives. Tarpa et al.
(1972) measured the UIBC in workers of the dye-stuff industry and noted that
exposed workers had UIBC values approximately 50% of the control subject
average (Table 81). This technique is far more sensitive, though not as
specific, as methemoglobin and sulfhemoglobin determinations as a measure of
exposure to toxic nitroaromatic derivatives. It seems that UIBC determinations
may be very useful as a part of medical surveillance and monitoring programs
in hazardous occupational situations.
Table 81. Mean Values of UIBC in Workers Exposed to Nitro- and
Amino-Aromatic Derivatives and in Control
(Tarpa et^ al., 1972)
Lot
Exposed to poisons
Control
' No. pers.
39
28
Sex
M F
• 31 8
18 10
UIBC (X+S)
131 + 35
246 + 34
Statistical
Significance
p<0.001
285
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a. Trinitrotoluene
By far the greatest number of poisoning cases related
directly to nitroaromatic chemical exposure has been among workers involved
with the production and handling of trinitrotoluene (TNT). The most serious
toxic effects from TNT absorption are liver damagej manifested by jaundice
which leads to acute yellow atrophy, and aplastic anemia (a persistent form
of anemia, generally unresponsive to therapy). Other major clinical symptoms
often include dermatitis, cyanosis, gastrointestinal disturbance, methemoglob-
inemia, and sulfhemoglobinemia.
A review of the early literature by Von Oettingen (1941)
described numerous studies on the incidence of adverse reactions to TNT among
industrial workers. He noted that over a 20 month period during World War I,
a single plant in the United States reported 7,000 cases of TNT poisoning with
105 fatalities; and another 7 1/2 month period produced 17,000 cases and 475
deaths, some of which were probably due to mixtures of toxic chemicals. Un-
protected TNT production workers are known to be very susceptible to intoxi-
cation, because TNT dust is readily absorbed through the skin, especially
when it is wet with perspiration.
Jaffe e_t^ al. (1973) have prepared an extensive literature
evaluation of TNT toxicity in which they noted that changes in the blood are
noted first with both acute and chronic exposures. Damage to the blood may
include reduction in the hemoglobin level and red cell count associated with
polychromasia (variation in hemoglobin content of the erythrocytes), poikilo-
cytosis (abnormally formed erythrocytes), anisocytosis (the presence of nu-
cleated erythrocytes), reticulocytosis (abnormal increase in reticulocyte
286
-------
number), and eosinophilia (abnormal increase in eosinophil number). Increases
are also observed in leukocyte and lymphocyte counts.
In a study of industrial workers by Soboleva (1969), ob-
servations were made of cataracts, cholecystitis (inflammation of the gall
bladder), hematological changes, neurasthenia (nervous exhaustion), poly-
neuritis (inflammation of many nerves at once), and hypotonic neurocirculatory
dystonia (loss of muscle tone). Gastrointestinal disorders resulting from
TNT poisoning were observed by Faerman (1957); they included hyperacidity in
those exposed for less than 10 years, and hypoacidity in those with exposures
from 10 to 30 years. Makienko and Karamanov (1973) examined the oral cavities
of TNT workers and found characteristic carious and non-carious tooth injury
and periodontal and oral cavity mucous membrane diseases. Hassman (197.1) noted
that alcohol ingestion affects the metabolism of TNT, causing reddening of the
face and cyanosis of the lips. He described the first symptoms of poisoning
as being toxic gastritis, reduced trypsin activity in the pancreatic secretion,
increased glomerular filtration, and hypo- or hypermenorrhea in women.
The early effects of exposure to TNT were described by
Stewart et^ al^. (1944) from a study involving 62 student volunteers working in
a filling factory. The major changes noted were in the blood picture, in
which over 80 percent of the students were affected. Hemoglobin was de-
creased by 10-15%, and a mild anemia was observed. Increased levels of
reticulocytes became quite pronounced after the worker had left contact with
TNT (Table 82, Figure 55). This was probably a compensatory phenomenon in-
1
dicative of a hematologic repair process which had been inhibited by the TNT.
287
-------
Table 82. Last Reticulocyte Count at Factory Compared with Value on
Return to Oxford Approximately 48 Hours Later
(Stewart et al., 1944)
Group
44 Females (I)
10 Males (II)
8 Males (III)
Reticulocytes
Average number per c.mm. blood (X 10 )
Before
exposure
3.8
3.6
4.3
Last count
at factory
7.5
8.1
15.0
Two to four days
after cessation
of contact
9.3
12.6
27.1
60-1
CO
a
Reticulocytes
During Exposure
Figure 55. Total Reticulocyte Count During and After TNT Exposure
(Stewart et al. , 1944)
288
-------
Clearly, the early effects of TNT exposure involve hemolysis of red blood cells
and interference with hematopoiesis. In addition, an erythroblastic reaction
in the bone marrow indicates further attempts to compensate for increased de-
struction of red blood cells. It was found, however, that only after the sub-
ject was removed from TNT exposure could the marrow exert full repair activity
by producing increased reticulocytes (immature red blood cells). This apparent
depression of bone marrow function may account for the cases of severe aplastic
anemia which have frequently been observed among TNT workers.
An occupational study conducted by McConnell and Flinn
(1946) reported on the cases of 22 fatalities which occurred in government-
owned ordnance plants during the period from 1941 to 1945. Of the 22 deaths
reported, eight died of toxic hepatitis; 13, of aplastic anemia; and 1 died
from a combination of both. While the main route of absorption was through
the skin, the lungs and gastrointestinal tract were also considered to be im-
portant. The authors pointed out that only a third of those who died were
exposed to excessive air concentrations of TNT, and that individual differences
such as nutritional state and other illness could alter susceptibility. The
influence of age on susceptibility was reflected by the fact that hepatitis
occurred more frequently among the younger age group (average age - 30 years)
and aplastic anemia among the older age group (average age - 45 years). The
median period of exposure to TNT for the hepatitis group was 63 days, as com-
pared to 216 days for the aplastic anemia cases. Diagnosis of TNT poisoning
in the early stages was said to be difficult, and clinical signs were often
not evident until severe damage had occurred. Among those with toxic hepatitis,
the signs of acute toxemia, including deep jaundice and stupor, developed rapidly
289
-------
and were followed shortly by death (median time - 34 days). In the anemia cases,
a rapid reduction in erythrocyte count and hemoglobin level signaled severe bone
marrow damage, and death shortly ensued (median time - 40 days). Pathological
examination of the liver in hepatitis victims revealed degeneration and great
reduction in size and weight, with advanced atrophy in the involved areas
accompanied by destruction of all parenchymal cells. The pathology of aplastic
anemia appeared to be independent of hepatitis and was characterized by multiple
petechiae (spots caused by hemorrhage) and diffuse hemorrhage of all organs,
including skin and membranes.
Several reports have been made concerning the development
of cataracts following occupational exposure to TNT (Soboleva, 1969, Logan et
al. , 1970). Zakharova and Manoilova (1971) studied the cases of 360 persons
exposed to small doses of TNT for a period of at least five years. They found
that 45.3 percent of the group developed a single cataract which appeared as
the only sign of TNT poisoning.
A case of peripheral neuropathy (disturbance of the peri-
pheral nervous system) and vasculitis (inflammation of the vascular system) in
a person sensitive to dynamite (nitroglycerine and TNT) has been reported by
Jacob and Maroun (1969). They described the salient neurological symptoms
which can accompany sensitivity to organic nitrates, including severe headache
due to vasodilation, behavioral disturbance, muscle weakness, and the precipi-
tation of acute violent mental disturbance upon the ingestion of alcohol. Among
persons with a strong sensitivity to organic nitrates, contact with clothing,
or even shaking the hand of a person who was exposed to these chemicals, could
produce clinical symptoms.
290
-------
In a study conducted in 1952 at Lone Star Ordnance Plant
in Texas (Goodwin, 1972), TNT dust and fume concentrations were found in most
3
cases to exceed the established threshold limit value of 1.5 mg/m of air, and
ranged as high as 9.5 mg/m . Results of this study were used to justify the
installation of modern processing equipment, and a reexamination of the situ-
ation since 1952 demonstrated that no fatalities or cases of permanent liver
damage had occurred. Pre-employment examinations, routine testing for liver
impairment, and transferral of persons with sensitivity to TNT are also credited
with reducing the incidence of TNT poisoning.
A report has recently been abstracted from the foreign
literature concerning the effects of chronic poisoning by nitro-containing
toluene compounds among 130 persons (Makotchenko, 1974). The major finding
was that a severe disturbance of adrenal cortex function had taken place. This
apparent loss of adrenal cortical hormone activity was manifested by such
symptoms as polyneuritis, gastric secretory disorders and loss of muscular
strength and energy.
b. Tetryl
Another nitroaromatic munitions compound of toxicological
significance, which has been produced in large quantities during wartime, is
tetryl (N-methyl-N-nitro-2,4,6-trinitroaniline). An account has been presented
by Hardy and Maloof (1950) of the cases of munitions factory workers during the
period from 1941 to 1945 who became ill or died from tetryl exposure. Yellowish
discoloration of the skin and hair affected nearly all workers, and tetryl-
induced dermatitis accounted for a high turnover rate in many high-exposure jobs.
Those who became seriously ill from tetryl intoxication usually displayed
291
-------
asthma-like attacks of wheezing and violent coughing, accompanied by anorexia
(loss of appetite), loss of weight, and degenerative changes of the liver. It
wfls frequently noted that users of alcoholic beverages were much more susceptible
to tetryl poisoning. While as many as 34 percent of exposed workers were
reported to develop local effects such as dermatitis and irritation of the upper
respiratory tract mucous membranes, many investigators noted pronounced systemic
toxicity as well. Removal of the patient from the workplace containing tetryl
did not always lead to remission of symptoms, and chronic debilitation may persist
for years, eventually resulting in death.
Qualitatively, the clinical picture of tetryl intoxication
is very similar to that of TNT. The insidious course of events resulting from
chronic exposure generally leads to irreversible damage of the liver and other
organs, producing permanent disability or death. Clearly, the need for measures
to control worker exposure is crucial in the case of munitions compounds.
c. Dinitrobenzene
Dinitrobenzene is generally regarded as the most potent
methemoglobin-forming agent of all the nitroaromatics. This characteristic
property is beneficial in the sense that intense cyanosis will occur with only
slight exposure and thereby serve as a warning of toxic absorption before severe
tissue damage can take place. This, unfortunately, is not the case with many
nitroaromatic compounds such as TNT, tetryl, or the nitrophenol derivatives.
It should be noted, however, that a review of the early literature (Von Oettingen,
1941) showed that 30 fatalities due to dinitrobenzene were recorded, 20 from
292
-------
acute exposures and 10 from delayed effects. The case reports indicated a
characteristic acute or subacute atrophy of the liver.
Industrial dinitrobenzene poisoning was a common occurrence
during both World Wars, when the chemical was manufactured as a constituent of
the explosive roburite (Von Oettingen, 1941). Several cases of poisoning by
m-dinitrobenzene were observed by Rejsek (1947), who noted that acute symptoms
appeared first as headache, pressure on the chest, general malaise, nausea, and
vomiting. Cyanosis soon became manifest and, in severe cases, liver function
was impaired, or atrophy developed. The case of a man was reported who had been
employed as a munitions worker handling m-dinitrobenzene and after six months
suddenly became cyanotic and was sent home. Four weeks after his symptoms had
disappeared, the man drank a glass of beer and within three hours became vio-
lently ill with nausea, vomiting, headache, and blue coloration. He was placed
in a hospital and more than one week later, when all symptoms had disappeared,
the patient was given a pint of beer (2% alcohol content). Within two hours,
the man became intensely cyanotic and shortly thereafter developed an extreme
episode of vomiting which persisted all day. One week later, the patient
discovered that while sunbathing for an hour his lips became cyanotic and he
developed a severe headache. Several other patients were subsequently seen
who had been exposed to m-dinitrobenzene and similarly developed a serious
relapse of acute symptoms upon exposure to sunlight or ingestion of alcohol.
It was remarkable to note that, even six weeks after the disappearance of toxic
symptoms, a complete relapse could occur.
Rejsek (1947) made the observation that the course of
poisoning could be affected by the patient's diet and genetic make-up.
293
-------
Beritic (1956), as well, pointed out that patients display a marked difference
in their response to dinitrobenzene, although they may have worked under iden-
tical conditions. He presented the cases of two women aged 22 and 25 years
who were employed in the manufacture of dinitrobenzene under very similar
working conditions. Both women developed classical symptoms of poisoning in-
cluding headache, fatigue, nausea, and cyanosis. Their clinical pictures, how-
ever, were quite different, with one woman developing methemoglobinemia,
moderate anemia, enlargement of the liver, and no Heinz bodies; the other woman
displayed anemia with Heinz bodies and no evidence of liver damage. It is
by no means clear why individuals vary in their response to nitroaromatic
exposure, even though it has been a common observation for many years. The
explanation most probably involves a complex interaction of factors which
apparently includes genetic make-up, nutritional state, body fat stores, and
general state of health governing the detoxification mechanisms of the body.
d. Nitrochlorobenzene
The effects of exposure to nitrochlorobenzene are similar
in many respects to dinitrobenzene and nitrobenzene poisoning. Cases of
occupational nitrochlorobenzene poisoning first began to appear at the be-
ginning of the 20th century. Reports clearly described the symptoms of nausea,
vomiting, cyanosis, shortness of breath on exertion, and mild anemia.
The most recent report of professional intoxication was
made by Saita and Moreo (1958), who presented the case of a 25 year old worker
accidentally poisoned by inhalation of ortho- and para-nitrochlorobenzene
vapors. The symptoms which he displayed were primarily related to toxic effects
294
-------
on the blood. These included cyanosis, methemoglobinemia, Heinz body forma-
tion, persistent sulfhemoglobinemia, and hemolytic anemia. Figure 56 details
the progression of the characteristic nitroaromatic-induced hematologic changes
in this case and illustrates the increases in reticulocytes and protoporphyrins
which are known to occur during the reparative stages of acute hemolytic anemia.
a
o
£ X
lOO-i 40-i
35-
30
25
*
80-
60-
40-
20-
15
10
5
-i 90 -»
10-
8-
6-
4-
2-
20 0J 0J 60
80-
70-
24/X
>Hb
Protop.
1/XI
10/XI 14/XI
Days
Figure 56. Hematologic Effects From Nitrochlorobenzene Poisoning*
(Saita and Moreo, 1958)
* Symbols: Protop. = protoporphyrins; H.B. = Heinz bodies;
SHb = sulfhemoglobin; Ret. = reticulocytes; Hb = hemoglobin.
295
-------
e. l-Chloro-2,4-dinitrobenzene (DNCB)
The action of DNCB as a potent skin sensitizer is well
documented (see Section III-B-4-b), although few cases of occupational ex-
posure have been encountered. A report of four cases of contact dermatitis
directly attributable to DNCB exposure has been made by Adams et^ al^ (1971).
Four persons employed as air-conditioning repairmen became sensitized to DNCB
through its use as an algicide in coolant water.
In one case, the patient developed a severe blistering
dermatitis of the left arm shortly after spilling a large quantity of an
algicide containing 22 percent DNCB on his skin and clothing. The man had
previously experienced many episodes of dermatitis following incidents where
he had been splattered with the algicide. Another worker developed a severe
dermatitis of the right face, neck, right arm, chest, abdomen, and both thighs
after an accident in which he was sprayed with the concentrated algicide. Two
weeks later, slight contact with water containing the algicide at a concen-
tration of one part per million produced a pruritic (itching) blistering der-
matitis of the right hand.
The authors commented that, even though the last previous
report of occupational exposure to DNCB occurred in 1928, the compound is
nevertheless one of the strongest primary skin irritants known and should be
handled with extreme caution. Exposure to DNCB, even in minute concentrations,
will almost certainly lead to allergic sensitization. Reactions from the
slightest further contact can range from a few pruritic papules or papulo-
vesicles to a widespread exfoliative dermatitis.
296
-------
Other Skin Sensitizers (see Section III-B-4-b)
Several additional incidents of skin irritation and
allergic contact sensitization in man have been reported involving nitroaromatic
derivatives.
In one case, a young man working with poultry feed
developed a chronic eczema of both hands which improved only during holidays
away from work (Bleumink and Nater, 1973). Patch testing with various feed
additive substances revealed that a positive allergic reaction was elicited
only by 3,5-dinitrotoluamide (Dinitolmide), an anti-coccidial chicken feed
additive.
A rare case of contact dermatitis from carbon paper
was reported by Calnan and Connor (1972). A woman developed seborrheic der-
matitis of the nose, eyebrows, forehead, ears, hands, and fingers after handling
a special carbon paper containing nigrosine. Nigrosine is a complex mixture
containing either nitrobenzene, nitrophenol, or nitrocresols. Patch testing of
the individual components of the carbon paper demonstrated that nigrosine alone
was responsible for the skin reaction.
In a controlled human study, Finnegan e.t al. (1958)
tested 50 subjects for sensitivity to pentachloronitrobenzene (PCNB). Thirteen
individuals developed positive skin irritation reactions from exposure to PCNB,
when patches were applied after an initial sensitizing application two weeks
earlier. Four of the persons developed delayed hypersensitivity reactions
ranging from eight hours to several days after removal of the PCNB-containing
patch from the skin.
297
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f. 2,4-Dinitrophenol
The early history of occupational dinitrophenol poisoning
was mainly related to its use in the manufacture of explosives during World
War I. The toxic manifestations of dinitrophenol exposure, as reviewed by
Horner (1942), included subacute symptoms such as gastrointestinal disturbances
(anorexia, nausea, vomiting, colic diarrhea), loss of weight, night sweating,
weakness, headache, and dizziness. Acute poisoning resulted in the sudden
onset of pallor, burning thirst, profuse sweating, agitation, dyspnea (dif-
ficult breathing), and a moderate elevation of temperature. Although prompt
removal from the workplace usually brought relief, the exposed alcoholic or
person with renal or hepatic disease might die within a few hours. The ad-
verse effects in humans caused by dinitrophenol can be closely linked to its
characteristic ability to uncouple oxidative phosphorylation (see Section III-
B-4-c).
A review of the early literature by Von Oettingen (1941)
revealed that 27 cases of fatal occupational dinitrophenol poisoning had been
reported for the years 1914 to 1916; 17 of which occurred in weighing and
melting operations, 8 in extraction, and one each in the warming and finishing
operations. During the year 1916 to 1917, 31 fatalities were recorded in France
and five more in the following 12 months.
Gisclard and Woodward (1946) reported two cases of fatal
exposure in workers involved with the manufacture of picric acid from dinitro-
phenol. The men handled open barrels into which was dumped dinitrophenol, which
generated considerable dust and fumes. In both cases, symptoms of fever, pro-
fuse sweating, and restlessness became evident after a few months of exposure.
298
-------
The men were returned to their jobs after palliative treatment and rest,
whereupon one worker collapsed and died within four hours after admission to
the hospital. One week later, the second man died in a similar fashion. Lab-
oratory investigation of their internal organs revealed a high degree of di-
nitrophenol absorption.
Several reports of toxic exposure have been summarized by
Saita (1949) involving workmen who impregnated wooden posts with a parasiticide
powder of dinitrophenol. These cases produced hematologic alterations including
hemolytic anemia in one worker, and neutropenia (decreased number of neutro-
philic leukocytes) and eosinophilia (increased number of eosinophils) in
others.
g. 4,6-Dinitro-ortho-cresol (DNOC)
The methyl derivative of dinitrophenol, DNOC, has been
widely used as a herbicide and anti-parasite, and has accounted for numerous
cases of poisoning and death among agricultural workers. The major hazard to
workers occurs during the mixing and application processes, when DNOC is sprayed
on cereal crops and orchard trees. The microscopic droplets, or dust, which are
formed by spray nozzles create a mist which is easily inhaled and has caused
many cases of poisoning.
The qualitative physiologic actions of DNOC in man are very
similar to those produced by dinitrophenol. Clinical symptoms which are ob-
served in acute poisoning cases (DiBosco, 1970) include fatigue, intense thirst,
profuse sweating causing dehydration, fever, heart failure, dyspnea, nausea,
vomiting, and abdominal pain. The skin becomes flushed and often dyed yellow
where exposure is greatest (hands, face, feet, knees), and the basal metabolism
299
-------
rate can rise to as high as 400 percent of normal. Additional symptoms can
be glucosuria (high glucose levels in the urine), hyperglycemia resulting from
the mobilization of carbohydrate reserves, and disturbances of the cardiac
rhythm. Actions on the nervous system may cause spasms (convulsive and vocal),
delirium, and coma. Death will often occur by respiratory failure resulting
from pulmonary edema. Subacute intoxication may become manifest as loss of
weight, headache, fatigue, anorexia, increased metabolic rate, dyspnea, and
abdominal pain. The characteristic yellow staining of the skin, hair, and
urine by DNOC is increased by repeated subacute exposures. Chronic exposure
to DNOC will produce mainly an increase in the metabolic rate, loss of weight,
fatigue, anxiety, and degeneration of the liver, kidneys, and heart.
A report summarizing eight fatal cases of DNOC poisoning
in Great Britain was presented by Bidstrup and Payne (1951). The authors noted
that, in nearly all cases, the early symptoms of poisoning such as excessive
sweating, unusual thirst, and loss of weight had been ignored by the patient
until he became acutely ill with symptoms of increased respiration, fever, and
tachycardia (increased heart rate). These acute symptoms were always followed
shortly by coma and death. They observed that among the cases of fatal poisoning
by DNOC reported throughout the world, many have occurred during unusually hot
weather. The relationship between high environmental temperature and the in-
cidence of poisoning may be partly due to the fact that workers remove pro-
tective clothing and thereby expose a greater skin surface for possible exposure.
Controlled studies in animals, however, have confirmed an increased suscepti-
bility with higher temperatures (see Section III-D).
300
-------
Bidstrup and Payne also reported a personal communication
on three cases of severe intoxication among men engaged in DNOC manufacture.
One patient who had worked with DNOC powder, pouring it from kegs into a
grinding mill, was employed only 17 days before becoming severely ill. His skin
was stained yellow within a few days of beginning work, but he remained on the
job until two days before his admission to the hospital. His most striking
symptom was profuse sweating which was described by the notation that "the
suprasternal notch filled with sweat as one stood and watched."
Two cases of polyneuritis, a condition known to occur with
other nitroaromatics, were observed by Stott (1956) in workers exposed to DNOC.
Both men were employed in servicing the spray equipment of aircraft used to
spray a 20 percent solution of DNOC in oil. It was determined that the major
route of DNOC absorption had occurred through the skin. The men presented
symptoms of a pins-and-needles sensation on the hands and fingers, accompanied
by partial loss of sensation to pin pricks on the hands and feet of one man.
The author postulated that a high local concentration of DNOC in the skin due
to prolonged contact with the oil solution might exert a local tissue effect
before more general symptoms could occur. This hypothesis seems reasonable in
light of the finding that localized symptoms and yellow staining occurred at the
areas of highest exposure, i.e., hands and feet.
h. Other Nitrophenol Derivatives
Studies were conducted in Washington State from 1956 to
1959 on the health hazards associated with the use of the triethanolamine and
isopropanolamine salts of 2-sec-butyl-4,6-dinitrophenol (dinoseb) as a weed-
control spray and the sodium salt of DNOC (Na-DNOC) as a blossom-thinning spray
301
-------
on tree fruits (Wolfe e_t^ al., 1961). Dermal exposure of spray-equipment
operators was determined by exposing absorbent cellulose pads and items of
cotton clothing on various parts of their bodies. Hands were rinsed with water
in a polyethylene bag to measure skin contamination. Results of the dermal
exposure studies are presented in Table 83. Individual variations were noted
among workers due to clothing (short-sleeved shirts, open collar, no hat or
protective gloves) and also where workers had accidentally spilled concentrated
material on themselves. The difference between careful and careless workmen
was illustrated by comparing the hand exposure to dinoseb where neither man
wore gloves. One worker had an exposure of 95.5 mg per hour, whereas the more
careful man had an exposure of 29.6 mg per hour. The value of wearing pro-
tective gloves was determined by noting an exposure of 22.4 mg per hour when
gloves were worn, and 91.1 mg per hour when they were not.
Determination of respiratory exposure revealed an average
inhalation potential of 0.12 mg per hour for dinoseb and 0.03 mg per hour for
Na-DNOC. The difference in the two values was probably due to the fact that
dinoseb was usually applied at the average rate of five pounds per acre, while
Na-DNOC was applied at about 1.3 pounds per acre.
A calculated estimate of the total potential daily exposure
for dinoseb was about 4.6 percent of the toxic level, and for Na-DNOC was about
0.9 percent of the toxic level. These figures compare to 43.2 percent of the
toxic level.for a sprayman using parathion under similar conditions.
A recent report by DiBosco (1970) revealed that 2,298 cases
of poisoning among agricultural workers in Italy were reported during the six-
year period from 1964 to 1969. Of these cases, four were attributed to
302
-------
Table 83. Dermal Exposure of Spraymen to DINOSEB and Na-DNOC (Wolfe et^ a^., 1961)
DINOSEB
A
Body part
Face
Front of neck; "V" of chest
Back of neck
Forearms
Total
Less hands
Thighs
Legs
Above ankles
Total
Pad Location
or
Area Rinsed
Shoulders
"V" of chest
Back of neck
Forearms
Hands (DNOSBP)
Forearms (NA-DNOC)
—
—
Thighs
Legs
Above ankles
—
Body
Area
(Sq. Ft.)
0.70
0.16
0.12
1.30
0.87
3.15
2.28
3.75
2.50
—
—
' No. Pads
or
Rinses
Analyzed
62
33
32
66
48
241
193
38
30
27
95
Toxicant
Recovery
(Mg/Sq.
Ft/Hr)
1
3
1
7
.6
.4
.0
.5
*
_
-
31
4
1
-
_
-
.7
.9
.5
-
Calculated
Exposure
(Mg/Body
Part/Hr.)
1.
0.
0.
9.
77.
88.
11.
118.
12.
—
131.
1
5
1
7
3
7
4
9
2
1
No. Pads
Analyzed
62
32
33
63
63
190
190
64
30
17
111
Na-DNOC
A
Toxicant
Recovery
(Mg/Sq.
Ft/Hr)
7
2
5
8
8
_
-
8
3
0
.0
.3
.6
.5
.5
„
-
.1
.7
.3
Calculated
Exposure
(Mg/Body
Part/Hr.)
4.9
0.4
0.7
11.0
7.4
24.4
17.0
30.4
9.2
.
39.6
*Bag rinse measurement for hand area.
-------
dinitrophenol derivatives, and one of these resulted in death. Two of the four
incidents resulted from exposure to 2-sec-butyl-4,6-dinitrophenyl-3-methyl-
crotonate (Binapacryl). The classical signs of dinitrophenol derivative
poisoning were observed, most notably headache, nausea, dyspnea, and profuse
sweating. The third poisoning case was caused by 2-capryl-4,6-dinitrophenyl
crotonate (Karathane), which caused an acute allergic dermatitis accompanied by
difficult respiration, thirst, and fever. The case which resulted in death in-
volved a man who had mixed an antiparasitic solution of 50 percent DNOC in
water. He became ill within 10 hours with vomiting, profuse sweating, muscular
tremors, and cerebral spasms. A progressive myocardial infarction and pulmonary
failure led to his death. Upon autopsy it was revealed that the man had ex-
tensive degenerative lesions of the heart, liver, kidneys and small intestine,
which were probably due in part to previous exposures to dinitrophenol compounds.
A generalized clinical picture of poisoning by dinitrophenol
derivatives was described by the above authors. Beginning with the uncoupling
of oxidative phosphorylation, metabolic processes are intensified, and energy
is produced and dissipated in the form of heat. An increase is seen in the
passive phase of protein metabolism, with massive mobilization of glucose and
fat reserves. Increased frequency of respiration and elevated oxygen consumption
are noted, along with the development of acidosis, which may cause tachycardia.
If death does not ensue quickly, degeneration of the liver, kidneys, and heart
usually takes place. Accumulation of dinitrophenol derivatives, particularly
DNOC, was shown to occur in man, which is in contrast to animal models where
cumulative effects were not conclusively demonstrated (see Section III-D).
304
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i. Nitroanilines
Of the three isomers of nitroaniline, para-nitroaniline is
regarded as the most toxic. Symptoms of £-nitroaniline poisoning are consistent
with the known effects of nitrobenzene derivatives in general. These include
primarily headache, restricted breathing, nausea, vomiting, and intense cyanosis
with violet coloration of the ears, lips, nose, tongue, fingers, and toes.
Methemoglobinemia is the main pathologic finding, which usually subsides spon-
taneously within a short time after removal from exposure.
A report of an incident of acute p_-nitroaniline poisoning
on a cargo ship was made by Anderson (1946). Seven dock laborers became ill
after sweeping up the contents of several kegs of £-nitroaniline which had been
broken in the ship's hold. One of the men became unconscious after the day's
work; he was admitted to the hospital and found to have enlargement of both
the liver and spleen, probably due to a previous malarial hepatitis and spleen-
itis. He was conscious by the following morning but had developed toxic jaundice
with fever, tachycardia, albuminuria (albumin in the urine) and hematuria (blood
in the urine). The patient eventually died 50 hours after his first exposure to
the _p_-nitroaniline. The diminished liver and spleen function, due to previous
illness, probably led to the development of severe jaundice and death because he
was unable to metabolize the poison.
2. Non-Occupational Exposures
The non-occupational exposure of humans to nitroardmatic compounds
has produced several important illustrations of their varied and potentially
lethal action. Incidents of fatal poisoning and severe intoxication have re-
sulted from both accidental and suicidal ingestion, as well as by administration
305
-------
as a drug in the case of dinitrophenol and DNOC. In the past, many individuals
were exposed to commercial products containing potentially harmful concentrations
of nitroaromatic ingredients. These cases have pointed out the necessity for
manufacturers to carefully consider not only the intended use of a product but
also the consequences of exposure resulting from misuse or accidental ingestion.
Failure to do so has led to considerable human suffering as well as criminal
charges against commercial producers.
a. 2,4-Dinitrophenol and Derivatives
One of the most striking examples of a highly specific
toxic effect due to chemical exposure was seen when an outbreak of cataracts
in young women occurred in an epidemic fashion during the mid-1930's. The
etiology of these incidents clearly indicated a common cause, the administration
of dinitrophenol as an anti-obesity agent. A comprehensive review by Horner
(1942) pointed out that dinitrophenol was received with overwhelming popularity
as a drug, in spite of warnings about harmful side-effects caused by disruption
of the metabolic rate. In one study, dinitrophenol treatment was successful in
achieving weight loss without dietary restriction in 89.4 percent of those
taking the substance at an average daily dose of 300 mg. It was estimated that
during the fifteen months following its introduction 100,000 persons were
treated with dinitrophenol. In addition, DNOC was also used for weight re-
duction, being considered three to five times as potent as dinitrophenol.
Toxic reactions to these weight-loss regimens soon began
to appear in the literature. Cutaneous lesions were noted in 8 to 23 percent
of the patients, as well as various gastrointestinal symptoms, agranulocytosis
of the bone marrow, neuritis, cardiovascular complications, and hepatic and
306
-------
renal damage. Nine deaths were reported from dinitrophenol administration and
one from DNOC. A narrative account by Parascandola (1974) described the tur-
bulent history and public concern over dinitrophenol in the United States. Con-
troversy was aroused by dramatic articles such as the one appearing in Newsweek
in 1933, entitled, "Diet and Die with Excess Alpha Dinitrophenol." This story
warned the public about the dangers of taking dinitrophenol by presenting the
case history of a physician who had been "literally cooked to death" by an
overdose of the drug, which produced extreme hyperthermia (see Section III-B-
4-c).
Reports of cataract development attributable to dinitro-
phenol therapy began to appear in 1935, and, during the next two months, seven
more cases were reported. The total number of persons affected with cataracts
was estimated at more than 164, before these incidents finally subsided during
1936-1937, when the drug was withdrawn from use. Horner summarized the charac-
teristic features of dinitrophenol-induced cataracts and concluded that, 1)
Occurrence was in young women of an age group not normally prone to cataract
development, 2) The lesions were bilateral and appeared after weight reduction
treatment with dinitro compounds, 3) An interval of months or years may elapse
between the last dose of drug given and the onset of symptoms, 4) The lenticular
changes occurred with striking similarity, 5) The changes progressed rapidly
until vision was obscured, 6) Treatment was not effective in halting their
development, and 7) Surgical removal of the cataract was uniformly successful in
restoring sight. The mode of action of dinitrophenol in producing cataracts
could not be clearly established, and investigators were hampered by the fact
that experimental cataracts could not be produced in laboratory animals (see
Section III-D).
307
-------
Acute exposure to dinitrophenol derivatives has produced
fatalities in several non-occupational incidents. A case of accidental fatal
poisoning by ingestion of a weed-killer containing alkanolamine salts of 2-
j3ec-Butyl-4,6-dinitrophenol (dinoseb) reported by Cann and Verhulst (1960),
illustrates the importance of proper labelling and storage of hazardous nitro-
aromatic compounds. In this case, a man had ingested and then ejected from his
mouth a small quantity of the liquid weed-killer, which he thought was grape
juice. His death ensued within 24 hours, and postmortem examination revealed
parenchymatous degeneration of the renal tubular epithelium.
Ingestion of concentrated solutions of dinitrophenol have
been shown to produce even more dramatic effects, as described by Swamy (1960)
in reporting a suicide case. His account was of a man who, after drinking a
solution of dinitrophenol with the intention of committing suicide, soon de-
veloped a burning sensation in the stomach and vomited yellowish matter.
He was taken to the hospital in a state of intense shock and incoherence with
feeble pulse and rapid respiration. Death occurred within five to six hours
of ingesting the solution. Physical examination revealed that the tongue and
mouth were eroded, pupils dilated, conjunctiva congested, and the patient
cyanotic with profuse sweating. On postmortem examination additional erosion
was found of the tissues of the esophagus and mucous membrane of the stomach.
A recent case of fatal exposure has been reported by
Buchinskii (1974) involving dinitro-ortho-cresol (DNOC). An ointment containing
DNOC was applied to the skin of a four year old child which caused death accom-
panied by tachycardia, convulsions, and mucosal hemorrhage.
308
-------
b. Nitrobenzene
The actions of nitrobenzene on the body can be quite varied,
but predominantly include toxic effects on the hematologic system, such as
methemoglobinemia and anoxemia due to a low level of normal blood hemoglobin.
Respiratory changes occur as the result of direct action on the respiratory
center of the brain, and from generalized cerebral anoxemia. Deprivation of
oxygen to the brain also leads to CNS reactions including coma, paralysis, in-
voluntary movements, headache, and behavioral changes. The lungs, liver, kid-
neys, and gastrointestinal system have also been shown to be damaged in certain
instances of nitrobenzene poisoning.
Accidental exposure to nitrobenzene has occurred in many
persons due to its past commercial use in common substances such as shoe polish
and dyes, inks, and even perfumes. Numerous deaths have also resulted when
women took nitrobenzene in order to induce abortion (Von Oettingen, 1941).
Contact with nitrobenzene has resulted in poisoning of babies wearing diapers
stamped with a laundry ink containing the substance. The largest number of re-
lated cases of accidental nitrobenzene poisoning, as summarized by Stifel (1919),
occurred when 17 persons developed severe cyanosis, headache, malaise, and
vertigo after wearing freshly dyed shoes.
An unusual incident of poisoning occurred among five infants
who suddenly became cyanotic after being breast-fed by their mothers (Dollinger,
1949). The women had each eaten a piece of cake flavored with an artificial
almond substance, presumably containing nitrobenzene, that had subsequently
passed into their milk and poisoned the infants.
309
-------
A case report by Wirtschafter and Wolpaw (1944) described
how a drunken man ingested 15 ml of laundry marker ink containing nitrobenzene
and became acutely ill. He was brought to the hospital with marked cyanosis of
the lips, nails, and ears; and his teeth, tongue, and mucous membranes were
stained dark blue. After four days, his color returned to normal, with his
pulse and respiration improving in three days. After 14 days, the patient was
sent home in good condition.
The authors pointed out that a considerable variation in
individual susceptibility has been evident from the past literature on nitro-
benzene poisoning. In some cases, as little as one gram of the substance may
be fatal, while in others a dose ranging from 3 to 100 grams may be followed
by recovery. For example, when nitrobenzene was used as an abortifacient by
a
16 women in doses of 15 to 100 grams, seven of them died and only one abortion
resulted.
Twenty-one infants in Egypt were poisoned by nitrobenzene
(Zeitoun, 1959); two cases resulted in death. They had been rubbed with a
supposed bitter almond oil, later found to contain 2-10 percent nitrobenzene.
The children all showed moderate to intense cyanosis which had developed between
four hours and four days after application of the false bitter almond oil. In
the cases where death occurred, it was preceded by intense cyanosis, shock,
vomiting, difficult respiration, and bronchopneumonia.
Suicidal ingestion of nitrobenzene has accounted for several
incidents of serious poisoning. The case of a 24 year old woman was presented
by Parkes and Neill (1953). She drank approximately 12 ml of nitrobenzene con-
tained in a bee mixture, in order to commit suicide. She became ill within an
310
-------
hour, with vomiting and dizziness. Intense cyanosis developed and persisted for
a week, along with evidence of methemoglobinemia and amino-aciduria (abnormal
excretion of amino acids in the urine). She recovered fully in four weeks, how-
ever, with no evidence of permanent tissue damage.
More recently, a case has been presented of a 19 year old
girl who survived a suicidal ingestion of about 50 ml of nitrobenzene (Myslak
e£ al., 1971). Within 30 minutes after the exposure she was hospitalized in an
unconscious state with marked cyanosis of the face, ears, palms, feet, and lips.
The peripheral blood level of methemoglobin was 82 percent. Prompt medical
treatment consisting of gastric lavage, oxygen inhalation, blood transfusion,
and drug therapy was probably responsible for saving her life. The clinical
course of hematologic effects and excretion of nitrobenzene metabolites is
presented in Figures 57, 58, and 59. The patient regained consciousness within
two hours, but cyanosis and poor health persisted for ten days along with en-
largement of the liver, vomiting, and severe headache. Transient tissue damage
to the bone marrow, heart muscle, and liver were noted due to severe hypoxia and
general toxemia, but recovery was rapid and complete.
c. Nitroaniline
An unusual outbreak of severe poisoning was recorded by
several investigators resulting from the ingestion of wax crayons (Rieders and
Brieger, 1953; Brieger, 1949; Jones and Brieger, 1947). The crayons were known
to contain a dye, para red, considered to be harmless and shown to be non-toxic
when administered to rats, dogs, and cats (Brieger et^ al., 1948). A careful
analysis of the crayons, however, revealed that unreacted £-nitroaniline, an
intermediate in the synthesis of the dye, was present in a number of the pigment
311
-------
and red crayon samples. The symptoms of methemogloblnemia following the in-
gestion of red wax crayons were, therefore, apparently due to contamination by
£-nitroaniline. This situation illustrated the need for careful analysis of
pigments prior to their use in products with a high potential for human exposure.
g% '
12-
11-
10-
9-
8-
7-
6-
5-
4-
3-
2-
1-
0
t Blood Transfusion [ 500 cm3
J Blood Letting [ 100 cm3 ]
Helthion Injection
Hb
HbO,
JIM
\ — i
i
i — i — \ — i — i — i — i
1 2 3 4 5 6 7 8 9 10 11 12
Hours After Injestion
Figure 57. The Level of Methemoglobin and Hemoglobin in Blood During
the First 12 Hours of Treatment
(Myllak et al., 1971)
312
-------
J Blood Trimf usion ( 500 cm1 )
1 Blood Letting I 100 on3 |
Helthion Injection
I I
8 9 10 11 12 13 14 18
Dlyi Afttr Ingntion
34 48
Figure 58. The Level of Methemoglobin and Hemoglobin During the
Entire Treatment Period
(Myllak et^ al., 1971)
mj/24 h
1000
100-
10-
1-
I ' I
2 4
I ' I
10 12
Dtv>
14 16 18
I
20
T
22
Figure 59. The Excretion Rate (mg/day) of Nitrobenzene Metabolites
£-Nitrophenol (PNP) and £-Aminophenol (PAP) During the
Entire Period of Observation
(Myslak et al., 1971)
313
-------
3. Epidemiological and Controlled Human Studies
Epidemiological investigations as they relate to the nitroaromatic
compounds have generally taken the form of experimental or intervention studies.
Major concern has been with measuring toxic effects in the industrial environ-
ment, where the potential for exposure is greatest.
a. Tetryl
A number of studies have been undertaken to define the extent
of occurrence and susceptibility to poisoning by the common nitroaromatic
munitions compounds. Emphasis has been placed on TNT and tetryl, because of
the large number of documented poisoning cases related to their handling.
A study was conducted over a period of 20 months among 203
workers who were employed in the manufacture of tetryl (Bonenti, 1956). The
most frequent symptoms of intoxication among these persons are summarized in
Table 84.
Table 84. Incidence of Disturbances of Occupational Origin Among 203 Tetryl
Workers (Bonenti, 1956)
Disturbance
Percent frequency
Pruritis (itching) of the face
Frontal headache
Epistaxis
Diarrhea
Restlessness, insomnia
Anorexia, nausea, labored digestion
Other (erythema, dermatitis, cholecystitis,
duodenal ulcer, anxiety, rhinbpharyngitis
33%
25%
18%
9%
8%
7%
15%
314
-------
The age of tetryl workers was not found to have an influence
on the incidence of disturbance. The number of intoxication episodes could be
reduced by limiting the work shift to three days with 15-20 day rest periods
in between. Workers who displayed allergic manifestations, or disturbances of
the liver or digestive system were removed from the work place due to the high
risk of their becoming seriously injured.
In a series of clinical and laboratory investigations con-
ducted by Parmeggiani et^ ol. (1956), over 200 workers who handled tetryl were
examined and followed up for two years. The results demonstrated a wide vari-
ation among responses to tetryl exposure. These included nosebleed, headache,
diarrhea, hyperexcitability, gastro-duodenitis, liver and gallbladder distur-
bances, itching, and dermatitis. Among 37 cases of dermatitis, only two were
found to be the result of allergic responses, the others apparently were due to
primary skin irritation. Adoption of measures for the prevention of over-
exposure to tetryl was stressed in their report; these included the reduction
of manual handling procedures and the use of special clothing and protective
creams to prevent the contact of tetryl dust with bare skin.
b. Trinitrotoluene
A recent investigation of the effect of TNT exposure on
the health of munitions factory workers has been made by El Ghawabi et al.
(1974). In their study of 35 workers who had been exposed to continuous TNT
3
concentrations of 0.1 to 1.2 mg/m in air, more than half the group had de-
veloped soine type of symptom (Table 85). It can be seen from Table 85 that
the symptoms displayed were characteristic of those resulting from a local
irritant effect rather than a systemic poisoning. Significant to note is the
315
-------
fact that no cases of dermatitis, cataracts, cyanosis, toxic jaundice, or
anemia had been discovered, even though these conditions had been reported
many times in the past (see Section III-C-1).
Table 85. Incidence of Different Symptoms in Exposed Workers Compared to the
Control Group (El Ghawabi £t al., 1974)
Symptom
Sneezing
Sore throat
Cough
Stomach-ache
Loss of appetite
Constipation
Flatulence
Headache
Lassitude
Nausea and vomiting
Exposed
Number of
workers
with
symptoms
21
18
16
15
10
13
12
4
2
12
group
Percent
of
total
60%
51.4%
45.7%
42.8%
28.4%
37.14%
34.28%
11.43%
5.71%
32.28%
Control
Number of
workers
with
symptom
1
1
4
1
2
2
3
2
1
1
group
Percent
of
total
5%
5%
20%
5%
10%
10%
15%
10%
5%
5%
It is postulated that modern standards of industrial hygiene
have accounted for the lack of severe TNT intoxication among the workers in this
study. In addition, a high-protein diet which was provided to the workers may
have afforded some protection, in light of evidence that a high-fat low-protein
diet rendered rats more susceptible to TNT and DNT poisoning.
Very recently, a report was made concerning the hemolytic
crisis which ensues from the exposure to TNT by persons with an inherited de-
ficiency of the enzyme glucose-6-phosphate dehydrogenase (G6PD) in the red blood
cells (Djerassi and Vitany, 1975). The case histories of three men were presented,
316
-------
all of whom held the same job in a TNT factory, were of Iraqi descent, and were
G6PD deficient. Upon exposure to TNT, each of the men developed paleness, en-
largement of the spleen and liver, and varying degrees of reticulocytosis and
increased urobilinogen in the urine. These symptoms are all indicative of
severe hemolysis of the red blood cells.
It should be anticipated that a hemolytic crisis may result
in persons with erythrocyte enzyme deficiences, when exposed to nitroaromatic
compounds other than TNT and tetryl. Pre-employment medical screening of persons
intended for high-exposure jobs will be necessary to identify individuals and
populations at high risk.
Several additional epidemiological TNT investigations have
been abstracted from the foreign literature. These reports have documented the
occurrence of a) liver lesions in 20.8% of a group of 79 TNT-exposed workers
(Poljak and Peljuskovic, 1969); b) characteristic carious and non-carious tooth
damage and disease of the peridontal and oral cavity mucous membranes (Makienko
and Karmanov, 1973); c) gastric disorders and impairment of the secretory
function of the stomach in 54% of TNT-poisoned patients (Faerman, 1957; and
d) occupational cataracts, toxic hepatitis, cholecystitis, peripheral blood
changes, neurasthenia, polyneuritis, and hypotonic neurocirculatory dystonia
(Soboleva, 1969; Zakharova and Manoilova, 1971; Manoilova, 1972).
c; Dinitrochlorobenzene (DNCB)
Among the epidemiological studies on non-munitions nitro-
aromatic compounds, an investigation has been conducted among a population of
normal Indian subjects to test skin sensitization by DNCB (Malaviya et al.,
1973). Patch testing of the skin for allergic sensitization by DNCB is a simple
317
-------
and commonly-employed method for the quantisation of cell-mediated immunity
(Catalona et_ jil. , 1972 a, b). Normally, it is possible to sensitize 85-95 per-
cent of the persons exposed to a single application of DNCB. Among 50 Indian
volunteers, however, a 100 percent rate of sensitization was achieved. In
addition, a relatively small dose of DNCB produced unusually severe spontaneous
flare reactions at the application site in 76 percent of the subjects. Further-
more, a very severe delayed hypersensitivity developed in 7 of the 50 persons.
These results suggest a higher level of immunoglobulins in
the Indian subjects, and is consistent with the theory that a high prevalence
of infectious disease in developing countries can lead to an overactive immune
system. The implication of this study is clearly to point out the hazard of
hyperreactivity to allergic sensitization by nitroaromatic chemicals among
populations with a high level of cell-mediated immunity.
d. Nitrobenzene
A controlled human study, to investigate the effects of
low nitrobenzene concentrations in air on several functions of the nervous
system, revealed its biological significance as an air pollutant (Andreyeshcheva,
1971). A determination of olfactory sensation to nitrobenzene by 29 volunteers,
aged 17 to 35 years, established that remarkably low concentrations of nitro-
benzene can be detected by smell in the air (Table 86).
Further studies demonstrated that the smell of subthreshold
concentrations of nitrobenzene could progressively decrease the sensitivity of
the visual system to light (Figure 60).
318
-------
Table 86. Results of Determination of Olfactory Sensation Threshold
of Nitrobenzene (Andreyeshcheva, 1971)
Number of Subjects
4
2
1
8
4
1
1
1
1
3
Nitrobenzene Concentration, mg/m
Threshold
0.0182
0.023
0.027
0.031
0.037
0.045
0.057
0.070
0.091
Subthreshold
0.0169
0.018
0.020
0.024
0.028
0.032
0.011
0.055
0.069
168
154-
142-
130-
118-
72-
60-
48-
38-
24-
12-
0
0 6 10 1B JO 26 JO 38 40
TtoM ol Eumiratloii (min )
Figure 60. Change in the Light Sensitivity of the Eye During Inhalation
of Nitrobenzene in Subject S
1 - pure air; 2 - concentration, 0.0118 mg/m ;
3 - 0.0157 mg/m ; 4 - 0.0169 mg/m . (Andreyeshcheva, 1971)
319
-------
Electroencephalographic measurements were also made to
determine the effect of nitrobenzene on reflex electrical activity of the brain.
In six healthy persons, aged 20 to 35 years, inhalation of nitrobenzene for six
minutes at levels below the threshold concentration for smell caused a dis-
ruption in the amplitude of intrinsic rhythm potentials of the brain (Figure 61).
\
10 12 14
Time of Examination ( min )
I
16
I
18
I
20
I
22
n
24
Figure 61. Changes in the Amplitude of Reinforced Intrinsic Potentials of the
Brain in Subject L (Andreyeshcheva, 1971) „
(1 - pure air; 2 - concentration, 0.008 mg/m ; 3 - 0.0129 mg/m ;
AP - period of gas inhalation)
The results of this study on nitrobenzene effects indicate
that all persons do not exhibit physiological responses at the same levels of
exposure. Furthermore, the actual biological consequences of low-level exposure
may be sufficiently subtle to avoid detection by conventional means and yet pose
a serious threat to health. Linch (1974) has commented, as well, that certain
individuals display a profound susceptibility or predisposition to the cyanogenic
effects of the nitroaromatic compounds. In reviewing 187 cases of cyanosis ,
320
-------
he found that 30 (21%) of the 143 employees involved had contributed 74 (40%)
of the cases. Moreover, eight persons, considered to be chronic repeaters,
had accounted for 30 cases and were removed from areas of potential exposure.
e. Dinitro-ortho-cresol (DN0C)
The effects of poisoning by DNOC have been studied by oral
administration of the compound to five human volunteers (Harvey e£ al., 1951)
(see Section III-B-1-a). The subjects were given 75 mg of pure DNOC daily for
five days, examinations were made of the blood and urine, and various physio-
logical responses were tested. These examinations failed to show the presence
of Heinz bodies or changes in the reticulocyte count, body weight, pulse rate,
respiratory rate, or blood pressure at the dosage level employed (equivalent to
0.92 - 1.27 mg/kg body weight). A yellow coloration of the sclera was evident,
however, in all subjects on the fourth day of the experiment. Additional symptoms
including headache, lassitude, and general malaise developed in two subjects,
which corresponded to the highest blood levels of DNOC achieved in each case.
Blood levels of DNOC in all subjects reached the 20 yg/g level after three to
five days. Temporary rises above that level were associated with symptoms of
poisoning.
In this study, the doses employed were necessarily low and
no attempt was made to produce toxic symptoms. The data presented from this
investigation demonstrated that DNOC gradually accumulates in the body when in-
gested at 24-hour intervals and is excreted slowly. Measurable blood levels of
DNOC persisted for more than three weeks after the treatment period ended. These
results established that DNOC can act as a cumulative poison in man, and imply
that other related dinitrophenolic derivatives may very well possess the same
cumulative properties.
321
-------
D. Toxicity - Birds and Mammals
1. Acute Animal Toxicity
Acute toxicity studies using animal model systems usually
involve exposures so massive tha£^they bear no practical relationship to
expected human environmental exposures. An analysis of the data as summarized
in Tables 103-107 (pp. 351-374) is very important, however, in evaluating the
potential dangers of nitroaromatic compounds, for at least three specific
reasons. One is that it is a means to identify compounds of such extremely low
toxicity that single dose exposures may be inconsequential (although chronic ex-
posure hazards may still be high). Secondly, a determination can be made of
the possible consequences of high exposures by misuse or accident, with maximum
permissible levels for short-term exposure being set accordingly. Finally,
compounds with unusual acute toxic properties or extreme toxic potency may be
identified and submitted for more extensive chronic studies at realistic sub-
acute dose levels. It is important to note here that acute toxicity studies
are of no value in estimating possible carcinogenic, mutagenic, or teratogenic
hazards associated with exposure to a chemical substance.
The symptoms of acute poisoning by the nitroaromatic compounds
in animals have been found to parallel their effects on humans. They are, for
the most part, directly attributable to: 1) action on the hematologic system,
2) the uncoupling of oxidative phosphorylation, 3) central nervous system
damage, or 4) stress to the major organs of foreign compound detoxification,
namely, the kidneys and liver. The only highly specific toxic effect noted
in animals has been the formation of cataracts by exposure to dinitrophenol
derivatives and dichloronitroaniline.
322
-------
The uncouplers of oxidative phosphorylation are evidently
limited to the dinitrophenol, dinitroaniline, and nitrosalicylanllide deriva-
tives. The severe hematologic poisons are among the nitrobenzene series of
compounds. Transient or irreversible damage to the kidneys and liver appears
to be a universal symptom in cases where nitroaromatic exposure is severe.
a. Dinitrophenol and Derivatives
The course of events in acute poisoning by 2,4-dinitro-
phenol (DNP), 2-sec-butyl-4,6-dinitrophenol (dinoseb), and 4,6-dinitro-ortho-
cresol (DNOC) has been well studied, receiving much attention because of their
widespread agricultural use. Characteristic symptoms induced in laboratory
animals by these compounds are increased respiratory rate, elevated body tem-
perature, increased heart rate, tremors, and the early onset of rigor mortis,
with the skeletal muscles becoming stiff just before the animal dies. Mice,
rats, rabbits, and dogs all react quite similarly to single doses of the DNP-
derivatives.
The time-course of events in poisoning by DNP, resulting
from its continuous intravenous infusion in a dog, was provided by Kaiser (1964).
This is presented in Figure 62.
The effect of increasing doses of DNP on body temperature
and mortality in rats is seen in Table 87 and Figure 63. These data indicate
a definite dose-related response to DNP exposure. Responses to acute administra-
tion of DNOC are very much the same as for DNP, although the hyperthermic re-
sponse may not be seen in all cases.
323
-------
280
240
700
. 160
120
80
40
Respiratory rale
\vs\\\\\\v
.'
/(limit of icala )
Body temp. I
'
t
Death
114
112
110
108
106
104
102
100
0 20 40 60 80 100 120 140 160
Infuiion Time (min.)
Figure 62. Infusion of Dinitrophenol in Conscious Dog - Rate: 0.4 mg/kg/minute,
right external jugular vein (Kaiser, 1964)
Time
(min)
Observation
20 Gums, red
25 Ears, moderately red
30 Skin, warm; tongue, purple red; profuse salivation; emesis, 10-15 ml,
greenish yellow
35 Nose,, warm
40 Tongue extended; emesis, 20 ml, greenish yellow
50 Ears, bright red; emesis, 20 ml, greenish yellow
60 Skin, hot
75 Nose, hot; skin, groin area, yellow
80-83 Dog became stiff and then relaxed
83 Emesis
84-85 Convulsions, death; dose, 36 rag/kg
85-86 All four limbs rigid
60-85 Temperature rose approximately 0.5°F per minute. Temperatures above
• 113°F are only estimates. Guard on instrument stopped the pointer at
an estimated temperature of 113.6°F.
(Reprinted with permission from Academic Press, Inc.)
324
-------
Table 87. Effect of Logarithmically Increasing Intraperitoneal Doses of 2,4-DNP on Rectal
Temperature and Lethality in Rats (Gatz and Jones, 1970)
Ol
Group
1
2
3
4
5
Number of
Animals
6
7
8
8
6
2,4-DNP,
mg/kg
16
20
25
31
39
A*
max
1.8 + 0
2.3 + 0
3.4 + 0
6.7 + 0
4.5 + 0
*
.2
.2
.3
.4
.3
Percent Mortality
0
0
25
100
100
Average
Lethal Time,
Minutes
•
94
77
12
* All data are expressed as mean values + S.E.
-------
u>
15
45
60 75
MINUTES
90 K>5 120
Figure 63. Effect of Logarithmically Increasing Intraperitoneal Doses of 2,4-DNP on Rectal Temperature
in Rats (Gatz and Jones, 1970) (Each point on a curve represents the mean of between 6 to 8
determinations of temperature at the specified times.)
(Reprinted with permission from the International Anesthesia Research Society.)
-------
Environmental temperature also affects acute toxicity of
the uncoupling compounds such that elevating the ambient temperature increases
the mortality from any given dose (Figure 64).
The addition of various groups to the basic DNP nucleus,
such as methyl, sec-butyl, or cyclohexyl, does not cause drastic alterations
in their relative animal toxicities. Addition or rearrangement of nitro groups,
on the other hand, may affect toxicity considerably, as shown in Tables 88 and
89.
The presence of a nitro group in a para-position to the
hydroxyl seems to be correlated with a higher toxicity by DNP and its various
derivatives; it also appears to be linked to the production of a hyperthermic
response. This characteristic may be due to an increased half-time of elimina-
tion from the blood for the para-substituted compounds.
In animal experiments, DNP, DNOC, dinoseb, and 2-cyclohexyl-
4,6-dinitrophenol did not produce significant skin irritation or primary sensi-
tization in rabbits and guinea pigs (Spencer et_ a!L., 1948). It was possible,
however, to produce death by skin absorption following a single application of
either DNP, DNOC, or dinoseb. The cyclohexyl derivative was not toxic by the
dermal route, probably due to its poor absorption across the skin (see Section III-B-1),
An analysis of dose-response relationships for the dinitro-
phenol derivatives has demonstrated a certain degree of unusual variability in
the effect of DNP on mortality from a single oral dose as compared to topical
exposure (Figure 65). However, DNOC, dinoseb, and 2-cyclohexyl-4,6-dinitro-
phenol are relatively consistent in conformance to a dose-related increase in
mortality (Figures 66-68).
327
-------
D
(M
T
5 10 15 20
Dose (mg/kg)
18-20°C
-10 °C
Figure 64. Effect of Environmental Temperature on Mortality in Rats Caused by
a Single Dose of Dinitro o-cresol (Parker et al., 1951)
328
-------
Table 88. Comparison of LD Values of Dlnitrophenols (LD for 2,4-DNP = 1)
(Harvey, 1959)
Dlnitrophenol
2,3- "
2,4- •*"
2,5-
2f • • • • • •
,t>-
3,4-
3,5-
Rats
5.4
i.o
4.3
1.1
2.8
1.3
Mice
5.5
1.0
7.6
1.3
3.1
1.4
Table 89. Comparison of
(Harvey, 1953)
Values in Rats for Dinitro- and Trinitrocresol
Substance
LD50 mg/kg
Dinitro-o-cresol
Dinitro-p-cresol
Trinitro-m-cresol
24.2
24.8
168.0
Given as 0.5 percent solutions in 0.5 percent NaC.l, 0.5 percent
NaHCO by intraperitoneal injection
329
-------
1000
•= 100-
Guinea Pig Dermal LD50 and 95 %
Confidence Limit! - 480 I 365-632 I
Slope > 1.47
Guinea Pig { Dermal
Rat (Oral I
LD50 and Confidence Limits
Cannot be Calculated
U01 0060' 07 06 1 7 5
20 » 40 60 GO 70 BO 90
Mortality (%)
98 99 99.8 99.9 99.99
10
Guinea Pig Dermal LD50 and 95%
Confidence Limit. • 345 { 278-427 )
Slope "1.28
Guinea Pig ( Dermal I -
Rat | Orel )
Rat Oral LD50 and 95% Confidence
Limits' 30.0(26.1-34.51
SI ope-1.38
001 0060.1 01 0612 5
M 30 40 SO 60 70 . BO M 95 9899 D9BM.9 9993
Mortality {% )
Figure 65. Mortality From a Single Dose of Figure 66. Mortality From a Single Dose
DNP by Oral and Dermal Administration* of DNOC by Oral and Dermal Administra-
(Spencer et^ al., 1948) tion* (Spencer et al., 1948)
_ 100-
1
~r
Guinea Pig Dermal LD50 and 95% Confidence
Limit>-189(129-277)
Slope -1.55
Guinea Pig ( Dermal I
Rat I Oral I
Rat Oral LD50 and 95% Confidence
Limiti « 36.5 I 30.7-43.4 I
Slope- 1.68
1000
001 0050102 05 I 2
20 30 40 SO 60 ;0 80
Mortality I % I
96 98 99 988999 9999
Rat Oral LD60 and 95% Confidence
Limiti ' 73 I 63.5-84.0 )
Slope » 1.763
001 0.060.102 00 1 2 5 10 20 30 40 M ft) In (HI 40 96 Hfl 1U 948
Mortality I % )
Figure 67. Mortality From a Single Dose of Figure 68. Mortality From a Single Oral
Dinoseb by Oral and Dermal Administration* Dose of 2-Cyclohexyl-4,6-dinitrophenol
(Spencer et_ al., 1948) in the Rat* (Spencer et al., 1948)
* Statistical analysis performed by the method of Litchfield
and Wilcoxon (1949)
330
-------
The above results indicate that, with the exception of
2-cyclohexyl-4,6-dinitrophenol, these compounds produce significant lethal
effects over a relatively narrow dosage range. This principle tends to hold
true among different mammalian species as well and is illustrated by Figure 69,
which depicts the effect on different species of a single oral dose of dinoseb.
Many authors have observed changes in the circulatory
system which were attributed to central nervous system stimulation by the
dinitrophenols. These effects include both the slowing and raising of the
pulse, as well as increased blood pressure. An increase in the respiratory
rate has also been a nearly universal observation in poisoned animals. It
is not entirely clear whether the effects of dinitrophenol substances on the
circulation and respiration are the result of a direct action on the central
nervous system or are a secondary response to anoxemia. It has been estab-
lished, however, that DNP, DNOC, and £-nitrophenol can directly stimulate both
the aortic and carotid chemoreceptors of the dog (Shen, 1962). This stimula-
tion produces a marked elevation of respiration, which parallels the hyperther-
mic action of the three compounds.
(i) Acute Cataract Development
The unusual phenomenon of cataracts of the eyes pro-
duced by acute exposure to DNP, DNOC, and their derivatives was first demonstra-
ted in animals almost ten years after the problem was known to exist in humans
(Robbins, 1944). Experimental cataracts, first produced in ducks and chickens,
differ from DNP-induced human cataracts in that they can be formed by acute
exposures and may appear in less than one hour. Furthermore, these lesions will
disappear spontaneously in animals within 24 hours.
331
-------
D
IT)
CM
a
CM
o
0 10 20 30 40 50 60 70 80 90 100
Dose ( mg/kg )
Figure 69. Acute Oral Toxicity of Dinoseb to Various Animal Species
(Bough et^ al., 1965)
332
-------
Buschke (1947) tested 37 compounds chemically related
to DNP for their cataractogenic activity by single-dose administration to adult
chickens. The results, summarized in Table 90, identified eight compounds which
were positive cataract-forming agents.
Table 90. Nitroaromatic Compounds with Cataractogenic Activity in Chickens
(Buschke, 1947)
Compound
2 , 4-Dinitrophenol
2, 6-Dibromo-4-nitrophenol
2 , 4-Dinitroanisole
2 , 4-Dinitrophenetole
i
2-Chloro-4 , 6-dinitrophenol
5-Chloro-2,4-dinitrophenol
2 , 6-Dinitrophenol
.4 ,6-Dinitro-o-cresol
mmol/kg
0.06
0.11
0.11
0.22
0.43
0.13
0.27
0.40
0.40
0.38
0.38
0.28
0.73
0.73
0.43
0.0125
0.02
0.025
0.10
Route
oral
oral
, I.M.
oral
oral
oral
oral
oral
' I.M.
oral
I.M.
oral
oral
. I.M.
oral
oral
oral
oral
oral
Time of
Onset (hrs)
3%
2
2
1
1
3
2
5
4
2
2
4
3/4
3/4
5
4-5
2
2
1
333
-------
In terms of structure-activity correlations, it is
important to note that the presence of ortho- and para- ring substituents is
essential to cataract-producing activity. A nitro group placed para to the
hydroxyl seems to be of great importance. Presence of the phenolic hydroxyl
group is a necessity, and any type of substitution for it abolishes activity.
The author explained that cataractogenic activity of 2,4-dinitroanisole and
2,4-dinitrophenetole may be due to the biotransformation of the ether to the
free phenol in the body.
Table 91 compares the relative cataractogenic potential
of various nitroaromatic compounds. The addition of a second ring system to
Table 91. Comparison of Cataract Producing Activities of Various Nitro Compounds
in Chickens (2,4-Dinitrophenol =1) (Buschke, 1947)
4,6-DN-Cresol
2,4-DN-Phenol
2,4-DN-Anisole
2,4-DN-Phenetole
2,6-Dibromo-4-Ni'tro- Phenol
2-Chloro-4,6-DN-Phenol
5-Chloro-2,4-DN-Phenol
2,6-DN-Phenol
4.8
1.0
>0.5
^0.5
^0.5
^0.22
<0.16>0.08
0.14
m-DN-Benzene
Mono-Nitro-Phenols
2-Chloro-4-Nitro-Phenol
2,4-DN-Naphthol
2,4-DN-Thymol
Nitro-Salicylic Acids
2,4-DN-Chlorobenzene
2,4-DN-Toluene
2,4-DN-Mesitylene
2,4-Dichlorb-Phenol
<0.125 or 0
334
-------
the molecule abolishes all activity. A fairly close correlation exists between
the ability to produce cataracts and the ability to raise the metabolic rate,
suggesting a possible relationship between effects on the eye and ability to
uncouple oxidative phosphorylation. The cataracts formed in these experiments
were all reversible, despite the continued presence of the drug in the diet,
and therefore did not precisely resemble the cataracts caused in humans from
chronic DNP ingestion (see Section III-C-2-a).
A more recent series of investigations by Gehring
and Buerge (1969) established that cataracts could be produced both in ducks
and rabbits by acute administration of DNP. Table 92 presents the results of
treating ducks by several routes with DNP, and reveals that a definite dose-
related response exists for cataract development.
Table 92. Incidence of Cataracts in Ducks Following the Administration of a
Single Dose of 2,4-Dinitrophenol (Gehring and Buerge, 1969)
Age Route of Dose
(days) Administration (mg/kg)
16-30a Oral 12
15
20
25
28
30
16-30 Intraperitoneal 3
6
9
12
14
16
C.
75 Intraperitoneal 4
6
9
12
No. with Cataracts
per No. Treated
0/4
0/4
3/8
3/4
3/3
4/4
3/10
6/10
10/10
5/8
9/10
9/9
2/5
4/5
5/5
4/4
Percent
Effect
0
0
38
75
100
100
30
60
100
63
90
100
40
80
100
JOO
a
ED5Q + 0.95 confidence limits equals 21.5 (17.9-25.8) mg/kg
b ED5Q equals 4.7 (3.05-7.24) mg/kg
C ED5Q equals 4.4 (3.12-6.20) mg/kg
335
-------
With oral administration, cataracts were first seen
within one to three hours, while similar opacities were often noted within 30
minutes after intraperitoneal injection.
The production of cataracts by intraocular injection
in ducks was very rapid, some appearing in less than 10 minutes. They could
be induced by very small doses of DNP ranging from 0.1 to 10.0 micrograms
(Table 93). Similarly, the treatment of rabbits by intraperitoneal or intra-
ocular injection, and by in vitro incubation of the lens with DNP, could produce
rapid cataract formation (Tables 94 and 95).
Table 93. Incidence of Cataracts in Ducklings Following a Single Injection of
2,4-Dinitrophenol Into the Posterior Chamber of the Eye
(Gehring and Buerge, 1969)
Dose
(yg)a
10.00
5,00
2.50
1.00
0.50
0.25
0.10
Control
No. with Cataracts
per. No. Treated
2/2
2/2
2/2
2/2
2/2
2/4
1/4
0/18
Percent
Effect
100.0
100.00
100.00
100.00
100.00
50.0
25.0
0.0
Q
The indicated dose of 2,4-dinitrophenol was contained in 10 yl of
sterile isotonic saline, pH 7.5 ED^g equals 0.20 (0.11-0.35) yg.
Controls were obtained by injecting 10 pi of isotonic saline, pH
7.5, into the posterior chamber of the eye contralateral to the
eye treated with 2,4-dinitrophenol.
336
-------
Table 94. Incidence of Cataracts in 90-Day-Old Rabbits Following a Single
Injection of 2,4-Dinitrophenol Into the Posterior Chamber of the
Eye (Gehring and Buerge, 1969)
Dose No. with Cataracts Percent
(yg)a per No. Treated Effect
50.0 2/2 100.00
10.0 2/2 100.00
5.0 2/4 50.0
2.5 0/2 0.0
Control13 0/10 0.0
The indicated dose of 2,4-dinitrophenol was contained in 10 pi of sterile
isotonic saline, pH 7.5. ED equals 5.0 (3.8-6.6) yg.
Controls were obtained by injecting 10 yl of sterile isotonic saline,
pH 7.5, into the posterior chamber of the eye contralateral to the eye
treated with 2,4-dinitrophenol.
Table 95. Cataract Production in Rabbit Lenses Incubated for 24 Hours at 37°
in KEI-4 Media Containing Varying Concentrations of 2,4-Dinitro-
phenol (Gehring and Buerge, 1969)
Concentration of
2,4-Dinitrophenol No. with Cataracts
Source of Lenses8 Ql) per. No. Treated
-3
Mature rabbits ...1.0 x 10
1.0 x 10~4
1.0 x 10~5
1.0 x 10~6
0
-4
Newborn rabbits 1.0 x 10
2.5 x 10~5
1.0 x 10"5
1.0 x 10~6
o
4/4
4/6
2/6
0/5
2/21
6/6
3/3
1/3
0/3
1/15
*a .
Mature rabbits were 90-119 days old; newborn rabbits were less than 5 days old.
337
-------
Table 96 illustrates that increasing age obviously
reduced the susceptibility of rabbits to cataract formation. The development
with age of mechanisms to rapidly metabolize and excrete DNP before it reaches
the lens may very well account for this fact. Further evidence is provided in
Table 95, which shows that isolated lenses of mature and newborn rabbits are
almost equally vulnerable to cataract formation when incubated in vitro with DNP.
Table 96. The Dose of 2,4-Dinitrophenol Causing a 50% Incidence of Cataracts
in Various Age Groups of Rabbits Following Intraperitoneal Admin-
istration (Gehring and Buerge, 1969)
a 0.95
b The I
Age
(days)
10 ± 1
18 ± 1
26 ± 1
32 ± 1
42 ± 2
62 ± 3b
Confidence limits
2050 value given for
ED50
(mg/kg)
6.6 (4.8^9U)a
9.6 .(7.6-12.1)
14.5 (11.2-18.7)
18.5 (14.8-23.1)
23.3 (19.4-27.9)
32.0 (27.1-37.4)
62-day-old rabbits is not
statistically valid because the dose necessary to
cause cataracts killed some of the rabbits.
An extrapolation of these results to the known cases
of DNP-induced cataract development in humans suggests that certain individuals
may be rendered more susceptible to the actions of DNP depending on their drug-
metabolizing capabilities. The unexpected observation of cataract formation in
dogs and pigs by chronic treatment with 2,6-dichloronitroaniline (DCNA) presents
further proof of mammalian susceptibility to lenticular damage by certain nitro-
substituted phenols (see Section III-D-2). The accidental observation that ex-
posure to sunlight was essential for the cataractogenic action of DCNA implies
that the underlying mechanism for this effect may depend upon several variables.
338
-------
b. Nitrobenzene and Derivatives
Clearly, the acute toxicity of nitrobenzene and its
chlpro-, methyl-, and amino-substituted derivatives can be most closely associa-
ted with hematologic alterations. Almost without exception, acute exposure of
animals to these compounds shortly results in the appearance of intracorpuscular
Heinz bodies (see Section III-B-4). A sign of severe erythrocyte damage, the
appearance of Heinz bodies in the blood signals the development of anemia.
Accompanying this reaction is a characteristic compensatory rise in the reticulo-
cyte count. In addition, the nitrobenzene-derived compounds are capable of pro-
ducing large quantities of methemoglobin and sulfhemoglobin. This feature is
in contrast to the apparent lack of hemotoxic effects caused by the dinitrophenol
derivatives.
A comparative study of the relative methemoglobin-forming
properties of the various nitrobenzene derivatives, when given by intraperitoneal
injection to cats, was undertaken by Bredow and Jung (1943). The data presented
in Table 97 clearly indicate the potent action of m-dinitrobenzene and 2,4,6-
trinitrobenzene in causing hematologic alterations. It is obvious from Table 102
that increasing the methyl-group substituents or decreasing the number of nitro
substituents tends to decrease the methemoglobin-forming properties of the com-
pound.
A comparison of dose-related methemoglobin responses to
several nitroaromatic chemicals (Table 98) presents strong evidence that the
formation of methemoglobin is probably not the primary factor in causing mor-
tality by exposure to these compounds. Note, however, that the oral LD_n for
nitrobenzene in rats is approximately three-fold less than that for £-nitro-
toluene (Table 98), and that methemoglobin formation by these compounds main-
tains a similar ratio at certain dosages. Therefore, the relative methemoglobin-
339
-------
Table 97. Comparative Toxicity of Some Nitroaromatic Compounds in Cats (Bredow and Jung, 1943)
Substance
Formula
Number of
Experiments
Amount Methemoglobin
Mole Substance
Time to Maximum Methemoglobin
Format Ion (hours)
Heinz Bodies
m-Dinitrobenzene
13
7.8 (max. 10. 3)
10
2,4,6-Trinitrobenzene
10
4.8 (max. 7.0)
LO
O
2,4,6-Trinltrotoluene
13
1.7
2,4-Dlnitrotoluene
,„
NO
1.4
-------
Table 97. Comparative Toxicity of Some Nitroaromatic Compounds In Cats (Bredow and Jung, 1943)
(Cont'd)
Substance
Formula
., , c
Number of
Amount Methcmog lobin
Experiments Mole Substance
Time to Maximum Methemoglobin
Formation (hours)
Heinz Bodies
Nitrobenzene
NQ
16
0. 86 (max .1.5)
2,6-Dinltrotoluene
23
0.55
4,6-Dinitro-l,3-xylene
£-Nitrotoluene
N0
CH
NO
0.1
0.05
-------
Table 97. Comparative Toxicity of Some Nitroaromatic Compounds in Cats (Bredow and Jung, 1943)
(Cont'd)
Substance
Formula
,.
Number of
Amount Methemuelobin
Experiments Mole Substance
Time to Maximum Methemoglobin
Formation (hours)
Heinz Bodies
m-Nitro toluene
0 . 04
£-Nitrotoluene
NO
CH
12
2,4-Dinitromesitylene
2,4,6-Trinitro-l,3-xylene
CH
-------
Table 98. Acute Effects of Several Nitroaromatic Compounds Given by Intraperltoneal Injection to
Rats (Magos and Sziza, 1958)
Nitrobenzene
Dose
(mMol/kg)
1.76
2.73
4.69
6.79
9.29
13.69
% Methemo-
g lob in
44.5
33.0
34.0
44.5
58.0
58.0
Time to
Death (hrs)
24-48
8-24
8-24
j3-Nitrobenzaldehyde
Dose
(mMol/kg)
1.19
1.85
2.78
4.10
6.22
9.26
% Methemo-
g lob in
38.6
47.9
50.0
Time to
Death (hrs)
2-3
2-3
2-3
p_-Nitrotoluene
Dose
(mMol/kg)
2.04
3.06
4.53
6.86
10.21
15.30
% Methemo-
g lob in
6.5
6.9
21.7
23.6
16.0
27.1
Time to
Death (hrs)
— -
24-48
24-48
24-48
OJ
-------
forming properties of the nitrobenzene derivatives may be a good indicator of
overall toxic potential.
The members of the nitrobenzene series differ significantly
from the nitrophenols in that they do not produce profound metabolic disruption
by the uncoupling of oxidative phosphorylation. Damage to the central nervous
system, however, is just as severe by the nitrobenzenes as with other nitro-
aromatic compounds. In chronic studies (see Section III-D-2), nitrobenzene-induced
damage to the central nervous system preceded hematologic effects.
c. Trinitrotoluene (TNT)
A noticeable lack of published information concerning the
acute toxicity and LD values for TNT in laboratory animals has been encountered
in the preparation of this report. Several lethality determinations were made
during the 1920's (Von Oettingen, 1941; Jaffe et^ al., 1973) which indicated that
considerable variation existed in the sensitivity to acute TNT poisoning among
the common mammalian species (Table 105). Although extensive comparative studies
have not been reported as yet, a number of investigations are now under way to
test the acute toxicity of munitions compounds, including TNT and dinitrotoluene
(Glennon, 1975).
d. Structure-Activity Relationships
A definitive statement probably cannot be made, based on
the currently available toxicity information, regarding the role of specific
nitroaromatic molecular structures in producing acute toxic effects; neither
has the mode of action of nitroaromatic chemicals at the target organ been
established. It is helpful to point out, however, that certain correlations
do seem to exist between the toxic potency, on a molar basis, of a nitroaromatic
compound and the presence or absence of various ring substituents.
344
-------
Among the nitroaromatic compounds, the dinitrophenol deriva-
tives possess the greatest degree of acute toxicity, as measured in terms of
lethality. Within this group, it is essential to have nitro substituents which
are ortho- and para- to the hydroxyl group for greatest toxicity to occur
(Table 104). Furthermore, the addition of a second ortho group, such as methyl
or butyl, will cause additional activity, while another nitro group or halogen
in that position will reduce toxic potency. Toxicity greatly diminishes when
either the hydroxyl or para-nitro group is replaced. These relationships are
illustrated in Table 99, which presents several nitroaromatic agricultural
chemicals in order of decreasing toxicity. The effect of various ring substitu-
ents in producing toxicity can be clearly seen as hydroxyl and para-nitro groups
are replaced and the number of substitutions on the ring increases.
Among the nitrobenzene derivatives, dinitrobenzenes are
more toxic than mononitrobenzenes, although neither group is as toxic as dinitro-
phenols. Further derivatization of nitrobenzenes into nitroanilines, nitro-
toluene, and nitrobenzoic acid will reduce toxicity significantly. In addition,
increasing the number of halogen substitutions (e.g., PCNB) and nitro groups
(e.g., TNT, tetranitroaniline, trinitrobenzene) in the molecule will also
greatly reduce toxicity. Throughout the entire series of nitroaromatic com-
pounds derived from a benzene skeleton, the importance of a para-positioned
nitro group for highest toxicity seems to stand out.
Attempts have been made to relate the acute toxicity of
the dinitrophenols to their relative potencies as uncouplers of oxidative phos-
phorylation with some degree of success (Ilivicky and Casida, 1969). A com-
parison was made of the levels for in vitro uncoupling in mitochondrial prepara-
tions and their relation to ID values (Table 100). These data reveal a fair
correlation between increasing uncoupling activity in liver mitochondria and
mortality produced in intact mice.
345
-------
Table 99. Comparative Toxicity of Various Nitroaromatic Structures in the Rat
Structure
Name
Acute Oral Rat LD
(mg/kg)
2,4-dinitrophenol
(DNP)
A,6-dinitro-o-cresol
(DNOC)
30
10-50
COCH
2-tert-butyl-5-methyl-
4,6-dinitrophenyl acetate
(medinoterb acetate)
42
2-sec-butyl-4,6-dinitro-
phenol
(dinoseb)
50-60
OCOCH
2-tert-butyl-4,6-dinitro-
phenyl acetate
(dinoterb acetate)
62
Berg, 1972
Schafer, 1972
346
-------
Table 99. Comparative Toxicity of Various Nitroaromatic Structures in the Rat
(Cont'd)
Structure
0
II
pCCH=C(CHj0
Name
2-sec-butyl-4 , 6-dinitro-
pheityl-3-methyl-2-
butenoate
Acute Oral LD
(mg/kg) 5°
161 + 25
NO,
0
II
OCCH=CHCH,
2,4-dinitro-6-
octylphenyl
crotonate
(dlnocap, karathane)
980
2,4-dichlorophenyl-
£-nitrophenyl ether
(TOK, nitrofen)
2,630
Berg, 1972
Schafer, 1972
347
-------
Table 99. Comparative Toxicity of Various Nitroaromatic Structures in the Rat
(Cont'd)
Structure
Name
Acute Oral LD
(mg/kg)
50
NIL
Cl
Cl
NO,
2,6-dichloro-4-
nitroaniline
(botran, diclpran)
>5,000
2,6-dinitro-N,N-di-
n-propyl-ot,a,a-
trifluoro-p_-toluidine
(trifluralin, treflan)
>10,000
pentachloronitro-
benzene
(PCNB, quintozene)
>12,000
Berg, 1972
Schafer, 1972
348
-------
Table 100. Potency of Various 2,4-Dinitrophenols as Uncouplers of Oxidative Phosphorylation In Vitro
and Their Toxicity to Mice, Houseflies, and Honey Bees (Ilivicky and Casida, 1969)
2, 4-Dinitrophenol
Derivative
Unsubstituted (DNP)
&-Cyclohexyl CDNOCHP)
6-Methyl (DNOC)
6-sec-Butyl Cdinoseb)
6-sec-Butyl-l-isopropyl
carbonate (Dessin)
Minimum Uncoupling Concentration, y[M],
with MltAchondrial Preparations from
Mouse
Liver Brain
50 1.0
1.5 1.0
20 20
1.0 0.5
80 80
Housefly
Thorax
50
0.2
20
0.8
100
Honey Bee
Head Thorax
30 50
0.8 1.0
20 30
0.5 0.8
100 200
LD,.0, Injected (ymoles/kg)
Mouse
141
95
94
42
383
Housefly
1,630
131
732
146
Honey Bee
108
3.5
18
11
-------
When the dinitrophenols were injected into mice, however,
a very good correlation was found to exist between uncoupling of brain mito-
chondria and the severity of overt poisoning symptoms (Table 101). The single
Table 101. Uncoupling and Inhibition of Brain and Liver Mitochondria After
Injection of Mice with Various Dinitrophenols (Ilivicky and
Casida, 1969)
Magnitude of Uncoupling
Compound
Dose
(umoles/kg)
Holding
Time
(min)
2,4-Dinitrophenol (DN)
DNP
6-Cyclohexyl-
DNP
Dinoseb
Dessin
Dinocap
269
38
94
63
100
150
181
181
275
550
20
20
20
20
20
20
30
60
30
30
Severity
of
a
Symptoms
uncouplers
2
0
2
0
1
2
1
2
1
2
or
Brain
0
0
2
0
1
2
1
2
1
2
Inhibition13
Liver
0
0
1
0
1
2
0
0
0
2
The severity of the symptoms is graded as follows: 0, not any, same control;
1, mild, survival of animal is probable; 2, severe manifestation, death of
animal is expected.
Effects on mitochondria are graded as follows: 0, not any, same as control;
1, partial uncoupling or inhibition; 2, complete uncoupling or inhibition.
exception to this observation was unsubstituted DNP, where severe toxic symptoms
were produced without significant uncoupling in brain mitochondria. The sug-
gestion was made that rapid potentiation of the metabolism and elimination
(continued on p. 375)
350
-------
Table 102. Acute Animal Toxicity of Various Nitroaniline Derivatives
Compound
2-Chloro-4-nitroaniline
4-Chloro-2-nitroaniline
4-Chloro-3-nitroaniline
C/J
Cn
2,6-Dichloro-4-Nitroaniline
2,4-Dinitroaniline
Species
Mice
Mice
Rabbits
Mice
Rabbits
Starling
Blackbird
Rats
Rats
Rats
Mallard
Duck
Rats
Rats
Dose
(mg/kg)
500
50
•
63
?
>100
100
>5000
418
1500 -
8QOO
>2QOO .
1800
250
. Route
of
Administration
I. P.
I.V.
Dermal
I.V.
Dermal
Oral
Oral
Oral
Oral
Oral
Oral
Oral
I. P.
Effects
Lethal dose
,
Lethal dose
Not corrosive to skin
LD50
Not corrosive to skin
LD50
LD50
LD5Q
"'so
LD50
LD ; regurgitation, ataxia, weakness, wing drop, falling
when walking; symptoms may persist for five weeks
LD Q; respiration inhibited; uncoupling of oxidative
phosphorylation
Lethal dose
-
Reference
Christensen and
Luginbyhl, 1974
Christensen and
Luginbyhl, 1974
Hanavan, 1975
Christensen and
Luginbyhl, 1974
Hanavan, 1975
Schafer, 1972
Schafer, 1972
Berg, 1972
Christensen and
Luginbyhl, 1974
Ben-Dyke et al . ,
1970
Tucker and Crabtree,
1970
Vasilenko et al . ,
1974
National Academy of
Science, 1953
-------
Table 102. Acute Animal Toxicity of Various Nitroaniline Derivatives (Cont'd)
Compound Species
2,4-Dinitro-6-bromoaniline Rats
N-Methyl-N,2,4,6-Tetra- Dog
nitroaniline
4-(Methylsulfonyl)-2,6- Mice and
dinitro-N,N-dipropylaniline rats
Rabbits
LO
l_n m-Nitroaniline Rats
N>
Rats
Mice
Mice
Rabbits
Dogs
Dose
(mg/kg)
4490
5000
>2000
>2000
900
535
450
308
500
70
Route
of
Administration
Oral
S.C.
Oral
Dermal
Oral
Oral
Oral
Oral
Oral
I. P.
Effects
LD50
Lethal dose
LD50
LD50
LD, ; methemoglobinemia and sulfhemoglobinemia at 450 mg/kg
LD50
LD,-n; hematologic changes ; severe degeneration ~of kidneys ,
liver and spleen
LDso
Methemoglobinemia, Heinz body formation, reticulocytosis,
decreased erythrocyte count
Death within three hours
Reference
Christensen and
Luginbyhl, 1974
Christensen and
Luginbyhl, 1974
Berg, 1972
Berg, 1972
Vasilenko et al. .
1974
MacEwen and Vernot ,
1972
Akahori, 1954
MacEwen and Vernot,
1972
Akahori, 1954
VonOettingen, 1941
-------
Table 102. Acute Animal Toxicity of Various Nitroaniline Derivatives (Cont'd)
Compound Species
£-Nitroaniline Rats
Rats
Mice
j>— Nitroaniline Rats
Rats
Co
(Ji
CO Rats
Mice
Mice
Guinea
Pigs
Starling
Blackbird
Dose
(mg/kg)
3564
3520
308
3249
1410
1500
.
812
250
450
>100
75
Route
of
Administration
Oral
Oral
Oral
Oral
Oral
Oral
Oral
I. P.
Oral
Oral
Oral
Effects
LD50
LD ; hepatotropic effects
LD50
LD50
LD,_; increased methemoglobin and su If hemoglobin
LD__ ; increased erythrocytes , reticulocytes , leukocytes »
Heinz bodies , hemoglobin ; spasms , lymphopenia
LD50
Lethal dose
LD,_: increased erythrocytes, reticulocytes, leukocytes,
Heinz bodies, hemoglobin; spasms, lymphopenia
LD50
LD50
Reference
MacEwen and Vernot,
1972
Vasilenko et al. ,
1974
MacEwen and Vernot,
1972
MacEwen and Vernot,
1972
Vasilenko et al. ,
1974
Moskalenko, 1966
MacEwen and Vernot ,
1972
Christensen and
Luginbyhl, 1974
Moskalenko, 1966
Schafer, 1972
Schafer, 1972
Tetranitroanlline
Dogs
2500
S.C.
Minimum lethal dose; death within 6 days
VonOettingen, 1941
-------
Table 103. Acute Animal Toxicity of Various Nitrobenzene Derivatives
u>
Ln
•IS
p_-Di nitrobenzene
Route
Dose of
Compound
l,2-Dlchloro-4,5-
dinltrobenzene
l,2-Dichloro-5-
nitrobenzene
2 ,5-Dlchloronitro-
benzene
3,4-Dichloronltro-
benzene
n-Dinitrobenzene
Species
Mice
Rats
Rats
Rats
Rabbits
Rats
Dogs
Dogs
Dogs
Rabbits
Rabbits
Black-
bird
Starling
(rag/kg)
125
643
1210
50
200
50
600 mg*
10
10-20
400-
500 mg*
200
42
>100
Admlnis .
i.p.
Oral
Oral
Oral
Dermal
Oral
Oral
i.v.
i.v.
Oral
Dermal
Oral
Oral
Effects
Lethal dose
LD50
LD50
No mortality produced
No mortality produced
No mortality produced
Minimum lethal dose
Serum iron rose sharply after one day and returned to normal in six
days
Methemoglobinemla , verdoglobinemia, Heinz bodies, liver damage,
cerebral paralysis, convulsions, anemia, increase white cells;
LD Q was about 10 mg/kg
Minimum lethal dose
No mortality produced
LD50
LD50
Reference
Chris tensen and Luginbyhl,
1974
Chrlstensen and Luginbyhl,
1974
,,
Hanavan, 1975
Hanavan, 1975
M II
VonOettingen, 1941
Cammerer e_t al. , 1949
Kiese, 1949
VonOettingen, 1941
Hanavan, 1975
Schafer, 1972
ii ii
Cats
29
Oral
Lethal dose
Christensen and Luginbyhl,
1974
-------
Table 103. Acute Animal Toxicity of Various Nitrobenzene Derivatives (Cont'd)
Compound
Route
Dose of
Species (mg/kg) Adminis.
Effects -
Reference
2,4-Dlnitrochloro- Rats
benzene
Rats
Rabbits
Dinitrotrichloro- Rats
benzene
l-Fluoro-2,4- Rats
dinitrobenzene
Mice
l-Fluoro-4- Rats
nitrobenzene
Nitrobenzene Rats
CO
Ui Rats
Cn
Rats
Rats
Rats
Mice
1593
500
193.6
500
50
100
250
640
664
836
100-
200
800
996
Oral
Oral
Dermal
Oral
Oral
s. c.
Oral
Oral
Oral
i.p.
s. c.
B.C.
i.p.
"'so
LD50
LD ; severe skin and corneal irritation
LD50
Lethal dose
Lethal dose
Lethal dose
"'so
"'so
100Z mortality, death within 24 to 48 hours; 44.51 methemoglobln
level
Formation of sulfhemoglobin, nltroxyhemoglobin, methemoglobin, Heinz
bodies
Lethal dose
100Z mortality within 24 hours; central nervous system Involvement
Smythe et al. , 1962
Edson e_t al. , 1964
Smythe et al. , 1962
Edson e_t al. , 1964
Christensen and Luginbyhl,
1974
n ii ii
II II M
II II II
Smythe et_ al. , 1970
Magos and Sziza, 1958
Vasllenko and Zvezdal, 1972
Christensen and Luginbyhl,
1974
Smith et_ al. , 1967
Mice
Mice
480
such as loss of righting reflex within 10 to IS minutes, coma,
shallow respiration, tremor, respiratory arrest
Lethal dose
482 mg* dermal Prostration, dyspnea; majority died within 24 hours
Mice 50-80 inhala- Toxic level
•8/1 tlon
Christensen and Luginbyhl,
1974
VonOettingen, 1941
Pislaru et_ al., 1962
-------
Table 103. Acute Animal Toxicity of Various Nitrobenzene Derivatives (Cont'd)
Route
Dose of
u>
Ln
Compound
Nitrobenzene
(Cont'd)
m-Nitrochlorobenzene
o-Ni troch lorobenzene
p_-Ni t roch lorobenzene
Species
Dogs
Dogs
Dogs
Dogs
Rabbits
Rabbits
Rats
Mice
Mice
Rabbits
Rats
Rats
Mice
Rabbits
Rats
Rats
Rats
(mg/kg)
750-
1000
500-
700
750
150-
250
600
600
555
390
400
520
50
268
135
200
812
670
420
Admlnis .
Oral
Oral
Oral
i.v.
Oral
Dermal
7
Oral
1
1
Oral
Oral
Oral
Dermal
Oral
Oral
Oral
Effects
Minimum lethal dose
Salivation, unrest, tremors, delirium, increased pulse, staggering
gait, clonic and tonic convulsions
Lethal dose
Minimum lethal dose
Lethal dose
Lethal dose
"'so
"'so
"V
"'so
No mortality produced
LD50
"'so
No mortality produced; not corrosive to skin
"'so
Approximate lethal dose
"'so
Reference
VonOettingen, 1941
ii it
Christensen and Luginbyhl,
1974
VonOettingen, 1941
Christensen and Luginbyhl,
1974
..
Davydova, 1965
Alishev and Osipov, 1966
Davydova, 1965
ii ii
Hanavan, 1975
MacEwen and Vernot, 1972
it it it
Hanavan, 1975
MacEwen and Vernot, 1972
Hanavan, 1975
Christensen and Luginbyhl,
i a~j/.
-------
Table 103. Acute Animal Toxicity of Various Nitrobenzene Derivatives (Cont'd)
Compound Species
j>-Nitrochlorobenzene Rats
(Cont'd)
Mice
Mice
Rabbits
Rabbits
Rabbits
Pentachloronitro- Rats
benzene
OJ
<~n Rats
Rabbits
l,2,4-Trichloro-5- Starling
nitrobenzene
Black-
bird
Dose
(mg/kg)
500-
600
1414
650
500
500
200
1650
1740
4000
>100
100
Route
of
Admlnis.
?
Oral
Oral
i.p.
s. c.
Dermal
Oral
Oral
Dermal
Oral
Oral
Effects
Metabolic disturbance of the brain, loss of adrenalin from the
adrenals, methemogloblnemia
"'so
"'so
Reduced blood pressure and myocardial glycogen level
Severe nethemoglobinemia (20Z of total hemoglobin) , Heinz bodies in
almost all erythrocytes within 16 hours; death within 24 hours
No mortality produced
"'so
"'so
No adverse effects were seen during a 14 day observation period when
applied to abraded skin
"'so
"'so
JV
Reference
Frenkel and Gordienka, 1958
MacEwen and Vernot,
Alishev and Osipov,
Labunskii, 1972
Nogawa, 1961
Hanavan, 1975
1972
1966
Chris tensen and Luginbyhl,
1974
Borzelleca et al. ,
n
Schafer, 1972
n n
1971
"
Trinitrobenzene
Rats 505 Oral LD5Q' depression, hyperpnea, gasping, cyanosis, salivation, tachy-
cardia, coma, loss of reflexes, hemorrhagic lungs, discoloration
of the blood
Fogleman et al., 1955
* Total dose
-------
Table 104. Acute Animal Toxiclty of Various Nitrophenol Derivatives
oo
Compound
2-Amino-4-nitrophenol
2-sec-Amvl-4 . 6-dinitrophenol .
2-Chloro-4, 6-dinitrophenol
2-Chloro-4-nitrophenol
3-Chloro-4-nitrophenol
2 ,4-Dichloro-6-nitrophenol
2 , 6-Diiodo-4-nitrophenol
>
Species
Mice
Mice
Rats
Mice
Rats
Mice
Rats
Rats
Rats
Rats
Rats
Dose
(rag/kg)
1280
4
500
125-500
100
125
100
170
105
105
122
Route
of
Administration Effects
Oral LD5()
I. P. Lethal dose
Oral Lethal dose
I. P. Approx. LD,g
Oral Lethal dose
I. P. Lethal dose
Oral Lethal dose
Oral LD__; tremors, prostration, increased respiratory
rate, tonic convulsions, rigidity of limbs prior
to or immediately after death
I.V. L^50' tremors> prostration, increased respiratory
rate, tonic convulsions, rigidity of limbs prior
to or Immediately after death
I. P. LD50' cremors> prostration, increased respiratory
rate, tonic convulsions, rigidity of limbs prior
to or immediately after death •
S.C. LD50' tremors> prostration, increased respiratory
rate, tonic convulsions, rigidity of limbs prior
Reference
Akahori, 1954
Christensen and Luginbyhl,
Christensen and Luginbyhl,
Doull et^ al. , 1962
Christensen and Luginbyhl ,
Christensen and Luginbyhl,
Christensen and Luginbyhl,
Kaiser, 1964
Kaiser, 1964
Kaiser, 1964
Kaiser, 1964
1974
1974
1974
1974
1974
to or immediately after death
-------
Table 104. Acute Animal Toxicity of Various Nitrophenol Derivatives (Cont'd)
VO
Compound Species
2,6-Diiodo-4-nitrophenor Mice
(Cont'd)
Mice
Mice
2,6-Dibutyl-4-nitrophenol Rats
Rats
Rats
Rats
Rats
Mice
Mice
Guinea
Pigs
Guinea
Pigs
Route
Dose of
(mg/kg) Administration Effects
212 Oral
88 I.V.
110 S.C.
450 Oral
500 Oral
260 I. P.
270 I. P.
300-600 I. P.
850 I. P.
700 I. P.
580 I. P.
800 Oral
LD ; tremors, prostration, increased respiratory
rate, tonic convulsions, rigidity of limbs prior
to or immediately after death
LD5Q; tremors, prostration, increased respiratory
rate, tonic convulsions, rigidity of limbs prior
to or immediately after death
LD,.; tremors, prostration, increased respiratory
rate, tonic convulsions, rigidity of limbs prior
to or Immediately after death
LDrn females
LD._ males
LD,Q females
LD, males
100Z mortality; histopathologic changes of the
liver, spleen, kidneys, heart, lungs, and
lymphold tissues
LD, females
LD males
"'so
JU
LDSO
ju
Reference
Kaiser, 1964
~
Kaiser, 1964
Kaiser, 1964
Vesselinovltch et al. ,
Vesselinovitch et al. ,
Vesselinovltch et al. ,
Vesselinovitch e£ al. ,
Vesselinovitch et al.,
Vesselinovltch et^ al. ,
Vesselinovltch e_t al. ,
Vesselinovltch et al. ,
Vesselinovitch et al. ,
1961
1961
1961
1961
1961
1961
1961
1961
1961
-------
Table 104. Acute Animal Toxicity of Various Nitrophenol Derivatives (Cont'd)
Compound Species
4 . 6-Dini tro-2-sec-butvlDhenol Rats
Rats
Rats
Rats
Rats
Rats
Rats
Rats
Rats
Rats
Mice
Mice
Mice
Guinea
Pig
Route
Dose of
(mg/kg) Administration Effects
40-60
50
30-60
60 "
25-40
50
21.4
200-600
80-200
80
20-40
10.1
100
20-40
Oral
Oral
Oral
Oral
Oral
Oral
S.C.
Dermal
Dermal
Dermal
Oral
I. P.
Dermal
Oral
LD50
"•so
LD50
100% mortality; largest dose survived by all
treated animals was 50 mg/kg
LD ; prostration, rapid respiration, convulsions
preceding death, death within 24 hours
LD50
LD50
"'so
"'so
LD50
LDcQ; prostration, rapid respiration, convulsions
preceded death, death within 24 hours
""so
20% mortality; 90% mortality at 500 mg/kg
LD.Q; prostration, rapid respiration, convulsions
nTpfoHoH Hojtfh H*»s»fh wit'h'fn 94 hniirR
Reference
Bailey and White, 1965
Ben-Dyke et^ al., 1970
Schafer, 1972
Spencer et al., 1948
Bough et_ al. , 1965
Edson et al. , 1964
Harvey, 1952
Edson et^ al. , 1964
Ben-Dyke et_ al. , 1970
Christensen and Luginbyhl, 1974
Bough elt al. , 1965
Ilivicky and Caslda, 1969
Bough et al. , 1965
Bough et_ al. , 1965
-------
Table 104. Acute Animal Toxicity of Various Nitrophenol Derivatives (Cont'd)
to
Compound Species
A .6-Dinitro-2-sec-butylphenol Guinea
(Cont'd) Pigs .
.Starling
4,6-Dinitro-i-cresol Rats
Rats
Rats
Rats
Rats
Rats
Rats
Rats
Rats
Route
Dose of
(mg/kg) Administration Effects
500
7.1
33
10-50
25-40
30
50
25-40
28.5
24.6
20
Dermal
Oral
Oral
Oral
Oral
Oral
Oral
Oral
I. P.
S.C.
s.c.
100Z mortality; largest dose survived by all
treated animals was 100 mg/kg
">50
LD50
LD50
"'so
""so
Minimum fatal dose
"'SO
"'so
LD,.; death usually occurred within two hours
Respiration rate increased rapidly after 10 to
Reference
Spencer et al. , 1948
Schafer, 1972
MacEwen and Vernot, 1972
Berg, 1972
Edson et al. , 1964
Bailey and White, 1965
Corti. 1953
Ben-Dyke et^ al. , 1970
Lawford e£ al. , 1954
Parker et_ a^. , 1951
Parker et^ al . , 1951
Rats
Rats
25.6
200-600
S.C.
Dermal
15 minutes and animals became prostrated, which
persisted for one to two hours followed by
gradual recovery; where death occurred, it was
preceded shortly by muscular rigidity which
was complete when the animal died and persisted
for some hours after death
LD_-; LDcQ range of four DNOC commercial
preparations was 26.2 to 27.5 mg/kg
'-D50
-Harvey, 1952
Edson et al., 1964
-------
Table 104. Acute Animal Toxicity of Various Nitrophenol Derivatives (Cont'd)
u>
N5
Compound Species
4,6-Dinitro-o,-cresol Rats
(Cont'd)
Mice
Mice
Mice
Mice
Mice
Mice
Rabbits
Rabbits
Guinea
Pigs
Guinea
Pigs
Mallard
Duck
Route
Dose of
(mg/kg) Administration
200-600
21
24
24.2
18.7
24.2
47 mg/m
10-40
23.5
22.5
200
22.7
Dermal
Oral
I.P.
I.P.
I.P.
S.C.
Inhalation
Oral
I.P.
I.P.
Dermal
Oral
Effects
LD50
LD50
U>5Q
LD ; sweating, .increased respiration, diminished
physical activity, marked rigor mortis
LD50
LD •, death usually occurred within two hours
50% mortality
Neurotoxic syndrome developed within 5 to 15
minutes ; decreased erythrocyte and increased
leukocyte counts; 45 mg/kg was fatal
U>50
LD50
Maximum tolerated dose; minimum fatal dose was
500 mg/kg
LD,.«; ataxia, wings crossed high over back, tall
Reference
Ben-Dyke et_ al. , 1970
MacEwen and Vernot, 1972
Lawford et^ al. , 1954
Harvey, 1953
Ilivicky and Caslda, 1969
Parker et al. , 1951
Chrlstensen and Luginbyhl , 1974
Arustamyan, 1973
Lawford et_ al. , 1954
Lawford et_ al^. , 1954
Corti, 1953
Tucker and Crabtree, 1970
tachypnea, dyspnea, unkempt feathers, tetany
with the legs extended posteriorly;.symptoms
persisted in some survivors for up to two weeks
-------
Table 104. Acute Animal Toxicity .of Various Nitrophenol Derivatives (Cont'd)
Co
Compound Species
2,4-Dinitro-6-octyl- Rats
phenylcrotonate (dinocap,
Karathane) Rabbits
2,6-Dinitro-£-cresol Mice
4,6-Dinitro-o_-cyclohexylphenol Rats
Rats
Mice
Mice
Guinea
Pigs
2,4-Dinitrophenol Rats
Rats
Rats
Rats
Rats
Rats
Rats
Dose
(mg/kg)
980-1190
>9400
24.8
180
250
100
25.3
1000
30
71
32.7
35
10-20
50
25
Route
of
Administration Effects
Oral
Dermal
I. P.
Oral
Oral
Oral
I. P.
Dermal
Oral
Oral
I. P.
I. P.
I. P.
I. P.
I. P.
LD50
LD50
LD50
100% mortality; largest dose survived by all
treated animals was 30 mg/kg
Lethal dose
Lethal dose
^50
No mortality produced
LD50
LD,_; tremors, prostration, increased respiratory
immediately after death
LD50
LD_- at 18-21° environmental temperature
Oxygen consumption increased 17 to 21 percent
100% mortality
25Z mortality; average time to death was 94
Reference
Edson et^ al. , 1964
Edson et al. , 1964
Harvey, 1953
Spencer et_ al. , 1948
National Academy of Science,
1953
Chrlstensen and Luginbyhl, 1974
Ilivicky and Caslda, 1969
Spencer et_ al. , 1948
Schafer, 1972
Kaiser, 1964
Lawf ord et_ al. , 1954
Harvey, 1959
Harvey, 1959
Obbink and Dalderup, 1964
Gatz and Jones, 1970
minutes
-------
Table 104. Acute Animal Toxiclty of Various Nitrophenol Derivatives (Cont'd)
Route
Dose of
Compound Species (mg/kg) Administration Effects
2,4-Dinltrophenol . Rats 31
(Cont'd)
Rats 39
Rats 60
I. P. 100% mortality; average time to death was 77
minutes
I. P. 100% mortality average time to death was 12
minutes
I. P. LDi;n' tremors> prostration, increased respiration,
. Reference
Gatz and Jones, 1970
Gatz and Jones, 1970
Kaiser, 1964
Rats
Mice
Mice
Mice
Mice
Mice
Mice
Mice
25
72
52
25.9
26
36
>5
56
S.C.
Oral
I.P.
I.P.
I.P.
I.P.
I.P.
I.V.
tonic convulsions, rigor mortis prior to or
immediately after death
U>,Q; 10 mg/kg caused no deaths while 50 mg/kg
produced 100% mortality
LD .; tremors, prostration, Increased respiratory
rate, tonic convulsions, rigor mortis prior to or
immediately after death
LD,-; tremors, prostration, increased respiration,
tonic convulsions, rigor mortis prior to or
immediately after death
LD
50
LD50
LD Q at 18-21°C environmental temperature
100% mortality at 39-41°C environmental
temperature
LD ..; tremors, prostration, Increased respiration,
tonic convulsions, rigor mortis prior to or
immediately after death
Talnter and Cutting, 1933
Kaiser, 1964
Kaiser, 1964
Ilivicky and Caslda, 1969
Lawford et^ al., 1954
Harvey, 1959
Harvey, 1959 •
Kaiser, 1964
-------
Table 104. Acute Animal Toxicity of Various Nitrophenol Derivatives (Cont'd)
-------
Table 104. Acute Animal Toxicity of Various Nitrophenol Derivatives (Cont'd)
Compound
4-Nitro-jijrcresol
2-Nitro-£-cresol
in-Nitrophenol
o-Nitrophenol
j>-Nitrophenol
Species
Mice
Rats
Rats
Mice
Dogs
Rats
Mice
Mice
Rabbits
Rabbits
Cats
Dogs
Rats
Rats
Rats •
Mice
Dose
(nig/kg)
500
3360
933
1414
83
2828
1297
600
1700
?
600
100
350
616
97
467
Route
of
Administration
I. P.
Oral
Oral
Oral
I.V.
Oral
Oral
I.M.
S.C.
Dermal
SvC.
I.V.
Oral
Oral
I. P.
Oral
Effects
Lethal dose
"SO
LD50 .
LD50
Lethal dose
LD50
LD50
Lethal dose
Lethal dose
Not corrosive to skin
Lethal dose
Lethal dose
"'so
LD50
LD50
LD -
Reference
Christensen and Luginbyhl,
Christensen and Luginbyhl,
MacEwen and Vernot, 1972
MacEwen and Vernot, 1972
Christensen and Luginbyhl,
MacEwen and Vernot, 1972
MacEwen and Vernot, 1972
Dittmer, 1959
Dittmer, 1959
Hanavan, 1975
Dittmer, 1959
Dittmer, 1959
Christensen and Luginbyhl,
MacEwen and Vernot, 1972
Dittmer, 1959
MacEwen and Vernot, 1972
1974
1974
1974
1974
-------
Table 104. Acute Animal Toxicity of Various Nitrophenol Derivatives (Cont'd)
u>
CT*
-J
Compound
_p_-Nitrophenol
(Cont'd)
Picric acid
3-Trifluoromethyl-4-
nitrophenol
2,4,6-Trinitro-m-cresol
Species
Mice
Mice
Rabbits
Dogs
Cats
Dogs
Rabbits
Rabbits
Rabbits
Rats
Mice
Mice
Mice
Dose
(rag/kg)
107.6
75
t
10
500 mg*
60
400 mg*
140-
250 mg*
350-
1000 mg
40
25-50
168
31
Route
of
Administration
I. P.
I. P.
Dermal
I.V.
Oral
7
Oral
*
I. P.
I. P.
I. P.
I. P.
Effect
Lethal dose
Not corrosive to skin.
Lethal dose
Nausea, vomiting, fatigue, pain, increased reflex
excitability within 45 minutes, tonic and clonic
convulsions, ascending paralysis causing death
by respiratory failure
Slowing of respiration and heart beat; death by
respiratory paralysis
Convulsions and death in 3 hours
Lowered body temperature, slowing of heart rate,
rise and subsequent fall in blood pressure
Severe damage to the kidneys
Lethal dose
Approx. LD
LD__; hair became erect, marked shivering,
occasional spasms followed by great nervous-
activity (e.g. running around cage), no marked
rig'or mortis upon death
Lethal dose
Reference
Lawford e£ al^. , 1954
Christensen and Luginbyhl,
Hanavan, 1975
Dittmer, 1959
VonOettingen, 1941
VonOettingen, 1941
VonOettingen, 1941
VonOettingen, 1941
VonOettingen, 1941
Christensen and Luginbyhl,
Doull e_t ai. , 1962
Harvey, 1953
Christensen and Luginbyhl,
1974
1974
1974
* Total dose
-------
Table 105. Acute Animal Toxicity of Various Nitrotoluene Derivatives
Compound
2-Amino-4-nitro toluene
2-Chloro-4-nitro toluene
2-Chloro-6-nitro toluene
2 , 3-Dinitrotoluene
LJ
00 2,4-Dinitrotoluene
2 , 5-Dinitrotoluene
Species
Mice
Rats
Guinea
Pigs
Rata
Mice
Rats
Rats
Rats
Mice
Rabbits
Cats
Rats
Mice
Dose
(rag /kg)
1800
3020
9
1122
1072
50
268
200 ppm
1625
200
27
707
1231
Route
of
Administration
Oral
Oral
Dermal
Oral
Oral
Oral
Oral
Inhalation
Oral
Topical
Oral
Oral
Oral
Effects
LD ; severe degeneration of liver, kidneys, and spleen
LD50
Mild skin irritation as a 20% solution in water or a 50%
solution in ether ; not a skin sensitizer
LD50
LD50
No mortality produced
LD50
No mortality produced when inhaled for one hour
LD50
No mortality produced; not corrosive to skin
Minimum lethal dose
LD50
LD
Reference
Akahori, 1954
Chrlstensen and
Luglnbyhl, 1974 ;
Hanavan, 1975
MacEwen and Vernot ,
1972
MacEwen and Vernot,
1972 [
Hanavan, 1975 j
MacEwen and Vernot ,
1972
Hanavan, 1972 :
MacEwen and Vernot,
1972 |
Hanavan, 1975 \
i
Spector, 1956
MacEwen and Vernot ,
1972
MacEwen and Vernot,
1972
-------
Table 105. Acute Animal Toxicity of Various Nitrotoluene Derivatives (Cont'd)
VO
Compound Species
2,6-Dinitrotoluene Rats
Mice
Cats
2,4- & 2,6-Dinitrotoluenes Rabbits
(mixed)
3,4-Dinitrotoluene Rats
Mice
m-Nitrotoluene Rats
Rats
Rats
Mice
Rabbits
Rabbits
Guinea
Pigs
Dose
(mg/kg)
177
1000
60
1000
1072
1414
2282
1072
200 ppm
330
2400
20
3600
Route
of
Administration
Oral
Oral
l.P.
Topical
Oral
Oral
Oral
Oral
Inhalation
Oral
Oral
Topical
Oral
Effects
LD
LD50
Lethal dose
Approximate lethal dose ; not corrosive to skin
LD50
LD50
LD50
W50
No mortality produced when inhaled for one hour
LD50
LD50
No mortality produced; not corrosive to skin
LD50
Reference
MacEwen and Vernot,
1972
MacEwen and Vernot,
1972
Spector, 1956
Hanavan, 1975
MacEwen and Verno t ,
1972
MacEwen and Vernot,
1972
Hanavan, 1975
Chris tensen and
Luginbyhl, 1974
Hanavan, 1975
Kosachevskaya, 1967
Kosachevskaya, 1967
Hanavan, 1975
Kosachevskaya, 1967
-------
Table 105. Acute Animal Toxicity of Various Nitrotoluene Derivatives (Cont'd)
Compound Species
^-Nitrotoluene Rats
Rats
Rats
Mice
Rabbits
£-Nitrotoluene Rats
Rats
Co
~vl Rats
O
Rats
Mice
Rabbits
3-Nitro-a,a,a-trifluoro- Rats
toluene
Trinitrotoluene Rats
Rabbits
Cats
Cats
Dose
(mg/kg)
2144
891
200 ppm
2462
200
2144
2144
939.4
200 ppm
1231
200
610
>700
500-700
480
200
Route
of
Administration
Oral
Oral
Inhalation
Oral
Topical
Oral
Oral
I. P.
Inhalation
Oral
Topical
Oral
S.C.
S.C.
Oral
S.C.
Effects
LD50
LD50
No mortality produced when inhaled for one hour
LD50
y p
^50
LD50
100% mortality within 24 to 48 hours; 23.6 methemoglobin
formation
No mortality produced when inhaled for one hour
LD50
No mortality produced ; not corrosive to skin
LD50
Lethal dose
Lethal dose
Lethal dose
Lethal dose
Reference
Hanavan, 1975
Christensen and
Luginbyhl, 1974
Hanavan, 1975
Christensen and
Luginbyhl, 1974
Hanavan, 1975
Christensen and
Luginbyhl, 1974
Hanavan, 1975
Magos and Sziza,
1958
Hanavan, 1975
Christensen and
Luginbyhl, 1974
Hanavan, 1975
Christensen and
Luginbyhl, 1974
Spector, 1956
Spector, 1956
Spector, 1956
Spector, 1956
-------
Table 106. Acute Animal Toxiclty of Miscellaneous Nitroaromatic Compounds
U)
Compound
2-tert-Butvl-4 , 6-dinitrophenyl
2-fi£c.-Butyl-4 , 6-dinitropheny 1-
3-methyl-2-butenoate
6-_t££t-Butyl-3-methyl-
2,4-dinitroanisole
(musk ambrette-artificial)
2-tert-Butvl-5-roethvl-
4, 6-dinitrophenyl acetate
2 , 5-Dichloro-3-nitro-
benzoic acid
2 , 4-Dichlorophenyl-4-
nltrophenyl ether
(Nitrofen) .
2 , 4-Dinit roanisole
4.6-Dinitro-2-sec-
butylphenyl acetate
2 , 4-Dinitro-6- tert-
butylphenyl methanesul-
fonate
2,6-Dinitro-N,N-dipropyl-
cumidine (tech)
Species
Rat
Rat
Rats
Rats
Rabbits
Rats
Rats
Rats
Rats
Rats
Rats
Rats
Mice
Mice +
Rats
Rabbits
+ Dogs
Dose
(mg/kg)
62
>2000
58-225
161 ± 25
1350
339
42
3500
3050
100
65
527
800
>5000
>2000
Route
of
Adminis.
Oral
Dermal
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Effects
U>
LD50
LD50
LD50
LD50
LD,-n; increased respiration and hypersensitivity
after 24 hours, scrawny fur and appearance, wet
posterior; time to death = 1 to 3 days
"'so
LD50
"'so
Lethal dose
"'so
J\J
100% mortality
100X mortality
""so
• JU
"'so
Reference
Ben-Dyke e_t_ al.
Berg, 1972
Ben-Dyke et al.
Berg, 1972
Ben-Dyke et al.
Jenner et al. ,
Ben-Dyke et^ al.
, 1970
, , 1970
, 1970
1964
, 1970
Bailey and White, 1965
Ben-Dyke ej^ al.
National Acad.
Berg, 1972
. , 1970
Sci., 1953
Tsubura and Kato, 1974
"
Berg, 1972
„
"
-------
Table 106. Acute Animal Toxicity of Miscellaneous Nitroaromatic Compounds (Cont'd)
u>
Compound
2,4-Dinitro-a-naphthol
2,4-Dinitro-6-octylphenyl
crotonate (Karathane)
2 , 4-Dinitrophenetole
2,6-Dinitro-N.N-di-n-
propyl-a,a,a-trif luoro-
_E-toluidine (Trif luralin)
Dinitroresorcinol
3 , 5-Dinitrotoluamide
3,5-Dinitro-j>-
toluidine
3 ' -Nitroacetophenone
5-Nitro-o-anisidine
Species
Rats
Mice
Dogs
Guinea
pigs
Rats
Rats
Rats
Rats
Rats
Rabbits
Dogs
Rats
Rats
Rats
Rabbits
Mice
Rats
Dose
(nig/kg)
47.5
55
30-60
80-100
980
250
5000
3700-10,000
>5000
>200
190
560
>500
3250
3.0 ml /kg
200-300
704
Route
of
Admlnls .
i.p.
i.p.
i.v.
s.c.
Oral
Oral
Oral
Oral
Dermal
Dermal
s.c.
Oral
Oral
Oral
Dermal
i.p.
Oral
Effects
"'so
LD50
Fatal, death within 30 minutes
Fatal, death within 15 to 30 minutes
LD50
J\J
Minimum lethal dose
^O
LD50
LD50
"'so
Fatal within 24 hours; oral administration of 1 to 3
grams had no effect
"'so
Minimum lethal dose
50% mortality after 14 days
. 50% mortality after 14 days
Approx. LD50
LD50
Reference
Lawford et^ al. , 1954
ii ii
Dittmer, 1959
., „
Berg, 1972
National Acad. Sci. , 1953
Bailey and White, 1965
Ben-Dyke et al. , 1970
"
Edson et_ al. , 1964
VonOettingen, 1941
Christensen and Luginbyhl, 1974
National Acad. Sci., 1953
Smythe e± al. , 1954
n ii
Doull e.t al. , 1962
Christensen and Luginbyhl, 1974
c>-Nitroanisole
£-Nitroanisole
Rabbits ? Dermal
Rabbits ? Dermal
Not corrosive to skin
Not corrosive to skin
Hanavan, 1975
-------
Table 106. -Acute Animal Toxicity of Miscellaneous Nitroaromatic Compounds (Cont'd)
Compound Species
p_-Nitrobenzaldehyde Rats
Rats
o-Nitrobenzamide Mice
pj-Nitrobenzenesulfonamide Rats
m-Nitrobenzoic acid Rats
Rats
Rats
Mice
Mice
Mice
OJ
t_j ^-Nitrobenzoic acid Mice
Mice
£-Nitrobenzoic acid Rats
Rats
Mice
Mice
Mice
o-Nitrobiphenyl Rats
Rabbits
£-Nitrobiphenyl Rats
Rabbits
1-Nitronaphthalene Dogs
Rats
Dose
(mg/kg)
545
619
500
500
1820
670
680
1290
610
640
3100
1920
1960
1210
1470
770
880
1230
1580
2230
1970
670
120
Route
of
Adminis
i.p.
i.p.
i.p.
Oral
Oral
i.p.
i.v.
Oral
i.p.
i.v.
i.p.
i.v.
Oral
i.p.
Oral
i.v.
i.p.
Oral
Oral
Oral
Oral
Oral
Oral
Effects Reference
LD - Christensen and Luginbyhl, 1974
100% mortality; death within 2 to 3 hours; a dose Magos and Sziza, 1958
of 420 mg/kg produced a methemoglobin level of 50%
Lethal dose Christensen and Luginbyhl, 1974
Lethal dose " "
LD ; hyperexcitability, immobilization, aggressiveness Caujolle et al. , 1966
LD ; hyperexcitability, immobilization, aggressiveness " "
LD ; hyperexcitability, immobilization, aggressiveness " "
LD ', hyperexcitability, immobilization, aggressiveness " "
LD .; hyperexcitability, Immobilization, aggressiveness " "
LD .; hyperexcitability, immobilization, aggressiveness " "
L^ert* hyperexcitability, immobilization, aggressiveness Caujolle et al., 1966
j(j — —
LD,...; hyperexcitability, immobilization, aggressiveness " " •
LD ; hyperexcitability, immobilization, aggressiveness Caujolle £t_ ai^. , 1966
LD,-; hyperexcitability, immobilization, aggressiveness " "
LD ; hyperexcitability, immobilization, aggressiveness " "
LD,Q; hyperexcitability, immobilization, aggressiveness " "
LD ; hyperexcitability, immobilization, aggressiveness " "
LD,_; survival time was 24 hours to 18 days Deichmann et^ al^. , 1947
LD ; survival time was 24 hours to 16 days " "
LD ; survival time was 24 hours to 10 days Deichmann et^ a±. , 1947
ID ; survival time was 48 hours to 7 days " "
Approximate lethal dose Hanavan, 1975
LD Christensen and Luginbyhl, 197'
-------
Table 106. Acute Animal Toxicity of Miscellaneous Nitroaromatic Compounds (Cont'd)
Compound
Route
Dose of
Species (mg/kg) Adminis.
Effects
Reference
2-Nltronaphthalene
o-Nitrostilbene
5-Nitro-o-toluidine
3-Nitro-£-toluldlne
4-Nitro-2 , 6-xy lenol
Tetranitroxylene
2,4, 6-Trinitroanisole
Rats
Rabbits
Mice
Rabbits
Rats
Starling
Blackbird
Mice
Dogs
Rabbits
Rats
4400
2650
500
9
574
32
3.2
500
5000
1000
>500
Oral
Oral
i.p.
Dermal
Oral
Oral
Oral
i.p.
s. c.
i.p.
Oral
LD,-; minimum lethal dose was 3200 to 4700 mg/kg;
degeneration of liver and kidneys
LD ; minimum lethal dose was 1400 to 2100 mg/kg;
con June tival discoloration occurred within several
hours, urine discolored, severe hepatic and renal
degeneration, formation of Heinz bodies and
methemoglobinemia
100% mortality; no lethal effects up to 250 mg/kg
Not corrosive to skin
^50
LD50
^50
Lethal dose
Minimum lethal dose
Minimum lethal dose
Lethal dose
Treon and Cleveland, 1960
11 ii
Dittmer, 1959
Hanavan, 1975
Chrlstensen and Luginbyhl,
Schafer, 1972
ii ii
Chris tensen and Luginbyhl,
Christensen and Luginbyhl,
11
National Acad. Sci., 1953
1974
1974
1974
11
-------
processes occurring in the intact cell or animal may have prevented sufficient
quantities of DNP from entering the brain mitochondria, thus explaining its
laCk of uncoupling activity. This argument seems plausible when one considers
the studies of Doggett and Spencer (1973), which demonstrated that DNP injected
directly into the cerebral ventricles of the brain in mice and rats caused an
uncoupling of oxidative phosphorylation. Furthermore, DNP, when administered
into the brain, can selectively reduce conditioned avoidance-response in rats
with no effect on unconditioned responses. This action of DNP on the brain
resembles that of chlorpromazine when centrally administered; this compound is
a known uncoupler in brain mitochondria.
In addition, it was found that dinoseb was an inhibitor
of mitochondrial respiration but not an uncoupler of oxidative phosphorylation.
The symptoms of poisoning by dinoseb differed greatly from those produced by
the uncouplers, most notably in that death was not followed by immediate rigor
mortis.
2. Subacute and Chronic Toxicity
For the sake of consistency in the following discussion and
in Table 107, all toxicity studies which involved more than a single dose and
lasted longer than 24 hours, but less than 90 days, will be referred to as
subacute. This classification covers the great majority of studies which were
obtained in preparing this section. Very few reports were found of studies
where nitroaromatic compounds were repeatedly administered for more than three
months.
The material presented in this section should be interpreted
with special consideration for the fact that target organs and responses of
375
-------
Table 107. Subacute and Chronic Animal Toxicity
•a
c
3
O
"
2-Cyclohexyl-4 ,6-
dinltrophenol
2,6-Dichloro-4-
nitroaniline
Q>
O
O-
Rats
Ducklings
Dogs
Dogs and
Swine
Dogs and
Swine
Monkeys
Rats
00
00
0>
cn
<§
0.10%
0.25%
24-48
192
48
160
400 and
1,000
c
o
4J
CD
cn
3 -5
3 6
O •*- T3
a: o <
Dietary
Dietary
Dietary
Dietary
Dietary
Oral
Oral
u
•a 3
0 01
•H O
p CL
0) ua X
PL. O W
6 months
4 days
Daily for
2 years
Daily
Daily
Daily
Daily for
3 months
cn
CJ
cu
w
45% mortality; significantly decreased weight gain
100% mortality; a 0.10% diet produced a 90% mortality
within 38 days
Irreversible cataracts developed after 55 days at the
48 mg/kg level and several weeks later at the 24 mg/kg
level; slight elevation of bilirubin and serum glutamlc
oxaloacetlc transaminase levels at the higher dosage
level; no other abnormalities were observed; natural
light was essential for cataract formation
All dogs died within 49 to 53 days and were extremely
emaciated; hemoglobin, hematocrit, and red blood cell
count were decreased; Heinz bodies and reticulocytosls
were detected in male animals; swine were not affected
in any manner
Irreversible corneal and lens opacities were seen in
dogs within 53 to 55 days; Heinz bodies were found in
dogs and swine; natural light was essential for eye
lesion formation; at a dose level of 24 mg/kg/day some
dogs developed eye lesions in 84 to 104 days, while at
75 mg/kg/day changes were observed in 13 days
Lethal within 3 months; more toxic to females than
males; caused structural alterations of the liver and
kidneys
Some mortality at only the 1,000 mg/kg level; liver en-
largement noted along with Increased hepatic demethylasc
and desulfurase activities; caused structural altera-
tions of liver and kidneys, and increased mitochondrial
oxygen utilization
0)
u
c
OJ
at
a:
Spencer e£ al^. , 1948
Spencer et al. , 1948
Bernstein et^ al. , 1970
Earl iet al_. , 1971
Earl et^ al., 1971
Serrone et. al. , 1967
Serrone et al., 1967 .
Co
-------
Table 107. Subacute and Chronic Animal Toxicity (Cont'd)
c
3
O
CL.
6
8 .
2,6-Dichloro-A-
nltroaniline
(continued)
m-Dinitrobenzene
4 . 6-Dinitro-o-sec-
butylphenol
Cfi
0)
-4 t
CJ
a.
C/l
Rats
Dogs
Rats
Dogs
Rats
Ducklings
00
^
00
E
01
Q
O
100 ppm
3,000 ppm
20, 100, and
3,000 ppm
0.2-6
0.05X
0.10Z
c
- 0
iJ
CO
w
-------
Table 107. Subacute and Chronic Animal Toxicity (Cont'd)
-a
c
3
O
a
1
4 , 6-Dinitro-o-sec-
butylphenol
(continued)
4,6-Dinitro-2-
(2-capryl)phenyl
crotonate
4,6-Dinltro-o-
cresol
2,4-Dlnitrophenol
en
o
eg
O-
cn
Mice
Rats
Rats
Ducklings
Rabbits
Rats
Rats
Rats
'oo
00
CD '
09
O
Q
16, 40, and
100
50 and 100
0.10%
0.25%
3% solution
in 95% EtOH
20
0.20%
0.10%
c
o
u
Id
09
to c
3 T=
o <~ -a
02 O <
Dermal
Oral
Dietary
Dietary
Dermal
S.C.
Dietary
Dietary
•a 3
S S
u a.
cu v x
cu O tu
5 days
weekly for
2 weeks
Daily for
10 days
10 days
2 days
Daily for
7 days
Daily for up
to 6 weeks
24 days
6 months
01
cj
CU
I4J
l*-l
U
Mice given the 40 and 100 mg/kg doses all died after
the first application; those given 16 mg/kg all sur-
vived and grew normally
Lowered protein utilization; decreased feed intake and
body weight gain; no effect on net energy utilization
50-60% mortality; rapid weight loss, enlarged spleen,
slight renal degeneration; rats fej a 0.05% diet for
6 months showed only decreased weight gain
100% incidence of bilateral cataracts within 24 hours;
100% mortality within 2 days
100% mortality
31% mortality within 17 days when animals were kept In
a warm laboratory; 8.5% mortality within 17 days was
obtained when rats were kept under cool environmental
conditions
40% mortality; emaciation, enlarged spleen, slight
renal degeneration, liver congestion, testicular
atrophy, cloudy swelling of the liver
Decreased rate of body weight gain by 10 to 15%
u
c
cu
cu
U-l
01
a.
Bough et_ al. , 1965
Salmowa e£ al. , 1974
Spencer et^ al. , 1948
Spencer et_ al. , 1948
Spencer e_t al. , 1948
Parker et_ al. , 1951
Spencer et al. , 1948
Spencer et al. , 1948
CO
^J
oo
-------
Table 107. Subacute and Chronic Animal Toxicity (Cpnt'd)
•a
c
3
o
a.
e
o
u
2,4-Dinitrophenol
(continued)
3,5-Dinitro-£-
toluamide
2,4- and 2,6-
Dinitro toluenes
(mixed)
j>-Nitroanlllne
j>-Nitroanisole
CO
Qj
U
CU
C.
VI
Rats
Ducklings
Guinea Pigs
Rats
Rats
Rabbits
Rats
Mice
00
00
Cj
CO
o
a
20
0.25Z
10
0.03Z
0.0125Z
1,000
5 . mg/m
0.01-
0.03 mg/1
e
o
4J
ca
CO
CU C
3 e
O iw T3
a: o <
l.V. and
I. P.
Dietary
I. P.
Dietary
Dietary
Dermal
Inhalation
Inhalation
TJ 3
O CO
•H o
14 a.
-------
Table 107. Subacute and Chronic Animal Toxicity (Cont'd)
c
3
O
O.
e
o
u
Nitrobenzene
£-Nitrochloro-
benzene
CO
•H
O
-------
Table 107. Subacute and Chronic Animal Toxicity (Cont'd)
•c
c
3
o
o.
1
j>-Nitrochloro-
benzene
(continued)
o-Nitrobiphenyl
K
-------
Table 107. Subacute and Chronic Animal Toxicity (Cont'd)
-o
c
3
C
O-
E
S
£-Nitrobiphenyl
£-Nitrophenol
Pentachloronitro-
benzene
Picric acid
c/l
111
O
O
O.
tn
Rabbits
Guinea Pigs
Rats
Rats
Dogs
Rabbits
Dogs
00
J^
00
41
a.
ft, O W
5 days a
week for
7 weeks
Daily
3 genera-
tions
3 months
2 years
Daily
Repeated
u
w
No effects on skin or body weight
Cataracts developed in vitamin C-deficient animals in
7 to 11 days
No effects seen on reproductive fertility, gestation,
viability, or lactation
Animals at the 5,000 ppm level were sacrificed after
2 weeks due to poor health; growth was depressed at
levels above 2,500 ppm for females and 1,250 ppm for
males; a significant liver-to-body weight ratio
Increase was seen at all levels except in females at
63.5 ppm; no hematologic changes were seen
No mortality; lowered hematocrlt values after 18 months
in dogs on the 30 and 180 ppm diets only; at the
1,080 ppm level higher serum glutamic oxaloacetic
transamlnase seen in females with elevated SAP levels
and slightly enlarged livers in both sexes
Jaundice, diarrhea, loss of weight; repeated doses of
180 mg/kg caused severe emaciation and death within
2 weeks
Kidney damage and increased nitrogen excretion
-------
Table 107. Subacute and Chronic Animal Toxicity (Cont'd)
-a
c
o
C-
E
O
Tetryl
Trinitrobenzene
Trinitrotoluene
VI
s;
2
£
Rats and
Rabbits
Rats
Dogs
Dogs
Dogs
Dogs
Dogs, Rats,
and Rabbits
oc
^
e
O
01
O
Q
1,000-
2,000
50
100
25
0.1-1.0
and 5-20
0.1 or 5-20
5-20
^
L!
^
-------
Table 107. Subacute and Chronic Animal Toxicity (Cont'd)
e
UJ 13
o <
Trinitrotoluene
(continued)
OJ
00
Dogs
Dogs
Rats
Rats
Rats
.20-50
100
10
100
s.c.
Dermal and
Inhalation
Oral.
Oral
Oral
Every other
day for
3 months
Up to
2 years
3-30 days
Daily for
100 days
Daily for
45 days
Decreased bile secretion and increased cholic acid and
bilirubin concentrations in the bile; during the third
month cholic acids decreased and cholesterol, increased
Disruption of the secretory and evacuating functions of
the stomach; changes in the volume of gastric secre-
tion; acid and enzyme-forming functions displayed a
wave-like character
Decreased levels of total protein and albumin and
increases in B- and y-globulins in the blood serum;
protein-fatty dystrophy of the liver; decreased level
of serotonin and increased activity of monoamine oxi-
dase in the liver and brain
Decreased leukocyte phagocytic activity which was
antagonized by treatment with 50 pg/kg vitamin B
or 5 mg/kg vitamin PP for the first 40 days of TNT
administration
Decreased leukocyte phagocytic activity which was
antagonized by treatment with 50 ug/kg vitamin B -
or 5 mg/kg vitamin PP for the first 40 days of TNT
administration
Kleiner, 1971 b
Kleiner, 1972
Mul'menko and Levina,
1974
Kuzovleva et_ al. , 1973
Kuzovleva et al., 1973
-------
acute bioassays may not be the same following repeated exposure to smaller
doses. The phenomena of storage, metabolic activation, and repeated damage to
organs and organelles are potential hazards of repeated exposure; they more
closely resemble the consequences to man from environmental contamination.
a. Dinitrophenol Derivatives
The possibility of chronic exposure to low levels of the
dinitrophenol compounds is relatively high, based on the patterns of agricultural
use for these substances. Recognizing this possible danger, several investi-
gators have undertaken to determine if long-term toxic effects can be produced
in laboratory animals.
The results of studies where DNP, DNOC, dinoseb, and cyclo-
hexyldinitropheriol were injected or fed in the diet indicated that cumulative
toxic effects did not occur at dosage levels below those which produce toxicity
as a single dose (Table 107). Symptoms of severe poisoning could be produced,
however, when DNOC was given by repeated injection with short intervals between
consecutive doses (Parker et_ al., 1951). A single dose in the rat or rabbit
of 5 mg/kg of body weight produced no deleterious effects, but hourly injections
of the same dose caused marked symptoms after the fourth or fifth injection.
The further observation was made that among rats given a long series of daily
20 mg/kg injections of DNOC, almost all deaths occurred after the first few
injections. This left a group of animals that were either functionally resis-
tant or had developed a tolerance to the effects of DNOC.
It seems evident from the above results and from the data
reported in Section III-B that DNP derivatives can be quickly eliminated from
the body with little or no cumulative poisoning occurring at dosages below the
385
-------
threshold for acute toxic responses. Furthermore, the apparent susceptibility
of some animals and the resistance of others may simply be a reflection of
individual variation in the rate of emzymatic detoxification of the administered
substance. Many of the reports encountered in preparing this review have demon-
strated that considerable inter- and intraspecies variations exist in suscepti-
bility to the toxic actions of DNP and its related compounds.
The potential for cumulative toxicity by the dinitrophenols
seems to be significant only for the alkylated derivatives, according to a report
by Burkatskaya (1962). He administered DNP, DNOC, dinoseb, and dinitro-o-
propylphenol to rats and cats by single oral dose of from 10 to 100 mg/kg body
weight. He found that DNP was completely eliminated within 24 hours, and that
DNOC cleared from the organs in three days and from the blood within five days.
Dinoseb was retained for at least five days, and dinitroiro-o-propylphenol per-
sisted for ten days.
One report has stated (Makhinya, 1969) that, unlike the
dinitrophenols, the ortho-, para-, and meta-mononitrophenols have distinct
cumulative properties. With these compounds, chronic administration to warm-
blooded animals caused alterations of neurohumoral regulation and pathological
changes including gastritis, enteritis, colitis, hepatitis, neuritis, and
hyperplasia of the spleen. Limiting doses were established for the disruption
of conditioned reflex activity and set at 3 mg/kg for ortho- and meta-nltrophenol,
and 1.25 mg/kg for para-nitrophenol.
(i) Chronic Cataract Development
An investigation of cataract development in guinea pigs
by subacute administration of various nitrophenols was conducted by Ogino and
Yasukura (1957). This phenomenon was studied with respect to the effects of a
386
-------
vitamin C-deficient diet upon susceptibility to cataract formation. It was
found that the daily oral intake of 10 mg of DNP could produce cataracts in
vitamin C-deficient guinea pigs within 14 to 18 days. Control animals, however,
which received ascorbic acid supplements along with the DNP-treatment did not
develop eye lesions. Among the other compounds testeds it was demonstrated that
cataracts could be formed by subacute administration of p_-nitrophenol, 2-nitro-4-
aminophenol, 2-amino-4-nitrophenol, and 4-nitro-6-cyclohexylphenol. Both
ortho- and meta-nitrophenol were inactive as cataractogenic agents.
A cataractogenic agent excreted in the urine of
rabbits given DNP orally was identified as 2s4-diaminophenol. Vitamin C-deficient
guinea pigs developed cataracts after injection for six days with 2-amino-p_-
quinoneimine, the oxidized form of 2,4-diaminophenol. Previous experiments
with cataractogenic substances have resulted in various quinoid substances
being identified as the proximate cataract-producing agent in animals given
naphthalene, tyrosine, and galactose. Therefore, the authors concluded that
DNP is metabolized to an active cataract-forming substance by the following
scheme:
NH-CO-CH,
2,4-Dinitro-
phenol
2,4-Diaminophenol
Figure 70.
2-Amino-
p-quin6neimine
(cataractogenic
substance)
Metabolism of Dinitrophenol and Production of a Cataractogenic
Substance (Ogino and Yaskura, 1957)
387
-------
The exact role of vitamin C deficiency could not be
clearly defined in this study. It was shown, however, that the presence of
hydroxyl and nitro groups in a para-position seems to be essential for activity.
In addition to the nitrophenols, the compound 2,6-
dichloro-4-nitroaniline (DCNA) is also known to produce eye lesions in dogs.
Although toxic specificity to the eye is a relatively rare occurrence with most
chemicals and drugs, the fungicide DCNA was an effective producer of phototoxic
corneal and lens opacities in dogs upon subacute administration (Bernstein et al.,
1970). DCNA was fed in the diet of dogs at levels of 0.75, 6.0, 24, 48, or 75 mg
per kg of body weight per day. Except for damage to the eye, dose levels up to
48 mg per kg per day produced none of the classical signs of chronic toxicity
when dogs were treated for up to two years (Table 107). Irreversible corneal
and lens opacities appeared within about 55 days at the 48 mg/kg dosage, and
several weeks later at the 24 mg/kg dose level. No abnormalities of any kind
were seen at dosages below the 24 mg/kg level.
It was found that exposure to normal outdoor sunlight
was essential for the development of eye lesions produced by DCNA. Furthermore,
administration of DCNA in the absence of light did not reduce the time required
to produce eye damage once exposure to light had begun. This observation indicated
that a cumulative drug deposition had not taken place. Eye lesion formation was
found to be dose-related, in that long periods of exposure to small quantities
of DCNA had no effects. As was the case with the nitrophenols, cataract forma-
tion by DCNA seemed to be related to the presence of para-nitro substituent,
and possibly an active quinone intermediate formed by metabolism of the parent
compound.
388
-------
Additional investigations by Earl et al. (1971) looked
further into the chronic toxicity of DCNA to both dogs and swine. Their findings
revealed that DCNA fed at 192 mg/kg/day was lethal to dogs within 49 to 53 days,
while dosages of 48 mg/kg/day or below produced no mortality. Swine were not
affected in any way, regardless of dose level. Corneal opacities were observed
in dogs within 77 days when given DCNA at 48 mg/kg/day and exposed to sunlight.
Changes in the eye could be seen in as little as 13 days when animals were given
75 mg/kg/day, thereby indicating a dose-related response. This compound provides
a good example of a case where the determination of mortality or LD values is
not sufficient to characterize its true toxic potential.
b. Nitroaniline
In contrast to the apparent non-cumulative properties of
DNP derivatives in animals, compounds of the nitrobenzene series can display
very pronounced cumulative effects. Vasilenko e_t^ _al_. (1974) noted that mono-
nitroanilines had weak cumulative effects, whereas the introduction of two
chlorine atoms into the molecule would increase their cumulative properties.
In addition, mononitroanilines were both hemotoxic and hepatotoxic, while
dinitroanilines could act as respiratory inhibitors and uncouplers of oxidative
phosphorylation.
c. Chloronitrobenzenes
The Chloronitrobenzenes are also known to be cumulative
poisons (Davydova, 1967), with the most active compound being the para-isomer
and the least active compound the ortho-isomer. Aside from their cumulative
toxicity to animals, the monochloronitrobenzenes have also been investigated
for allergenic action, due to their similarity with 2,4-dinitrochlorobenzene,
the extremely potent skin sensitizer in both animals and man (see Section III-B-4),
389
-------
Rusakov et^ al. (1973) exposed rats to concentrations of
ortho- and para-chloronitrobenzene in air at levels which had been detected
in the vicinity around industrial plants. Animals were made to inhale, over a
five month period, either the para- or ortho- isomer at a concentration of
3
0.008 mg/m . A third group was subjected to a combination of both substances,
3
each present at 0.008 mg/m . The state of sensitization was determined by the
presence or absence of circulating antibodies at the end of the treatment.
The results demonstrated that para-chloronitrobenzene produced a strong state
of sensitization, as also did the mixture of both isomers. ortho-Chloronitro-
benzene produced sensitization as well but was the least potent substance in
that regard. The authors found that it was possible to achieve a passive
transfer of allergy by injecting a leucocyte mass and blood serum from sensi-
tized rats into the skin of recipient guinea pigs.
Both the ortho- and para-isomers of chloronitro-
benzene were also established as contact skin sensitizers, although their
activity was considerably less than 2,4-dinitrochlorobenzene. It was noted
that three drops of a one percent solution of 2,4-dinitrochlorobenzene applied
to the skin of guinea pigs would sensitize all guinea pigs tested, whereas the
same dose of ortho- or para-chloronitrobenzene was not effective. Increasing
the concentration to 10 percent resulted in sensitization of all animals re-
ceiving para-chloronitrobenzene but was effective in only half of those treated
with ortho-chloronitrobenzene..
d. Nitrobenzene
A comprehensive study on the effects of chronic poisoning
by nitrobenzene in rabbits was undertaken by Yamada (1958). His observations
390
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amply illustrate the pronounced development of toxic symptoms that occurs with
increasing time of nitrobenzene exposure.
Daily subcutaneous injections of 0.7 ml (840 mg) per kg body
weight of nitrobenzene were given to eight rabbits over a six month period. This
treatment evoked a three stage response; an initial response stage, a resistance
stage, and a final exhaustion stage. Anemia occurred during the first stage
which disappeared during the resistance stage, only to reappear again before
death. Increasing reticulocyte counts progressed throughout the entire experi-
ment. In the last stage of poisoning, a breakdown of metabolic detoxification
processes was evidenced by reduced capability for amination, acetylation, and
hydroxylation reactions, and heavy output of urinary metabolites. Loss of
appetite and emaciation occurred during the final stage of poisoning, and exten-
sive histopathological damage was noted (Table 108).
As was the case with chronic poisoning by the dinitro-
phenols, several of the nitrobenzene-treated rabbits died early in the experi-
ment, while most of the remaining animals survived until being sacrificed
after six months. Pathological comparisons of the animals dying early with
those that survived revealed that the only difference was in damage to the
adrenal cortex. It is not clear whether individual differences in adrenal
function can account for variation in resistance to chronic nitrobenzene
poisoning. However, the role of the adrenal gland in response to environ-
mental stresses and adaptation through compensatory physiological mechanisms
is clearly vital to survival.
391
-------
Table 108. Chronic Effects of Subcutaneous Nitrobenzene Injections on Tissues
of the Rabbit (Yamada, 1958)
Liver
Yellowish appearance, intense stasis.
Fatty degeneration, irregular cellular cord, turbid swelling and
karyolysis of liver cells.
Slight hyperplasia of connective tissues, considerably severe
cellular infiltration.
These findings are slight in degree in 3 of 8 rabbits.
Kidney
Swelling and adhesion of glomeruli, scanty fluid in glomeruli,
slight cellular infiltration.
Slight hyaline droplet degeneration of tubular cell.
These findings are more remarkable in 4 of the 8 rabbits.
Spleen
Enlargement is seen only in 3 of the 8 rabbits and which also
survived more than six weeks.
Intense stasis, opening of sinus and appearance of megakaryocytes,
hypertrophy and hyperplasia of reticuloendothelial cells,
yellowish brown pigments are seen in many cells.
Hypertrophia and hyaline degeneration of vascular wall.
Malpighian follicle is clearly observed.
Adrenal
In 5 rabbits: The fascicular layer is narrow in width.
Degenerated darkish and small cellular groups with pyknotic
nucleus, disorder of cellular cord and the lack of intracellular
minute vacuole are seen in the fascicular layer.
In 3 rabbits: The findings described above are rare.
Formation of submembranous cortical nodules is seen.
In both groups of rabbits: Few findings in the reticular and
glomerular layer and in the medulla.
Heart
Slight wax and vacuole degeneration, atrophy of muscle.
Lung
Hypertrophy of alveolar wall and megakaryocytes in alveolar
vessel are seen only in one rabbit.
Bone-Marrow
Increase in megakaryocytes.
Pancreas
Degeneration of Langerhans' Insule is seen in high grade only
in one rabbit.
392
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e. Nitrotoluenes
In terms of chronic toxicity, the mono-, di-, and tri-
nitrotoluenes are probably less hazardous than their corresponding nitrobenzene
derivatives. Kovalenko (1973) noted that the hemotoxicity resulting from oral
administration of these compounds to rats for one to three months decreased in
the order trinitrotoluene > dinitrotoluene > m-nitrotoluene > p_-nitrotoluene >
£-nitrotoluene.
Studies by Shils and Goldwater (1953) indicated that diet
was an important factor in the susceptibility to dinitrotoluene poisoning. They
found that a high-fat diet increased the resistance of rats to the lethal effects
of 2,4-dinitrotoluene when administered by injection but not when it was given
in the diet. This discrepancy may have been due to a higher food intake on
the high-fat diet, which caused a greater ingestion of the chemical when incor-
porated in the food. A high-protein diet, on the other hand, reduced the inci-
dence of mortality by 2,4-dinitrotoluene, regardless of the mode of administration.
A previous report by Shils and Goldwater (1950) stated
that susceptibility of rats to trinitrotoluene poisoning was not influenced by
either the amount or type of fat in the diet. Protein content in the diet was
likewise ineffective in altering TNT toxicity.
f. Trinitrotoluene (TNT)
Much of the published work concerning the subacute toxicity
of TNT to animals was reviewed by Von Oettingen (1941, 1944), Dacre and Rosenblatt
(1974), and Jaffe et^ al. (1973). Early studies indicated that a marked varia-
tion in susceptibility to TNT poisoning occurred among the different mammalian
species. When administered to dogs, the most significant features of subacute
poisoning were anemia and red blood cell destruction, as well as reduced hemoglobin
393
-------
levels and a compensatory increase in the reticulocyte count. The feeding
of TNT to dogs also produced ataxia, incoordination, diarrhea, and darkened
urine after two to three doses at 5 to 100 mg/kg body weight. Damage to
the central nervous system developed with subacute feeding of 50 mg/kg/day
for 12 weeks. Repeated injections of TNT to dogs has produced serious dis-
turbances of the gastric and pancreatic secretions, as well as changes in bile
secretion (Kleiner, 1969, 1971, 1972; Kleiner et al., 1974).
The rat and rabbit are clearly less affected by TNT
exposure, although the reason for this is unknown. Rabbits given 200 mg/kg
body weight of TNT by subcutaneous injection every other day would survive for
17 to 57 days (Jaffe et_ a±., 1973). However, when cats were given daily doses
of 50 mg/kg body weight by subcutaneous injection, they died in four to nine
days. The treatment of rats with daily oral doses of TNT at 30 mg/kg body
weight for six days produced only a decrease in phagocytosis, which could be
prevented by niacin administration.
A great deal of further investigation remains to be com-
pleted with respect to the toxicity of the munitions-related nitrotoluene
derivatives. An extensive series of investigations have been undertaken by
the U.S. Army Medical Research and Development Command to determine the toxicity
of TNT in different animals by various routes of exposure (Glennon, 1975).
These studies are aimed at establishing environmental quality standards for
unique munitions water and air pollutants. They include aquatic and mammalian
toxicity determinations for TNT and dinitrotoluene, as well as inhalation tox-
icity studies on the mononitrotoluenes.
394
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3. Sensitization
There is little doubt that several nitroaromatic chemicals
are active skin-sensitizing agents. Most notable among this group is 2,4-
dinitrochlorobenzene (DNCB), which is considered to be one of the most potent
contact allergens and primary skin irritants known to man (see Sections III-B-4-b
and III-C-1-e). Documented evidence of accidental human exposures to DNCB
is very limited, but controlled studies with humans have demonstrated without
question that DNCB is a nearly universal skin sensitizer. Contact with DNCB,
even in minute concentrations, must certainly be avoided at all costs.
A recent study (Krawiec and Gaafar, 1975) has compared the
allergenic and primary skin-irritant properties of DNCB in dogs. Although
dogs are generally resistant to experimental allergic contact dermatitis,
sensitization was established in all of 14 pups following DNCB challenge.
As expected, seven non-sensitized control pups could not be made to react
to a DNCB challenge dose. The pups were sensitized, in most cases, by intra-
dermal injection with 0.1 ml of 0.1% DNCB every other day for a total of 10
injections. All pups were challenged two weeks after the last sensitizing
injection by placing six to eight patches containing DNCB on various skin
locations. The reactions to DNCB challenge involved slight erythema and edema
in two days, moderate erythema and edema after eight days, with severe erythema
and edema at the patch application site in two animals.
To determine the primary skin irritation caused by DNCB,
closed patches were applied to the ventral skin of non-sensitized dogs, which
contained solutions of 1%, 5%, and 10% DNCB in ethanol. Reaction to DNCB was
395
-------
most severe at the 10% dosage and involved complete necrosis and loss of structure
of the epidermis after one to five days of exposure. The dermis became ede-
matous, and massive infiltration with polymorphonuclear cells also took place.
Microscopically, the lesions of DNCB-induced primary irritant dermatitis were
said to be similar to those produced in guinea pigs and man. In contrast
to the cellular changes seen in primary skin irritation by DNCB, the lesions
of allergic contact dermatitis produced infiltration by mononuclear cells
and a much milder dermal edema. The allergic reaction to DNCB reached a peak
in intensity three to four days.post challenge, and by seven days the inflam-
matory response had subsided. In primary skin irritation by DNCB, regeneration
of the epidermis seemed to begin within 72 hours of the application.
Considerable evidence has been accumulated from occupational
studies among munitions factory workers that dermatitis and skin sensitization
are common health hazards. Many reports have been made which link exposure
to trinitrotoluene (TNT), picric acid, and trinitroanisole to the development
of severe dermatitis and skin irritation (Von Oettingen, 1941; Schwartz, 1944).
It is difficult to distinguish from the evidence presented, however, whether
all cases have resulted from true allergic sensitization or may have involved
primary skin irritation as well. Schwartz has stated that sensitization to
TNT, ammonium picrate, and picric acid is known to occur in chronically exposed
workers. These reactions are most common for TNT at the hands, wrists, and
forearms, as well as at points of friction on the body such as the collar
and belt lines. With anmonium picrate and picric acid, the site of dermatitis
usually involves the face, especially around the mouth and sides of the nose.
Edema, papules, and vesicles develop, which are subsequently followed by desquamation.
39f
-------
Allergic sensitization to tetryl is a well-known fact, sup-
ported by a number of extensive occupational studies. A study on a working
population of 800 to 900 employees engaged in tetryl manufacture (Probst et al. ,
1944) revealed that four percent of the workers had dermatitis. A review
of 404 cases of tetryl dermatitis indicated that age, sex, and race did not
affect susceptibility. They observed that most cases occurred among new workers
one to two weeks after their introduction to tetryl. This evidence is clearly
suggestive of the mechanism of delayed contact hypersensitivity to DNCB, which
produces a state of sensitization in humans within two weeks after their initial
exposure (see Section HI-B-4-b). Schwartz (1944) also reported that tetryl
dermatitis was probably the most common cutaneous hazard associated with munitions
manufacture. He cited a working population of 6,394 persons exposed to tetryl
in which 1,904 (30%) developed tetryl dermatitis in the first six months of
operation. Dermatitis cases generally reached a maximum number after about
the third week. Most workers could become "hardened" to tetryl one to four
weeks after the development of dermatitis and no longer be affected by exposure.
In one shell-loading plant, 85% of the workers who had been affected became
non-reactive, which may suggest a possible state of chronic immunosuppression,
due to continuing tetryl exposure.
An extensive review on tetryl toxicity has been prepared by
Bergman (1952) which presented evidence for tetryl dermatitis being due to
allergic sensitization rather than local skin irritation. The author sum-
marized the results of animal studies whereby guinea pigs were sensitized to
397
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tetryl following intradermal injection, subcutaneous implantation, or smoke
inhalation. Animals become reactive to tetryl ten to fourteen days after
the initial exposure. It was noted that guinea pigs sensitized to tetryl
were also cross-sensitized to picryl chloride and 2,4,6-trinitrophenetole.
Several occupational studies reported by Bergman were concerned with the in-
cidence of dermatitis among tetryl workers. Table 109 summarizes ten years of
experience at the Picatinny Arsenal and shows that a combined average of
6.05% of exposed workers developed dermatitis.
Table 109. Tetryl Exposure at Picatinny Arsenal; Incidence of Dermatitis Treated
(Bergman, 1952)
Year
1941
1942
1943
1944
1945
1946
1947
1948
1949
1950
Exposed
... 2,710
4,410
4,140
2,500
2,669
917
507
649
767
.... 1,182
Dermatitis
5.0%
7.6%
7.4%
7.7%
6.9%
4.8%
6.0%
5.1%
5.8%
4.2%
Additional studies were cited by Bergman, which revealed the remarkably high
prevalence of dermatitis as the principal toxic reaction to tetryl. One report
involved 1,258 cases of tetryl poisoning, 75% of which were due to dermatitis
alone. Another report was made of 3,807 cases of dermatitis, none of which
involved any systemic poisoning.
398
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A few isolated reports of human exposures to various
nitroaromatic substances causing allergic dermatitis have appeared in the
literature. One incident was noted where a man developed contact dermatitis
from handling 3,5-dinitrotoluamide (Bleumink and Nater, 1973; see Section III-C-1),
Other reports have described allergic skin reactions in humans to pentachloro-
nitrobenzene (Finnegan et^ a!L., 1958; see Section III-C-1-e) and nigrosine (Calnan
and Connor, 1972; see Section III-C-1-e). Sensitization may also be achieved by
exposure to nitrobenzene and nitrotoluenes (Mayer, 1954). Studies with animals
have clearly demonstrated that contact sensitivity may be produced in response
to dinitrofluorobenzene (Schneider, 1974), as well as to picric acid and picryl
chloride (Chase and Maguire, 1974).
4. Mutagenicity
Information is very limited with regard to the mutagenic potential
of most nitroaromatic compounds. Mutagenicity data are particularly important,
however, not only in assessing environmental chemicals as potential hazards to
reproduction, but also in predicting carcinogenicity. Kriek (1974) has stated
that all carcinogens are also mutagenic (but not necessarily vice-versa).
Furthermore, the International Agency for Research on Cancer now includes muta-
genicity data in its monographs on the evaluation of the carcinogenic risk of •
chemicals to man.
A study has recently been conducted on the effect of various
phenolic compounds, including DNP, on chromosomes of bone marrow cells from
mice (Micra and Manna, 1971). Mice were injected intraperitoneally with
varying doses of DNP and bone marrow tissue collected 24 hours after treat-
ment. The results, summarized in Table 110, showed that DNP produced mainly
chromatid type breaks. There was no linear relationship, however, between
the frequency of chromosome aberrations and the dose of DNP.
399
-------
Table 110. Frequency of Chromosomal Aberrations Induced by Saturated Solution
of DNP After 24 Hours of Treatment (Micra and Manna, 1971)
No. of
Dose in ml Metaphases
Counted
0.25 290
0.50 230
1.0 250
No. of Meta-
phases with
Aberrations
20
34
42
No. of
Chromatid
Breaks
20
45
57
% of
Aberrations
6.9
14.5
17.2
In Affected
Cell Break
per Chro-
mes ome
0.025
0.033
0.034
The commonly employed herbicide dinoseb acetate was tested
for mutagenicity by measuring its effect on the induction of mitotic gene con-
versions in a diploid strain of the yeast Saccharomyces cerevisiae (Siebert
and Semperle, 1974). This test is regarded as a sensitive indicator of com-
pounds which produce base-pair substitutions and frame-shift mutations. The
results demonstrated that dinoseb acetate did not significantly increase con-
version frequency over the control level.
400
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Using a bacterial mutational system based on reversion to
tryptophan independence (try ) in Escherichia coll, Clarke (1971) tested the
ffiutagenic activity of pentachloronitrobenzene (PCNB). He found that PCNB
caused a ten-fold increase in try to try revertant numbers, but only in the
her (excision repair deficient) strain. PCNB was not found to be mutagenic
in the her (excision repair competent) strain.
When tested for mutagenic activity in mice, PCNB did not sig-
nificantly increase the mutation rate in studies by Busselmaier and coworkers
(1973). In addition, they demonstrated that p-nitrophenol was also ineffective
as a mutagenic agent in the same test system.
A report abstracted from the foreign literature (Romanova and
Rapoport, 1971) detailed the results of treating spores of Actinomyces sphaeroides
for two hours with various nitro compounds. Among these compounds were meta-
and para-nitroaniline, meta- and para-nitrophenol, meta- and para-nitrobenzalde-
hyde, ortho-chloronitrobenzene, para-nitrotoluene, and nitrobenzene. At. con-
centrations of 0.001 to 0.004 M these chemicals caused a decrease in viability
of 6 to 80%. The greatest number of morphological alterations were caused by
meta-nitrobenzaldehyde (25%), and the fewest by para-nitroaniline (12.6%).
The nitroaniline isomers and meta-nitrobenzaldehyde produced marked mutagenic
effects at 0.001 M.
More recently, mutagenicity tests have been conducted on hair
colorants and constituents containing nitrophenylenediamines (Searle et al., 1975)
The two compounds, 2-nitro-p_-phenylenediamine (2-NPPD) and 4-nitro-o-phenylenedi-
amine (4-NOPD) were tested in bacteria which detected either base-substitution
401
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or frame-shift mutations. Negative results were obtained using those
bacteria which reverted by base substitution, but both 2-NPPD and 4-NOPD
were found to be mutagenic when tested against the frame-shift mutant detecting
strains. 4-NOPD was about three times more potent as a mutagen than 2-NPPD at
equivalent doses (Table 111).
Table 111. Mutagenicity of 2-Nitro-p_-Phenylenediamine and 4-Nitro-o-Phenylene-
diamine in £. typhimurium TA1538 With or Without Liver Microsomal
Activation (Searle et al., 1975)
Dose
Applied
Test Compound
2-NPPD
4-NOPD
Induced his
5 yg per Elate
43 27
133 188
Revertants per Plate
50 yg per
I -(S-9)
335
883
Plate
213
727
Samples were assayed in the presence or absence of benzo(a)pyrene-induced
rat liver supernatant (S-9 mix).
Because many mutagens are known to be chromosome-breaking agents,
both 4-NOPD and 2-NPPD were added to cultures of human peripheral blood lymphocytes
to test for the production of chromosome damage. In this system, 4-NOPD failed
to show any chromosome damage in cultures at concentrations up to 100 yg/ml
during incubation for 48 and 72 hours. Similar experiments with 2-NPPD, however,
produced a considerable number of chromosome and chromatid gaps and breaks
at concentrations between 50 yg/ml and 100 yg/ml (Table 112).
402
-------
Table 112. Effect of 2-Nitro-£-Phenylenediamine on Cultured Human Lymphocytes
(Searle et al., 1975)
Number of Cells with Aberrations
Total Cells Total
Examined B Cells C Cells Abnormal
2^-NPPD present from Time
Zero (ug ml *)
75
50
25
2-NPPD Present for 24 h
before Harvesting
( yg ml"1)
75
50
25
Control
100
100
100
100
100
100
100
10
8
5
21
17
2
3
1
1
0
2
0
1
2
11
9
5
23
17
3
5
Human peripheral blood lymphocyte cultures were collected at 48 h. Cells
were stained with orcein and scored for aberrations but not fully analyzed.
B cells have chromosome or chromatid gaps or breaks only, whereas C cells
have stable or unstable chromosome rearrangements
5. Teratogenicity
Only a few reports have appeared in the literature concerning
teratogenic effects from exposure to nitroaromatic compounds. The concept
of teratogenesis used in searching the literature was applied in its broadest
sense and included (1) structural and/or functional abnormalities occurring
during gestation, (2) embryotoxicity or fetal death and resorption, and (3) fetal
growth retardation. Nevertheless, the material which follows in this section is,
for the most part, limited in its scope and suggestive of the need for further
research.
a. 2,4-Dinitrophenol
Hagstrom and Lonning (1966) conducted a detailed analysis
of the morphogenetic effect on the sea urchin embryo of 2,4-dinitrophenol (DNP).
In their study, direct observations were made on the relative rates of cleavage
and course of development both during and after periods of treatment with DNP.
403
-------
They found that pretreatment of unfertilized eggs with DNP
had little effect on the subsequent development of the egg following insemina-
tion. On the other hand, there were several distinct effects on the cells of
-4
developing larvae subjected to DNP treatment. At concentrations above 10 M
a cytostatic effect on cleavage takes place, such that cells remain in the inter-
phase portion of mitosis until DNP is removed. Even though mitosis could be
reinitiated, developmental irregularities were noted in the embryo, which were
dependent upon the duration of DNP treatment. In addition, it was found that
the effects of DNP were more persistent on embyros which had been exposed
during an early developmental stage (e.g. 16-cell stage) rather than during
the late blastula or gastrula phase.
-4
While concentrations of DNP at about 10 M stopped cell
division, development of the larvae progressed even in the continued presence
-4
of DNP at concentrations below 10 M. These larvae, however, showed signs of
inhibited differentiation, and development of the gut, skeleton, and arms was
inhibited in relation to controls.
Cytological examination of the mitochondria of sea urchin
blastomers revealed a definite swelling and aggregation following treatment in
DNP. The mitochondria appeared to be paralyzed and lacked the characteristic
"jerking" movements in the cytoplasm displayed by controls. Upon removal of DNP,
new mitochondria began to form, but the aggregated clusters from the DNP treat-
ment persisted. Pathological changes noted in the development of the larvae
exposed to low concentrations of DNP were attributed to the failure of mito-
chondrial populations to recover from the cytostatic effects of DNP.
404
-------
A report of teratogenic synergism following the combined
administration of DNP and insulin to chicks was made by Landauer and Clark
(1964). Insulin is a well-known teratogen, causing various structural abnor-
malities when administered to chicks during the first two days of incubation.
The injection of 100 yg/egg of DNP was non-toxic and non-teratogenic after
96 hours of incubation. However, the combined administration of 1.5 I.U. of
insulin with 100 yg of DNP dramatically raised the incidence of embryo mortality
and shortened upper beak (Table 113).
Table 113. Results of Experiments in Which Either Insulin or 2,4-Dinitrophenol
or Both Compounds Were Injected into the Yolk Sac of Eggs of White
Leghorn Fowl After 96 Hours of Incubation (Landau and Clark, 1964)
Insulin (I.U.) 1.5 1.5
2,4-Dinitrophenol (yg) — 100 100
Treated '184 281 185
Mortality percent
to end sixth day 17,9 53.0 3.2
7-13 3.8 0.7 2.2
14-22 26.1 23.5 18.3
Hatched percent 52.2 22.8 76.2
Survivors of thirteenth day
Normal percent
Micromedia percent
Parrot or short lower
beak percent
Short upper beak percent
Miscellaneous defects percent
144
84.0
15.3
4.2
1.4
2.1
130
70.8
15.4
3.8
18.5
3.8
175
97.7
0.0
0.0
0.0
1.7
The combination of insulin with sodium salicylate, another uncoupler of
oxidative phosphorylation, also caused great synergistic increases in embryo
mortality and other malformations. These results suggest a possible en-
hancement of teratogenic potency by certain compounds via the concomitant
405
-------
uncoupling of oxidative phosphorylation. It may likewise be postulated that
other nitroaromatic uncouplers (e.g., DNOC, nitrosalicylanilides) would also
magnify the teratogenic effects of certain substances.
When Gibson (1973) treated pregnant mice during,early
organogenesis with oral or intraperitoneal doses of DNP, no significant morpho-
logic defects were noted in the fetuses (Table 114).
b. 2-sec-Butyl-4,6-dinitrophenol
The teratogenic potential of 2-sec-buty1-4,6-dinitropheno1
(dinoseb) was studied in considerable detail by Gibson (1973). Dinoseb was
administered daily to groups of pregnant mice by intraperitoneal or subcutane-
ous injection and by oral intubation. Treatments were given either through-
out organogenesis (days 8-15 of gestation), during early organogenesis
(days 10-12), or during late organogenesis (days 14-16).
When given intraperitoneally at 17.7-20.0 mg/kg/day,
dinoseb produced hyperthermia in the dams and some maternal deaths. Those
that survived bore litters of smaller number and size than control dams. The
incidence of gross soft tissue and skeletal anomalies produced by dinoseb
during early organogenesis is given in Table 115. When administered through-
out organogenesis at 5 mg/kg/day, dinoseb produced no fetal anomalies; this
dose was considered to be the no-effect level.
The subcutaneous administration of dinoseb produced
maternal toxicity at similar doses to intraperitoneal injection but dinoseb
was not as effective by this route in producing fetal malformations. The
no-effect level for dinoseb by this route of administration was fo\ind be about
10 mg/kg/day.
406
-------
Table 114. Effect on Resorption Rate and Fetal Size of 2,4-Dinitrophenol (DNP) Administered to Mice
.During Early Organogenesis (Days 10-12 of Gestation) (Gibson, 1973)
*-
o
Molar Equivalents Number of
Dose Level" of DNP of Dinoseb " Pregnant Number of Number of
(mg/kg/day) and Route (mg/kg) Mice Treated Implantations Fetusest
0 9 13 + 1 12 + 1
7.7 (ip) 10 8 13+1 12 + 0
13.6 (ip) 17.7 8 13+1 11 + 1
25.5 (oral) 32 7 14+1 13 + 1
38.3 (oral) 50 7 13+1 12 + 1
- Mean response/litter + SEM.
* Values marked with an asterisk differ significantly from those of controls: *P<0.05.
Fftal
Fetal Crown-Rump
Resorptionst Body Weightt Lengtht
(%) (g) (cm)
4.4 + 2.0 1.409 + 0.036 2.7 + 0
5.6+1.8 1.383 + 0.034 2.6 + 0
14.1 + 7.1 1.307 + 0.038* 2.6 + 0*
9.6+3.0 1.351+0.029 2.7+0
6.1 + 2.9 1.366 + 0.037 2.6 + 0
-------
Table 115. Gross, Soft-Tissue, and Skeletal Anomalies in Offspring of Pregnant Mice Given Dinoseb
by I.P. Injection During Early Organogenesis (Days 10-12) (Gibson, 1973)
o
00
0
Anomalies No. of litters examined ..; 8
Gross
Oligodactyly
Imperforate anus
Acaudia
Microcaudia
Brachygnathia
Amelia
Micromelia
Open eyes
Soft-Tissue
Internal hydrocephalus
Hydronephrosis
Cleft palate
Enlarged bladder
Adrenal agenesis
Skeletal
Ribs : supernumerary
fused
absent
Sternebrae: fused
absent or not ossified
Vertebrate: fused
not ossified
absent
Long bones absent or not ossified
0
0
0
0
0
0
0
0
14.6 + 8.4
5.4 + 5.4
1.8 + 1.8
3.6 + 2.4
0
27.2 -1- 12.2
0
0
0
6.6 + 4.3
0
0
0
0
Incidence of
10.0
11
0
0
0
0
0
0
0
3.6 + 3.0
92.0 + 3.0*
15.6 + 4.5
1.3 + 1.3
0
0
13.1 + 6.8
0
0
0
11.2 + 3.6
0
0
0
0
Anomalies Following Treatment at
12.5
7
1.3 + 1.3
0
0
0
0
0
0
1.1 + 1.1
97.1 + 2.9*
23.4 + 6.3
0
0
0
23.4 + 13.6
22.1 + 9.4
0
0
19.7 + 7.2
4.4 + 2.9
0
0
4.8 + 3.1
15.8
2
2
8
55
18
9
20
14
34
2
7
9
.9
.9
.3
.9
.9
.4
.7
.3
.0
.0
.1
.1
7
0
+
+
0
0
0
0
+
+
+
0
+
0
+
+
0
0
+
+
0
+
+
2.9
2.9
5.9
11.5*
10.2
4.6
6.1
14.3
13.9
2.0
7.1
7.1
Dose Levels (mg/kg/day)
17.7
13t
35.9 + 9.8*
19.2 + 8.7* .
17.5 + 7.7*
25.3 + 8.0*
2.5 + 2.5
16.3 + 8.7*
5.5 + 3.9
0
76.2 + 8.6*
31.6 + 8.1*
0
1.4 -1- 1.4
16.2 + 9.9
26.0 + 8.9
54.5 + 11.6*
12.5 + 7.7*
15.6 + 7.8*
56.5 + 9.8*
76.2 + 8.1*
19.5 * 8.8*
30.8 + 10.5*
41.3 + 10.7*
of
18.8
15
6
1
7
8
20
18
8
10
24
37
25
37
13
6
.0 +
.7 +
.7 +
.8 +
0
0
.3 +
0
.5- +
.5 +
0
.2 +
.0 ±
.7 +
.2 +
0
0
.8 +
.2 +
0
0
.3 +
13.1
6.7
1.7
6.5
8.3
5.5
5.7
5.3
10.0
5.9
13.9*
13.6
13.9*
9.9
Only 12 litters examined for soft-tissue anomalies.
Values are the mean percentage responses/litter + SEM and those marked with asterisks differ significantly from those of controls:
*P<0.05.
-------
Doses of dinoseb as high as 50 mg/kg/day were given by
oral intubation during early organogenesis but had no effect on fetal size or
survival. Oral treatment at any stage of gestation did not produce statistically
significant gross or soft tissue anomalies, but skeletal defects were noted in
some groups at very high doses levels (20-32 mg/kg/day). The no-effect level
for teratogenicity and embryotoxicity throughout organogenesis was set at
20 mg/kg/day.
The author concluded that these results indicated a dose-
response relationship for teratogenesis by dinoseb which was dependent on the
route of administration. Furthermore, a threshold level seemed to exist such
that high doses produced terata and a lower dose could be established below
which teratogenicity did not occur.
In a later study, Preache and Gibson (1974 a) investigated
the effect of maternal food deprivation on teratogenicity by dinoseb. Pregnant
mice were deprived of food for 24 hours on the ninth day of gestation and given
intraperitoneal injections of dinoseb at 15.8 mg/kg/day for the next three days.
Litters from the food-deprived, dinoseb-treated mothers had a higher incidence
of anomalies than those from mothers given dinoseb alone. Although the inci-
dence of anomalies was increased, the types of defects produced were qualita-
tively the same from food-deprived and non-food-deprived mothers.
The effects of environmental stress were also investigated
in combination with dinoseb treatment to produce teratogenicity in mice (Preache
and Gibson, 1974 b). Dinoseb was administered at 0-17.7 mg/kg/day to pregnant
mice on days 10, 11, and 12 of gestation. By forcing the mice to swim for two
hours after the day 11 treatment, the incidence of external, soft tissue, and
409
-------
skeletal anomalies was reduced significantly. Reducing the ambient temperature
to 4°C for two hour periods had no effect, but raising the temperature to 32°C
on day 11 lowered the dose required to produce maternal death and embryo toxicity.
c. Pentachloronitrobenzene
Jordan and Borzelleca (1973) treated pregnant rats by oral
intubation of from 100 to 1,562 mg/kg/day of pentachloronitrobenzene (PCNB)
on days 6 through 15 of gestation. On day 20, when dams were sacrificed and
fetuses removed, examination revealed no significant incidence of skeletal
or soft tissue anomalies in PCNB-treated rats as compared to controls.
In another study, however, (Courtney, 1973) PNCB was
found to inhibit kidney formation in the mouse fetus. Renal agenesis was pro-
duced in 80% of mouse litters by oral administration of 500 mg/kg daily from
day 7 to 11 of gestation.
More recently, a reevaluation of PNCB teratogenicity was
conducted in the rat by Jordan et^ al_. (1975). Pregnant rats were treated dur-
ing the most teratogen-sensitive period of gestation (days 6-15) with daily
oral doses of PCNB at 8, 20, 50, or 125 mg/kg body weight. The treatment had
no significant effect on the number or position of implantations, incidence
of dead and resorbed fetuses, viable litter size, fetal sex ratios, and birth
weights. Fetal examinations were performed on day 20 of gestation, both
visually and by histopathologic analysis. External, skeletal, and soft tis-
sue malformations in the offspring of PCNB-treated rats did not differ sig-
nificantly from the incidence of defects in negative controls.
410
-------
6. Carcinogenicity
The production of cancer by most nitroaromatic compounds has
not been definitely established as an occupational hazard to man. However, a
number of nitroaromatic chemicals are active tumor initiators, tumor promoters,
or complete carcinogens in various animals. Furthermore, because metabolic con-
version of nitro groups to amino, nitroso, and hydroxylamino substituents can
occur, nitro compounds which are initially inactive may generate highly car-
cinogenic substances.
The nitroaromatic compounds which are known to produce tumors
fall into three general categories: 1) derivatives of 4-nitroquinoline-N-oxide;
2) certain mono-ndtro and heterocyclic nitro compounds such as polychlorinated
nitrobenzenes, and derivatives of 5-nitrofuran, 5-nitroimidazole and 4-nitro-
benzene; and 3) nitro analogs of classical aromatic amine carcinogens such as
4-nitrobiphenyl, 2-nitrofluorene, 2,7-dinitrofluorene, 4-nitrostilbene, and
derivatives of nitronaphthalene.
All of the known tumor-producing nitroaromatic substances appear
to owe their activity to a highly reactive metabolic intermediate rather than
to the parent compound. Recent studies by Poirier and Weisburger (1974) and
Sternson (1975; see Section III-B-3) support the belief that the carcinogenic
potential of many aromatic nitro compounds is probably caused by their biological
reduction to hydroxylamino intermediates by the scheme:
RN02 -> RNO -»• RNHOH -> RNH
These intermediates would presumably share the common properties of all car-
cinogens as suggested by Miller and Miller (1971) and Kriek (1974), in that
they 1) are generated in their active form by reaction with intracellular
macromolecules at the tissue site where they initiate carcinogenesis, 2) are
411
-------
transient intermediates if formed by metabolic interconversion, 3) are highly
electrophilic, and 4) are all strong mutagens.
Cancer formation in both animals and man induced by the aromatic
amines has been known for many years and was recently reviewed by several authors
(Kriek, 1974; Arcos and Argus, 1974). The nitroaromatic compounds, on the other
hand, have not received extensive treatment in the cancer literature, even though
it is clear that their metabolism may involve many of the same active nitroso
and hydroxylamino intermediates. Generally speaking, the nitro analogs of aromatic
amine carcinogens, however, will be considerably less active as tumor-producers.
In all likelihood this is due to the fact that they must first be enzymatically
reduced to active forms, a process which is species-dependent and may occur
very slowly jin vivo.
In terms of structure-activity relationships among aromatic
carcinogens, a fundamental observation is that unsubstituted polycyclic aromatic
hydrocarbons with less than four condensed rings do not generally possess any
carcinogenic activity (Arcos and Argus, 1974). The addition of amino groups
or other so-called "amine-generating" groups (nitro, nitroso, hydroxylamino) to
the inactive polycyclic nucleus can produce significant carcinogenic activity.
The most important aromatic skeletons which become carcinogens by amino sub-
stitution are pictured in Figure 71. For maximum carcinogenic activity, the
amino substituent(s) must be introduced at the terminal carbon atom(s) of the
longest conjugated chain, as illustrated in Figure 71.
412
-------
0
©
Naphthalene
Anthracene
Q< >=< >© 0
3(\=CH-CH=/}© ©/)=N-N=/)©
\=/ \ / \ / \ /
Stilbene
Azobenzene
Figure 71. Polycyclic Aromatic Carcinogen Skeletons (Arcos and Argus, 1974)
413
-------
Several nitro derivatives of the above structures have already
been identified as tumorigens in animals. However, many of the nitroaromatic
compounds have yet to be tested for carcinogenicity, while others have been
found to be inactive as tumor-producing agents. Several of the nitroaromatic
chemicals which have been tested for carcinogenicity are presented in Table 116.
A discussion follows concerning individual nitroaromatic compounds of particular
interest to the cancer problem.
a. 4-Nitroquinoline-N-oxide (4NQO)
The carcinogenic properties of 4NQO have been the subject
of many intensive studies in the past (Endo ^t^ al. , 1971). It is one of the
more potent carcinogens used in experimental tumor studies, causing cancer
at the site of application in cutaneous or subcutaneous tissues. In addition,
4NQO can cause lung carcinomas in the rat (Mori, 1962)., as well as increased
hepatoma occurrence in rats fed dimethylaminoazobenzene as part of the diet
(Takayama, 1961).
While 4NQO is predominantly an experimental tool and
not an industrial compound with environmental contamination potential, it
is important nevertheless to consider the biological fate of this classic
nitroaromatic carcinogen and its relationship to the metabolism of nitroaromatics
in general. Sugimura et al. (.1966) studied the activity of an enzyme in rat
liver and lung that reduces 4NQO to 4-hydroxyaminoquinoline-N-oxide (4HAQO)
by the following scheme:
NHOH NH,
4NQO 4HAQO
Figure 72. Metabolic Conversions of 4NQO (Sugimura et al., 1966)
414
-------
Table 116. Nitroaromatic Compounds Tested for Carcinogenicity
Ln
Compound
2-Amino-4- (p-nitro-
phenyl) thiazole
l-Chloro-2,4-
dtnitronaphtha-
lene
2,6-Dichloro-4-
nitroaniline
2 , 4-Dlnitroanisole
Species
Rats
Rats
Rats
Rats
Rats
Rats
Rabbits
Strain
or
Type
Sprague-
Dawley
Sprague-
Dawley
Sprague-
Dawley
Fischer
Fischer
Fischer
7
Number and
Sex
35 (F)
___
20 (F)
20 (F)
3 (M) + 2 (F)
14 (M) + 15 (F)
12 (M) + 12 (F)
7
Preparation
dnii
Administration
.011 in the diet for
weeks 0-6; then .025Z
for weeks 6-20; then
.0501 for weeks
20-21; then .010%
for weeks 21-25
500 mg oral suspen-
sion-single dose
10 doses every 3 days
for 30 days bv
gastric intubation
total dose = 3,000 mg)
100 mg orally
5 times a week for
52 weeks
30 mg orally 5 times
a week for 52 weeks
0.3-10 mg orally
5 times a week for
52 weeks
3% in propylene
glycol daily on the
skin
On ration
lit"
r.x.per ini'jnt.
50 weeks
6 months
9 months
18 months
18 months
18 months
90 days
Et f eccs
23 of 35 rats developed adenocarci-
noma of the breast
Of 19 survivors, 2 developed breast
cancer , 1 developed adenocarcinoma
of the lung
Of 17 survivors, 2 had carcinoma,
2 had fibroadenoma and 1 had
hyperplasia of the breast
One male rat developed a testlcular
interstitial cell tumor
Six males developed testicular
interstitial cell tumors; one female
developed an adenocarcinoma of the
breast
Six males developed testicular
interstitial cell tumors, one had a
thyroid adenoma and one had a fibroma ;
one female developed a lymphoma
Reference
Cohen e* al. , 1975
Griswold et al., 1966
Griswold et^ al. , 1968
Hadidian et^ £l. , 1968
Hadidian et_ al . , 1968
Hadidian et^ al. , 1968
Oraize et al., 1948
-------
Table 116. Nitroaromatic Confounds Tested for Carcinogenicity (Cont'd)
Compound
4,6-Dinitro-o-
cresol
.3,4-Dinitro-
dimethylanlllne
2,5-Dinitro-
f luorene
2,7-Dinltro-
f luorene
2 , 4-Dinitrophenol
1,2,3,4,5,6-
Hexachloro-7-
nltronaphthalene
2,2' , 4, 4', 6,6'-
Hexanitrostilbene
Species
Rabbits
Rats
Rats
Rats
Rats
Rats
Rats
Rats
Rats
Strain
or
Type
White
Dow and
B & L
Albino
Holtzman
Holtzman
Holtzman
Dow and
B & L
Sprague-
Dawley
Sprague-
Dawley
Number and
Sex
7
102 (?)
7
10 (M)
10 (M)
8 (M) + 8 (F)
70 (M)
20 (F)
20 (F)
Prepare cinn
and
Admin isc ration
5% in olive oil,
20 applications to
the skin
0.002-0.05% in the
diet
200 ppm in the diet
(15 mg/kR/day)
2.67 ma/kg of diet
for 12 months
1.62 mJl/kg of diet
for 8 months
1.62 mM/kg of diet
for 8 months
0.01-0.10Z in the
diet
500 mg oral
suspension as a
single dose
10 doses every
3 days by gastric
intubation (total
dose = 800 mg)
Durat ton
of
Experiment
4 weeks
182 weeks
18 weeks
12 months
10 months
10 months
179 days
6 months
9 months
Effe.-.ts
No tumors produced
No tumors produced
No tumors produced
No tumors. produced
One female developed mammary cancer ;
one female had a f ibroadenoma of the
breast
Eight females and one male developed
mammary cancer; two rats had cancer
of -the small intestine
No tumors produced
Of 19 survivors, none developed
tumors
All animals survived, no tumors were
produced
Reference
Spencer et al. , 1948
Spencer et^ al., 1948
Parker et al. , 1951
Miller et al., 1957
Miller et al., 1962
Miller et al., 1962
Spencer et_ al. , 1948
Griswold et al., 1966
Griswold et al., 1968
-------
Table 116. Nitroaromatic Compounds Tested for Carcinogenicity (Cont'd)
Compound
2-Hydrazino-4-
(g-nitrophenyl)-
thlazole
N-Methyl-N-2,4,6-
tetranltroaniline
(tetryl)
£-Nitrobiphenyl
2-Nitrof luorene
5-Nitro-2-furalde-
hyde semicarbazom
Species
Rats
Rats
Dogs
Dogs
Rats
Rats
Rats
Strain
or
Type
Sprague-
Dawley
Sprague-
Dawley
Mongrel
Mongrel
Holtzman
Sprague-
Dawley
Sprague-
Dawley
Number ;md
Sex
35 (F)
20 (F)
4 (F)
4 (F)
20 (M)
20 (F)
10 (F)
Pr.?D:ir:: r ion
.mo
Adm i n 1 s r t a t ion
0.10Z In the diet
for weeks 0-46;
(total dose =
540 mg/rat)
10 doses every
3 days for 30 days
by gastric intubation
(total dose - 400 mg)
300 mg orally
3 times a week
300 mg orally each
day
1.62 mM/kg of diet
10 oral doses every
3 days for 30 days
(total dose - 500 mg)
10 oral doses every
3 days for 30 days
(total dose = 350 mg)
Ourat ion
of
Expe.r :.r:enll
75 weeks
9 months
33 months
33 months
12 months
9 months
9 months
Effects
29 rats developed tumors; 11 were
adenocarcinomas and 9 were fibro-
adenomas of the breast
Of 19 survivors, 1 had hyperplasia. of
the breast f 1 had an adenoma of the
stomach
2 dogs developed urinary bladder
cystic carcinomas at 25 and 33 months
1 dog developed invasive cystic
epithelial tumor masses of the
bladder at 33 months
3 of 4 dogs developed urinary bladder
cystic squamous cell carcinomas
17 rats had squamous cell carcinomas
of the forestomach; 13, 4, 2, and 1
had tumors of the liver, ear duct,
small Intestine, and breast.
respectively
Of 5 survivors, 1 had carcinoma of
the breast
All animals survived; 1 had
hyperplasia of the breast
Ret erunce
Cohen et, a^l. , 1973
Griswold et_ al. , 1968
Deichmann et al. , 1965
Coplan, 1960
Miller et^ a_l . , 1955
Griswold, 1968
Griswold, 1968
-------
Table 116. Nitroaromatic Compounds Tested for Carcinogen!city (Cont'd)
Compound
N-[4-(5-Nitro-2-
furyl)-2-
thlazolyl]
acetamlde
2-Nitronaphthalene
£-Nitroperbenzoic
acid
c^-Nitrophenol
£-Nitrophenol
£-Nitrostilbene
Species
Rats
Monkey
Mice
Mice
Mice
Mice
Mice
Rats
Strain
or
Type
Sprague-
Dawley
Rhesus
ICR/Ha
Swiss
Millerton
ICR/Ha
Swiss
Millerton
CFN
(Swiss
Webster)
Sutter
Sutter
BDI and
Wistar
Number and
Sex
35 (F)
1 (?)
15 (F)
16 (F)
31 (F)
31 (F)
27 (F)
Preparac ion
and
Admin istrat ion
0.199% in the diet
for 46 weeks
(total dose a
10.3 g/rat)
242 mg/kg by stomach
tube 6 days a week
0.05 mg given s.c.
once weekly for
26 weeks
1 mg given s.c.
once weekly for
26 weeks
0.05 mg given s.c.
once weekly for
26 weeks
20% solution in
dioxane given twice
weekly on the skin
for 12 weeks
20% solution in
dioxane given twice
weekly on the skin
for 12 weeks
0.01%; then 0.04%
in the diet
Duration
of
Expt?r linent
66 weeks
54 months
21 months
21 months
21 months
12 weeks
12 weeks
Over 400
days
Effects
Of 34 survivors, 20 had flbroadenoma
of the breast
Multiple papillary tumors and
1 fungating tumor of the bladder
2 mice developed sarcomas at the
injection site
No tumors observed; 9 of -16 mice had
died by 6 months
1 mouse developed a sarcoma at the
injection site; 4 other tumors were
also observed
Among 30 survivors, no tumors were
seen
Among 30 survivors , no tumors were
seen
11 rats had papillomas and
forestomach squamous .cell carcinomas;
2 had otic carcinomas
Reference
Cohen et al. , 1975
Conzelman et_ al. , 1970
VanDuuren and Katz,
1972
VanDuuren and Katz,
1972
VanDuuren and Katz,
1972
Boutwell and Bosch,
1959
Boutwell and Bosch,
1959
Druckrey et al. , 1955
.p-
M
co
-------
Since it has been shown that 4HAQO is a more potent carcinogen than 4NQO itself
(Endo and Kume, 1965; Shirasu, 1965) it was suggested that 4NQO is carcinogenic
by virtue of its reduction to 4HAQO. Furthermore, its specific carcinogenic
activity in the lung and liver, the sites where a 4NQO-reducing enzyme has
been located, supports the argument that hydroxylamine derivatives of nitroaroma-
tic compounds are the true proximate carcinogens involved in tumor-production.
b. Nitrobenzene Derivatives
Several of the polychlorinated nitrobenzenes are active
both as tumor initiators and complete carcinogens. Searle (1966 a) treated
the skin of male and female mice twice weekly with 0.2 ml of a 0.3% solution
in acetone of pentachloronitrobenzene (PCNB) to test its tumor-initiating
ability. He also tested 2,3,4,5-, 2,3,4,6-, and 2,3,5,6-tetrachloronitrobenzene
(TCNB) under similar conditions. After 12 weeks of treatment, all mice received
applications of croton oil, a well-known tumor-promoting agent, for 20 weeks
(a tumor-initiating substance is one that produces tumors or carcinomas only
after the subsequent application of a tumor-promoting chemical such as croton
oil or phorbol ester). The results clearly indicated that all of the substances
tested were active tumor-initiators (Table 117, Figure 73). Multiple skin
papillomas began to show in all groups after five to eight weeks of croton
oil treatment and progressed until five to ten weeks after cessation of trts-it-
ment, whereupon some tumors regressed. The males in this experiment clearly
showed a tendency to develop tumors earlier and in greater number than did
females, except in the case of PCNB, where the reverse was true. This apparent
sex difference in tumor development could not be explained by the results of
the study.
419
-------
Table 117. Incidence of Skin Tumors on Mice During and After Treatment with Croton Oil, Following
Q
Applications of Some Chloromononitrobenzenes (Searle, 1966 a)
TEST COMPOUND
Acetone controls
Pentachloronitrobenzene
2,3,4, 5-Tet rachloro-
nitrobenzene
2,3,4, 6-Tetrachloro-
nitrobenzene
2,3,5, 6-Tet rachloro-
nitrobenzene
NO
SEX
(15)M
(15)F
(15)M
(15)F
(15)M
(15)F
(15)M
(15)F
(15)M
(15)F
SURVIVORS AT3
10
WK
10
10
10
10
8
8
10
10
10
10
20.
WKb
9
10
10
9
7
5
8
7
8
8
30
WK
8
8
9
7
6
3
7
4
7
6
40
WK
7
7
8
4
5
3
6
1
6
5
MICE WITH TUMORS AT*
10
WK
1
0
2
6
6
4
4
3
7
3 .
20,
WKb
6
3
6
7
7
7
6
5
8
6
30
WK
5
4
8
7
7
7
6
5
7
5
40
WK
1
4
7
7
7
7
6
5
7
5
TOTAL TUMORS AT
10
WK
1
0
4
13
26
5
12
4
18
6
20,
WKb
7
5
23
27
54
14
31
10
41
13
30
WK
6
7
29
33
61
19
39
11
39
14
40
WK
3
7
26
28
57
22
37
11
36
11
WKa TO
50%
TUMOR
INCI-
DENCE
16
13
10
7
14
13
19
8
15
N)
O
Time is measured' from start of croton oil treatment
End of croton oil treatment
-------
35 4O
WEEKS
Figure 73. Average Number of Skin Tumors/Surviving Mouse During and After
Treatment with Croton Oil (Searle, 1966 a)
KEY: Previous treatment with: acetone (-
PCNB (<
2,3,4,5-TCNB (Q-
2,3,4,6-TCNB (X-
2,3,5,6-TCNB (Q-
-O)
-x)
D)
(Reprinted with permission from the American Association for
Cancer Research.)
421
-------
Histologic examination of the papillomas formed revealed
that the tumors were generally non-malignant, the exception being a single
squamous-cell carcinoma on a mouse treated with PCNB, and one basal-cell
carcinoma on a mouse due to 2,3,5,6-TCNB treatment. In subsequent studies,
Searle (1966 b) established that these compounds were indeed active as complete
carcinogens when administered subcutaneously to mice.
Searle (1966 a) pointed out that the metabolic reduction
of the nitro group to the hydroxylamine has been shown to occur in the rabbit
with all compounds in this series (Bray et^ ail. , 1957). This process is probab-
ly an important mechanism in skin tumor initiation, just as it may be in the
activation of other nitroaromatic carcinogens.
The tumorigenic activity of PCNB was further studied by
Innes et al. (1969). Male and female mice were given a daily oral dose of
464 mg/kg body weight of PCNB from 7 to 28 days of age and thereafter received
the compound at 1206 ppm in the diet for the remainder of the 18 month study.
Their results (Table 118) showed that PCNB can act alone in producing tumors
at several different sites.
Strict pathological interpretation of the individual
tumors encountered was not attempted in this investigation. For the most part,
however, pulmonary tumors consisted mainly of adenomas, and lymphomas were
usually Type B reticulum-cell sarcomas. The use of the term "hepatoma" in
reporting results was not meant to imply that these tumors were benign, however.
The authors felt that a distinction between malignant and benign liver tumors
in the mouse could not be made, but that most hepatomas had malignant potential.
422
-------
Table 118. Tumor Formation in Male and Female Mice Receiving PCNB (Innes et al., 1969)
K>
Co
Compound
PCNB
Strain*
X
Y
Number of
Mice at Term
M
14
16
F
18
17
Total Mice
Necropsled
M
18
17
F
18
17
Weeks at
Term
M
78
78
F
78
78
Mice with
Hepatomas
M
2
10
F
4
1
Mice with
Pulmonary
Tumors
M
2
1
F
1
0
Mice with
Lymphomas
M
2
1
F
0
1
Total Mice
with Tumors
M
5
11
F
5
2
*Strain X = (C57BL/6 X C311/Anf)F ; strain Y = (C57BL/6 X AKR)F .
-------
Another nitrobenzene derivative, l-fluoro-2,4-dinitrobenzene
(DNFB), was found to be a powerful tumor promoter in female mice (Bock et al.,
1969). Well-known as a potent skin-sensitizer (see Section III-D-3), DNFB was
tested both as a tumor initiator and as a tumor-promoter in separate mouse skin-
painting studies. Animals were painted five times a week for 32 weeks with
0.25 ml of acetone containing DNFB in concentrations ranging from 0.03% to 3.0%.
Twenty-one days before DNFB treatment, selected groups of mice had been exposed
to 125 yg of 7,12-dimethylbenzanthracene (DMBA) as a tumor-initiating stimulus.
Another group received DMBA followed by painting with 0.03% croton oil and
served as positive controls. The results presented in Tables 119 and 120 show
that DNFB, while inactive as a tumor-initiator, was a very effective tumor-promoting
agent. The first skin tumors produced by DNFB appeared after four weeks of
treatment, as compared to the croton oil-treated positive control group, where
tumors first appeared after six weeks. By the end of the experiment, however, a
larger number of tumors had been produced by the croton oil treatment.
The authors concluded that, aside from the phorbol esters
(the active agents of croton oil), DNFB is one of the most potent tumor-
promoting agents known. While the mechanism of this observed tumor-promoting
activity is unknown, it has been postulated that a relationship may exist between
tumor promotion and disturbance of the immune system. The powerful ability of
DNFB and other skin-sensitizers (e.g., l-chloro-2,4-dinitrobenzene) to form
nonspecific conjugates with amino acids, peptides, or proteins in the body may
be important in the expression of this tumorigenic activity. Evidence has
not been encountered, however, which demonstrates any tumor-producing capa-
bilities for other nitroaromatic skin-sensitizers, such as l-chloro-2,4-
dinitrobenzene.
424
-------
Table 119. Tumor Promotion in Female Mice by l-Fluoro-2,4-dinitrobenzene (DNFB) (Bock et ^1., 1969)
fO
VJi
Initiating
S timulus
None
2 X 2500 yg DNFB
125 yg DMBA
Promoting
Stimulus
0.03% croton oil
0.03% croton oil
None
0.03% croton oil
0.3% DNFBC
0.1% DNFB
0.03% DNFB
No. of Mice
at Riskb
56
38d
30
30
24
30
21
Mice
No.
1
1
0
15
6
21
9
with Tumors
2
3
0
50
25
70
38
Total »o.
of Tumors
1
1
0
79
8
41
11
No. of Mice in
Which Tumors
Regressed
1
1
0
0
2
1
0
0.25 ml of acetone solution 5 times a week for 32 weeks.
ICR Swiss mice surviving at least 2 weeks of promoting stimulus.
6 of 30 mice died after only 8 applications of 0.3% DNFB; further treatment was discontinued.
22 of 60 mice died after the second DNFB treatment.
-------
Table 120. Tumor Promotion by l-Fluoro-2,4-dinitrobenzene (DNFB) in Various Stocks of Female Mice
(Bock ^t al., 1969)
Strain of Promoting
Mouse Stimulus3
Swiss 0.1% DNFBb
C57BL/6
BALB/c
Swiss Acetone only
£ C57BL/6
<^ BALB/c
Swiss 0.03% croton oil
C57BL/6
BALB/c
Swiss 0.5% dinitrophenyllysine hydrochloride
Duration of
Promotion
(weeks)
50
50
14
50
50
14
50
50
14
34
Mice with Tumors
No, of Mice
at Risk
50
30
30
50
30
30
50 .
30
30
50
No.
35
6
5
2
0
0'
49
20
5
0
%
70
20
17
4
0
0
98
67
17
0
Total No.
of Tumors
55
8
7
2
0
0
346
35
8
0
Tumors per
Tumor-bearing
Mouse
1.6
1.3
1.4
1.0
7.1
1.8
1.6
0.25 ml of acetone solutions, applied 5 times a week beginning 21 days after a single application of 125 yg of DMBA
in 0.25 ml of acetone.
50 Swiss mice treated with 0.1% DNFB, but not with DMBA, developed no tumors in 38 weeks.
-------
c. Heterocyclic Nltro Compounds
Several nitrofuran and nitroimidazole compounds have been
synthesized as anitbacterial agents and are widely used as feed additives in
animal meat production. Studies by Cohe.n et al. (1973, 1975) demonstrated that
several nitrofuran and nitroimidazole compounds were effective carcinogens
(Table 116).
d. Nitro Derivatives of Aromatic Amine Carcinogens
Recently, a number of nitro derivatives of naphthalene,
biphenyl, and fluorene have been shown to produce cancer in laboratory animals.
It is well-known that many of the amino derivatives of these same aromatic
structures are proven carcinogens in humans as well as animals.
Para-aminobipheny1 (xenylamine) is a powerful bladder
carcinogen in humans (Arcos and Argus, 1974; Kriek, 1974) whose activity has
been recognized for a number of years. A study by Deichraann et_ _al. (1965) has
indicated that para-nitrobiphenyl (PNB), an intermediate in the manufacture of
P ar a-aminob i pheny1, is just as potent a carcinogen in dogs as the amino deriva-
tive. Furthermore, they state that the bladder tumors found in humans who were
occupationally exposed to para-aminobiphenyl may well have been partially caused
by PNB. Their data indicated that 7 to 10 g/kg body weight of PNB fed to dogs
over a period of 25 to 33 months produced tumors of the bladder (Table 116).
This compared with 8.2 to 14.1 g/kg body weight of para-aminobiphenyl fed over
a period of 29 to 33 months to produce similar tumors. Obviously, both com-
pounds were about equal in their tumorigenic potency.
In another investigation, CopIan (1960) compared the
pathology of PNB-induced bladder tumors in dogs with those resulting from para-
aminobiphenyl (Table 116). The dog was chosen as the animal model to study
427
-------
because of the close similarity in the histological and functional structure of
the urinary bladder in both man and dog. It was found that the bladder tumors
induced by the feeding of either compound were always medium-grade squamous-cell
carcinomas and identical in every respect. From the above evidence, PNB must
be regarded as carcinogenic to humans and handled with the same precautions
accorded to para-aminobiphenyl.
Another agent known to produce bladder tumors among men
in the dyestuff industry is 4,4'-diaminobiphenyl (benzidine). The carcinogen-
icity of benzidine has been extensively reviewed by several authors (Hueper,
.1969; Scott, 1962; Arcos and Argus, 1974). A report has also been made by
Laham jjt _al. (1964) stating that the closely related compound 4,4'-dinitrobi-
phenyl is similarly carcinogenic to laboratory animals.
2-Acetylaminofluorene is a potent carcinogen over a wide
range of tissues and species when administered systesnically (Kriek, 1974). The
addition of nitro groups to the fluorene molecule also produces compounds with
carcinogenic activity (Miller et_ aj.. , 1955). The feeding of 2-nitro fluorene
i
to rats for a period of eight months produced a high incidence of multiple
tumors in the forestomach, particularly squamous-cell carcinomas (Table 116).
This carcinogenic effect of 2-nitrofluorene was particularly interesting due
to the high incidence of cancers (17 of 18 rats) and the unusual specificity
for gastric tumor formation. The stomach is a site where most carcinogens
generally do not produce malignancies.
Another derivative of fluorene which possesses carcino-
genic activity is 2,7-dinitrofluorene. The activity of this compound was
studied by Miller £t al. (1962) along with that of 2,5-dinitrofluorene. Only
the 2,7-isomer exhibited strong carcinogenic properties when fed to rats. These
effects were manifested mainly as mansnary cancers (Table 116). In tissues
other than the liver, 2,7-dinitrofluorene was found to be equally carcinogenic as
2-acetylaminofluorene.
428
-------
Bladder cancer in man has also been reported to be caused
by exposure to 2-naphthylamine (Arcos and Argus, 1974). Several nitro analogs
of this potent carcinogen have likewise exhibited tumorigenic activity in animals.
One of these compounds, 2-nitronaphthalene, has been reported to induce numerous
benign papillomas in the urinary bladder of a Rhesus monkey when it was fed for
almost five years (Conzelman jajt ai_. , 1970, Table 116). On the basis of these
findings, the author suggested that the same precautions be observed when handling
2-nitronaphthalene as with 2-naphthylamine or 4-nitrobiphenyl. Treon and
Cleveland (1960) were unable to induce tumors in either of two monkeys by the
single dose feeding of 2.1 or 4.7 gm/kg of 2-nitronaphthalene.
A nitro derivative closely related to 2-naphthylamine is
3-nitro-2-naphthylamine, which was tested for carcinogenicity in rats by
Weisburger £t _al. (1967). The compound was administered by oral lavage five
times weekly for 52 weeks at a dose of 10 mg per day (one-third the maximally
tolerated level). .Twelve tumors of the forestomach developed in 8 of 60 male
rats after 555 days, while 7 of 60 females developed a total of 12 tumors after
420 days; 4 lesions of the forestomach, 6 of the mammary gland, and 2 at other
sites.
Weisburger and coworkers (1967) also tested 1,2-dichloro-
3-nitronaphthalene for carcinogenicity in rats and found it to be less active
than 3-nitro-2-naphthylamine. After treatment with 30 mg five times weekly
for 52 weeks, 3 of 60 males and 5 of 60 females developed tumors by the end
of the experiment (564 days). The males developed two fibromas and one pitui-
tary adenoma, while four of the five females developed mammary tumors.
A more detailed examination of the carcinogenicity of
l,2-dichloro-3-nitronaphthalene at different dose levels in rats was made by
429
-------
Hadidian ett^ _al. (1968). As in the above study, the compound was found to be
only weakly tumorigenic when compared to controls (Table 121). Comparison of
l,2-dichloro-3-nitronaphthalene with 3-nitro-2-naphthylamine again found the
latter compound to be a much more active carcinogen and, in fact, more potent
than 2-naphthylamine under the conditions employed. As was seen with the case
of 2-nitrofluorene, an unusual propensity for the induction of forestomach
tumors was exhibited by 3-nitro-2-naphthylamine (Table 121).
Another naphthalene derivative, l-chloro-2,4-dinitro-
naphthalene, has shown weak carcinogenic activity in animals. Griswold et al.
(1966) studied the compound because it has a nitro group in the important 2-
position of naphthalene and is used for various insecticide, fungicide, and
synthesis applications. Administration of a single 500 mg/kg body weight dose
by gastric intubation in female rats produced carcinomas in three of twenty
animals. Two lobular carcinomas of the breast and one adenocarcinoma of the
lung were observed (Table 116).
In a subsequent study, the effects of multiple intragastric
doses of l-chloro-2,4-dinitronaphthalene were tested for carcinogenicity
(Griswold et al., 1968). The dosages were selected on the basis of preliminary
determinations to find the maximally tolerated dose. A total of 3,000 mg of
l-chloro-2,4-dinitronaphthalene was administered to each of 20 rats over a
period of just under 45 days. Of the 17 survivors of the acute and chronic
effects from exposure to the chemical, two developed carcinomas of the breast
and two developed fibroadenomas (Table 116). The high total dosage of compound
administered in this study to produce two malignant tumors indicates that 1-
chloro-2,4-dinitronaphthalene is probably not one of the more potent carcino-
genic threats to man.
430
-------
Table 121. Carcinogenesis by l,2-Dichloro-3-nitronaphthalene and 3-Nitro-2-naphthylamine (Hadidian et al. ,
1968)
Compound
Number
and Sex
Dose (mg)
Neoplastic Growths,
Number of Rats with Tumors
Non-Neoplastlc Lesions,
Number of Rats with Lesions
3-Nitro-2-naphthylamine
OJ
3 M
3 F
15 M
15 F
3 M
3 F
3 M
3 F
3 M
3 F
30
30
10
10
100
10
0.3
0.3
Interstitial cell tumor - testis, 1
Papilloma - stomach, -2
Squamous cell carcinoma (ear, stomach), 2
Adenoma (pituitary, lung), 2
Adenocarcinoma - breast, 1
Hepatotoxicity, 1
Papilloma - stomach, 2
Adenocarcinoma - breast, 2
Squamous cell carcinoma - ear, 1
Metastasis, 1
Interstitial cell tumor - testis, 10
Papilloma - stomach, 8
Lymphoma, 2
Mesothelioma - testis, 1
Adenoma - adrenal, 1
Metastasis, 2 '
Adenocarcinoma - breast, 5
Papilloma - stomach, 4
Fibroadenoma - breast, 2
Fibrosarcoma - cervix, 1
Interstitial cell tumor - testis, 2
Papilloma - stomach, 1
Fibroadenoma - breast, 2
Papilloma - stomach, 3
Interstitial cell tumor - testis, 1
Papilloma - stomach, 1
Basal cell carcinoma - stomach, 1
Papilloma - stomach, 2
Interstitial cell tumor - testis, 3
Papilloma - stomach, 1
Lymphoma, 1
Hepatotoxicity, 1
Hepatotoxicity, 1
Hepatotoxicity, 1
Hyperplasia - endometrium, 1
Polyp - uterus, 1
Compounds were administered by gastric intubation, in a volume of 1.0 ml or less, 5 days per week for 52 weeks. Observation of animals continued for
6 months past the end of treatment (total duration of study = 18 months).
-------
Table 121. Carcinogenesis by l,2-Dichloro-3-nitronaphthalene and 3-Nitro-2-naphthylamine (Hadidian et al. ,
1968) (Cont1d)
Compound
Number
and Sex
Dose (mg)
Neoplastic Growths,
Number of Rats with Tumors
Non-Neoplastic Lesions,
Number of Rats with Lesions
1,2-Dichloro-3-nitronaphthalene
3 M
100
Interstitial cell tumor - testis, 2
Adenocarcinoma - lung, 1
Fibroadenoma - breast, 1
Mesothelioma - testis, 1
3 F
100
None
14 M
30
Interstitial cell tumor - testis, 7
Fibroma , 2
Adenoma - pituitary, 1
CO
N>
15 F
30
Fibroadenoma - breast, 4
Fibrosarcoma, 1
Hyperplasia - endometrium, 1
Hepatotoxicity , 2
Hyperplasia - breast, 1
Polyp - uterus, 1
Compounds were
6 months past
3
3
3
3
3
3
3
2
M
F
M
F
M
F
M
F
administered by gastric intubation,
the end of treatment (total duration
10
10
3
3
1
1
0.
0.
in
of
Interstitial
Fibroadenoma
Interstitial
None
Interstitial
None
3 Interstitial
. 3 None
a volume of 1.0 ml or les
study = 18 months) .
cell
tumor -
testis
- breast, 2
cell
cell
cell
!S, 5
tumor -
tumor -
tumor -
days per
testis
testis
testis
week
, 2
, 3
, 1
Polyp - uterus, 1
, 2
for 52 weeks. Observation of animals continued for
-------
e. Miscellaneous Nitroaromatic Carcinogens
A carcinogenic bioassay of £-nitroperbenzoic acid was
recently conducted in two laboratories and reported by Van Duuren et^ al. (1972).
Mice were injected subcutaneously once weekly for 26 weeks and observed for
subsequent tumor development. In one laboratory, £-nitroperbenzoic acid at
0.05 mg injected subcutaneously once weekly for 26 weeks (total dose, 1.3 mg)
produced two sarcomas at the injection site in a group of 15 mice. In a sep-
arate study, the same dose produced one sarcoma and four tumors at other sites
in a group of 16 animals (Table 116).
Recent concern over the toxic properties of certain hair
dye constituents has led to investigations of their long-term adverse effects,
including carcinogenicity. Two hair dyes have now been tested, one of which
contains 2-nitro-p_-phenylenediamine (2-NPPD) and 4-nitro-£-phenylenediamine
(4-NOPD), and the other containing an aminonitrophenol (Searle et^ jal., 1975).
Commercial hair dye preparations containing these substances were tested by
repeated topical applications in aqueous acetone solutions to the skin of mice.
These tests were intended to resemble actual patterns of human usage. Pre-
liminary results demonstrated the formation of malignancies in 5 out of 48
mice receiving the preparation containing the two isomers of nitrophenylene-
diamine. A second strain of mice developed cancers in 4 out of 52 animals
receiving the same preparation. Two out of 32 mice of one strain and 3 out
of 32 mice of a different type developed malignant tumors when exposed to the
hair dye component containing aminonitrophenol.
All tumors in these experiments were of the lymphoid system;
no tumors were seen on the treated skin. Three mice developed lymphosarcomas,
while the rest had malignant lymphomas involving the spleen, with infiltration
to the liver and other organs. The possibility of oral ingestion of the compound
.' 433
-------
by mice during the process of grooming must be considered here, as well as the
potential role of oncogenic viruses which are known to cause lymphomas in mice.
Since these results are preliminary, a final judgement regarding the carcino-
genic hazard of certain hair dyes must be reserved until more extensive tests
have been completed.
An observation was made by van Esch and coworkers (1957)
that rats being fed for long periods with hexanitrodiphenylamine developed
large multiple mammary tumors. In a subsequent study they treated female rats
with hexanitrodiphenylamine in the diet at 500 ppm for about two and one-half
years. In addition, they also fed groups of rats with the closely related
compounds picramide and picric acid at the same concentration.
Their results, presented in Table 122, show a high inci-
dence of tumor formation in all treated groups. In the control animals, however,
Table 122. Incidence of Mammary Tumors in Female Rats Treated with Hexanitro-
diphenylamine (van Esch et al., 1957)a
Group
Controls
Hexanitrodiphenylamine
Picric acid
Picramide
No. of
Rats
19 (83)
10
10
8
No . Alive
After
2 Years
12 (63)
1
7
7 .
No. With
Mammary
Tumors
8 (18)
10
4
4
Average Age of
Appearance of
Tumors
25 months (.26)
19 months
22 months
29 months
aDosage, 500 ppm mixed in dry food. The numbers in parentheses indicate
total incidence as found in untreated rats, including that from other
experiments during the same period.
434
-------
a very high frequency of tumor development was also noted which tends to discount
the results seen with picramide and picric acid. Hexanitrodiphenylamine, on
the other hand, caused the formation of multiple mammary tumors (average, three
per animal) in all treated rats, whereas control animals rarely developed more
than a single tumor. All tumors in this experiment, including those in un-
treated rats, were non-malignant fibroadenomas, adenofibromas, or adenomas of
the breast.
7. Possible Synergisms
Exposure to chemical substances often results from contact with
a mixture of compounds rather than a single pure source. Even though the
majority of occupational, domestic, and environmental exposures to nitroaro-
matic chemicals will probably involve the concomitant exposure to any number
of synthetic organic substances, little attention has been given to the dangers
of synergistic and joint toxic actions. A report has been made by Smyth et al.
(1969) which characterized the relative synergistic toxic effects of nitro-
benzene in combination with 26 industrial chemicals. These results are presented
in Table 123.
Overall, nitrobenzene paired with various chemicals resulted in
11 pairs where the ratio of predicted/observed LD,-n exceeded 1.00 and 14 pairs
where the ratio was less than 1.00. The median unadjusted ratio was 0.98, which
does not tend to indicate a high potential for synergistic toxicity under the
conditions employed. While this relationship may hold true for other deriva-
tives of nitrobenzene, these results should not be extrapolated to the dinitro-
phenols, which produce their toxic effects by an entirely different mechanism.
435
-------
Table 123. Unadjusted Ratios of Predicted to Observed LD Values of Nitro-
benzene Mixed by Volume with Various Chemicals (Smythe et al., 1969)
Nitrobenzene Mixed With Predicted/Observed LD
Acetone 1.47
Acetonitrile 0.85
Acetophenone 0.98
Acrylonitrile 0.82
Aniline 1.32
Butyl Cellosolve 0.85
Butyl Ether 1.28
Carbon Tetrachloride 1.47
Diethanolamine 0.70
Dioxane 1.39
Ethyl Acetate 0.97
Ethyl Aerylate 1.07
Ethyl Alcohol 0.97
Ethylene Glycol 1.07
Formalin 1.20
Isophorone 0.62
Morpholine 0.86
Phenyl Cellosolve 1.12
Polyethylene Glycol 200 0.72
Propylene Glycol 1.00
Propylene Oxide 0.87
Tergitol XD 0.92
Tetrachloroethylene 0.82
Toluene 0.78
Ucon 50HB260 0.99
Ucon LB250 1.26
436
-------
In studying the affects of chronic poisoning in rats with
m-dinitrobenzene, Baede and Kiese (1949) noted a marked synergism with ethanol.
While dogs are not affected, chronically ethanol-poisoned rats are more suscep-
tible to the effects of m-dinitrobenzene. Furthermore, in chronically m-dini-
trobenzene- poisoned rats, ethanol has a more pronounced and long-lasting effect.
In addition, propanol, isopropanol, and acetic acid, but not butanol, produced
a similar synergism with m-dinitrobenzene.
E. Toxicity to Lower Animals
Several studies have been encountered which demonstrate selective
toxicity for various species of fish and aquatic organisms by nitroaromatic
compounds. The most prominent group of chemicals in this class which have
been tested for toxicity are the mononitrophenols (metabolite of parathions),
especially those containing halogens. Additional studies have also been con-
ducted to determine the effects of nitroaromatic herbicides, nitrosalicylanilides,
and TNT wastes on mortality among fish. Only very limited information is availa-
ble concerning either the toxicity of non-phenolic nitroaromatics or the toxicity
of nitroaromatic compounds to non-aquatic lower animals.
1. Nitrophenols
The problem of water-borne phenolic wastes has long been recog-
nized as a serious threat to many species of fish (Blyth and Blyth, 1920).
Lammering and Burbank (1960) conducted an investigation to look specifically
at the adverse effects of oj-nitrophenol on bluegill sunfish in order to deter-
mine the role of ortho-substitution of inorganic radicals on the phenol ring.
Fish were maintained under standard static conditions and exposed to o_-nitro-
phenol at various concentrations. Reactions of the fish to the substance were
observed over a 48 hour period. Plots of survival versus time of exposure to
pj-nitrophenol at four different concentrations are shown in Figure 74.
437
-------
CM
a
CM
<
100
90-
- 80-
o>
o
70-
-------
A loss of equilibrium occurred in fish at concentrations equal
to or greater than 49 mg/1 during the second 24 hour period. The median tolerance
limit (concentration that kills 50 percent of the fish) for o-nitrophenol was
determined to be 66.9 mg/1 for a 24 hour period and 46.3-51.6 for a 48 hour
period.
The difference between the 24 hour and 48 hour tolerance limits
indicates that a certain degree of cumulative toxicity is possible in bluegills
exposed to o-nitrophenol. These results are in contrast to those obtained by
exposing fish to phenol or chlorophenol, where little difference was seen between
24 hour and 48 hour tolerance limits. Even though the acute toxicity of o-nitro-
phenol is three-fold less than for phenol, the possiblity of chronic poisoning
by its cumulative action may create serious environmental problems.
The actual symptoms of poisoning in fish exposed to phenol,
chlorophenol, and ^-nitrophenol were quite similar in all cases. These included
nervous twitching with continuous quivering of the fins and jaws, suggesting an
increased rate of respiration. Rapid paroxysmal swimming was noted in response
to slight movements near the fish tanks, indicating that the fish were very
excitable. The loss of equilibrium preceded death by a considerable length of
time and occurred either as a temporary partial loss or, more commonly, a
permanent loss of function. These reactions of bluegills to phenolic contaminants
were so characteristic as to be potentially useful as a qualitative means of
bioassay for their detection.
2. Halogen-substituted Nitrophenols
Between 1953 and 1957 a total of 4,346 chemicals were screened
for activity as selective toxicants in controlling the parasitic sea lamprey of
the Great Lakes (Applegate et_ al_. , 1966). From all these compounds, a single
439
-------
substance, 3-bromo-4-nitrophenol, was found to have the desired biological
effect of being more toxic to sea lamprey larvae than to native fish species.
This finding led to a series of exhaustive tests on halogen-substituted mono-
nitrophenols for specific larvicidal activity. Results of these tests indicated
that a small group of nitroaromatic chemicals possessed the desired property
(Applegate et al., 1967). These compounds all contained a nitro group in the
4-position on a phenolic nucleus and halogens or a trifluoromethyl group sub-
stituted directly on the ring.
Six compounds were initially identified as effective larvicides
and tested further for toxicity to fish (Applegate jet al., 1958). The results
of these acute toxicity tests under static conditions are presented in Table 124.
Table 124. Differential Toxic Effects Among Larval Lampreys and Fishes of
Certain Mononitrophenols Containing Halogens (Applegate et al., 1958)
(Sodium salt is expressed in parts per million of free phenol.)
Concentration
Required to
Name and Form Kill All
of Compound Lamprey
Larvae
(pom)
2-Bromo-4-nitrophenol
Free phenol
Na salt
3-Bfomo-4-nitrophenol
Free phenol
5-Chloro-2-nitrophenol
Free phenol
2 , 5-Dichloro-4-nitrophenol
Free phenol
Na salt
3,4, 6-Trichloro-2-nitrophenol
Free phenol
Na salt
3-Trifluorjomethyl-4-nitrophenol
Free phenol
Na salt
5 .
7
5
3
3
5
5
13
2
2
Concentration Required to Cause
Significant Mortality t Among Fishes
(ppm)
Rainbow Brown Bluegill
trout trout sunfish
13 11
15
11 15
5 5
13 7
17
17 15
23
9 7
7 7
Mortality of approximately 10 percent of all test animals,
a,a,a-Trifluoro-4-nitro-m-cresol
440
-------
All of the trifluoromethyl, monohalogen, and dihalogen deriva-
tives of 4-nitrpphenol in this study were significantly more toxic to lamprey
larvae than to trout. The tri- and tetrahalogen-4-nitrophenols, on the other
hand, were more toxic to the fish than to lampreys. Furthermore, these com-
pounds were more toxic at lower concentrations than were any of the mono- and
disubstituted 4-nitrophenols.
Moving the halogen from the 2- to the 3-position in the mono-
nitrophenols did not greatly affect toxicity. The two isomers of trifluoro-
methyl-4-nitrophenol, however, were quite different in their species-specific
and lethal effects. Placing the trifluoromethyl group in the 3-position of
the ring increases the differential toxicity to 4.5 (see Table 125). 3-Tri-
fluoromethyl-4-nitrophenol is now widely used in the Great Lakes for sea lam-
prey control.
Additional studies were carried out on unsubstituted mononitro-
phenols, halogenated di- and trinitrophenols, and one polyhalogenated nitro-
phenol (Applegate £t ail. , 1966, 1967). Table 126 presents the results of tests
where fish and lampreys were exposed to these chemicals at concentrations up
to 5 ppm.
Earlier investigations had determined that aniline and nitro-
anilines were generally not toxic to fish unless a halogen was present. Simi-
larly, the alkylated phenols, such as the cresols, were not highly toxic when
a nitro group was added. On the other hand, the dinitroalkylphenols (e.g.,
dinitrocresol) were quite toxic to both fish and lampreys. In addition, the
alkylated phenols containing both nitro groups and a halogen were also toxic.
Several investigators have reported that fish are unable to
conjugate "foreign phenols" (Maickel £t al., 1958, 1959) and, in fact, that
443
-------
Table 126. Biological Activity of Phenol and Some Substituted Phenolic Compounds
a,b
Other Than Mononitrophenols Containing Other Halogens
(Applegate et. al. , 1966)
Compound
Biological
Activity
Phenol
Phenol (liquified USP XIV)
Toxic to RBT; not toxic to BG 5"L
Mononitrophenols
x-nitrophenol
2-nitrophenol
3-nitrophenol
4-nitrophenol
No toxic effect on any species
ditto
ditto
ditto
Dinitrophenols
2,4-dinitrophenol
Not toxic to L
Halo-dinitrophenols
4-fluoro-2,6-dinitrophenol (40 ppm)
4-chloro-2,6-dinitrophenol (40 ppm)
2-chloro-4,6-dinitrophenol
2,5-dichloro-4,6-dinitrophenol
3-bromo-2,4-dinitrophenol
3-bromo-4,6-dinitrophenol
Not toxic to RBT & L
ditto
Toxic to RBT & BG; not toxic to L
Not toxic to RBT, BG, or L
Not toxic to RBT or L
ditto
Halo-trinitrophenols
3-bromo-2,4,6-trinitrophenol
Not toxic to RBT or L
Poly-halo-mononitrophenols
4-bromo-2-chloro-6-nitrophenol
More toxic to RBT & BrT than to L
Maximum concentration tested: 5 ppm (except where otherwise noted after name
of chemical)
Abbreviations: RBT - rainbow trout; BrT = brook trout; BT = brown trout;
BG = bluegills; L = larval lampreys
444
-------
the major route of their excretion is outward diffusion via the gills. More
recently, however, Lech (1974) has demonstrated that conjugation of TFM in
rainbow trout and excretion in the bile is an extremely important factor in
determining the fish's survival. The significance of glucuronide conjugate
formation was measured by treating trout with salicylamide, a known inhibitor
of glucuronide formation, before injecting the fish intraperitoneally with TFM.
The results, depicted in Figure 75, clearly show that inhibiting the conjuga-
tion process increased the acute toxicity of TFM. This study indicates that,
in the case of water-borne phenolic compounds, the capacity of fish for glucuro-
nide conjugation may be an important factor in determining their survival.
o
n
s
CVJ
100-1
80-
^ 60H
o^
J-
"ro
o 40-
20-
Salicylamide
/
ide/
25 mg/l
i
25 )•
L
25
I
4
\
6
TFM Concentration ( mg/l)
Figure 75.
Enhancement of the Acute Toxicity of TFM to Rainbow Trout by Salicylamide
(Lech, 1974)
Solid line = controls; broken line = toxicity curve in presence of
25 mg/l of salicylamide. Numbers in parentheses indicate the number
of animals at each point. Horizontal bars indicate 95% confidence
limits.
-------
3. Nitrosalicylanilide
In the United States today, the sea lamprey toxicant which is
most commonly employed contains both TFM and 2*,5-dichloro-4'-nitrosalicylani-
lide in a 98:2 combination (Kawatski and Bittner, 1975). The nitrosalicylani-
lides are known to be among the most potent uncouplers of oxidative phosphoryla-
tion ever tested (see Section III-B-4). The toxicity of these compounds to
rainbow trout and sea lampreys was investigated by Starkey and Howell (1966).
The general structure-activity relationships governing selec-
tivity and lethality by the salicylanilides are given in Tables 127, 128, and
129.
Table 127.
Comparison of Molecular Requirements for Substituted Mono-halo-
nitrosalicylanilides Exhibiting Selective Toxicity to Larval Sea
Lamprey and Fingerling Rainbow Trout (Starkey and Howell, 1966)
V \\4-HH-
R
Compound
benzanilide
4 ' -chlorobenzanilide
salicylanilide
3-nitrosalicylanilide
4 ' -chloro-3-nitrobenzanilide
Substituents
R RI
. . . .
. . . .
-OH
-N02
-N02
4' -chloro- 3-nitrosalicylanilide -N02 -OH
4-chloro-3-acetamidosalicyl- -NH _QJJ
anilide COCH3
R2
. .
-Cl
••
• •
-Cl
-Cl
-Cl
Lamprey
LD100
(ppm)
>10.0
>10.0
9.5
3.0
>10.0
0.3
3.0
Trout
LD25
(ppm)
>10.0
>10.0
9.51
3.0
>10.0
0.7
2
3.0
LD25 at 9.5 ppm
1
LD100
446
-------
the major route of their excretion is outward diffusion via the gills. More
recently, however, Lech (1974) has demonstrated that conjugation of TFM in
rainbow trout and excretion in the bile is an extremely important factor in
determining the fish's survival. The significance of glucuronide conjugate
formation was measured by treating trout with salicylamide, a known inhibitor
of glucuronide formation, before injecting the fish intraperitoneally with TFM.
The results, depicted in Figure 75, clearly show that inhibiting the conjuga-
tion process increased the acute toxicity of TFM. This study indicates that,
in the case of water-borne phenolic compounds, the capacity of fish for glucuro-
nide conjugation may be an important factor in determining their survival.
D
o
CO
CNJ
<
100-1
80-
60H
o 40-
20-
Salicylamide
25 mg/l
35
25
i
• ( 25 )
20)
1
3
\
4
\
6
TFM Concentration ( mg/l
Figure 75.
Enhancement of the Acute Toxicity of TFM to Rainbow Trout by Salicylamide
(Lech, 1974)
Solid line = controls; broken line = toxicity curve in presence of
25 mg/l of salicylamide. Numbers in parentheses indicate the number
of animals at each point. Horizontal bars indicate 95% confidence
limits.
.445
-------
3. Nitrosalicylanilide
In the United States today, the sea lamprey toxicant which is
most commonly employed contains both TFM and 2",5-dichloro-4'-nitrosalicylani-
lide in a 98:2 combination (Kawatski and Bittner, 1975). The nitrosalicylani-
lides are known to be among the most potent uncouplers of oxidative phosphoryla-
tion ever tested (see Section III-B-4). The toxicity of these compounds to
rainbow trout and sea lampreys was investigated by Starkey and Howell (1966).
The general structure-activity relationships governing selec-
tivity and lethality by the salicylanilides are given in Tables 127, 128, and
129.
Table 127. Comparison of Molecular Requirements for Substituted Mono-halo-
nitrosalicylanilides Exhibiting Selective Toxicity to Larval Sea
Lamprey and Fingerling Rainbow Trout (Starkey and Howell, 1966)
V V!(_-_
Compound
benzanilide
4 ' -chlorobenzanilide
salicylanilide
3-nitrosalicylanilide
4 ' -chloro-3-nitrobenzanilide
Substituents
R Rj
• • • •
• • • *
-OH
-N02
-N02
4' -chloro- 3-nitrosalicylanilide -N02 -OH
4-chloro-3-acetamidosalicyl- -NH _QH
anilide COCH3
R2
. .
-Cl
••
• •
-Cl
-Cl
-Cl
Lamprey
LD100
(ppm)
>10.0
>10.0
9.5
3.0
>10.0
0.3
3.0
Trout
LD25
(ppm)
>10.0
>10.0
9.51
3.0
>10.0
0.7
2
3.0
LD25 at 9.5 ppm
I
LD100
446
-------
Table 128. Comparative Toxicity of Halonitrosalicylanilides to Larval Sea
Lamprey and Fingerling Rainbow Trout as a Function of Substituent
Loci (Starkey and Howell, 1966)
Compound
3 ' -chloro-3-nitrosalicylanilide
4 ' -chloro-3-nitrosalicylanilide
3 ' -iodo-3-nitrosalicylanilide
4 ' -iodo-3-nitrosalicylanilide
3" -bromo-3-nitrosalicylanilide
4 ' -bromo-3-nitrosalicylanilide
4" -chloro-5-nitrosalicylanilide
3 ' -f luoro-3-nitrosalicylanilide
4 ' -iodo-5-nitrosalicylanilide
4 ' -bromo-5-nitrosalicylanilide
2 ' -chloro-5-nitrosalicylanilide
2 ' -iodo-3-nitrosalicylanilide
2 ' -bromo-3-nitrosalicylanilide
4 ' -f luoro-3-nitrosalicylanilide
2 ' -f luoro-3-nitrosalicylanilide
2 ' -chloro-3-nitrosalicylanilide
4 ' -f luoro-5-nitrosalicylanilide
3 ' -chloro-5~nitrosalicylanilide
Lamprey
LD100
(ppm)
0.3
0.3
0.3
0.3
0.3
0.3
0.5
0.5
0.5
0.5
0.9
1.0
1.0.
1.0
3.0
3.0
3.0
15.0
Trout
LD25
(ppm)
0.9
0.7
1.0 '
0.7
1.0
1.0
1.0
0.9
1.0
1.0
3.0
3.0
l.O1
3.0
3.0
7.0
-
15.0
^100
447
-------
Table 129.
Selective Toxicity of Polysubstituted 3-Nitrosalicylanilides to Larval Sea Lamprey and
Flngerllng Rainbow Trout as a Function of Atomic Loci (Starkey and Howell, 1966)
oo
Compound
-3-nitrosalicylanilide
2', 3'-dimethyl-
2 ' -methyl-3 ' -chloro-
2',4'-dimethyl-
2 ' -methyl-4 ' -chloro-
2 ',5 '-dime thy 1-
2 ' -methyl-5 ' -chloro-
2',5'-dichloro-
2 ' -methoxy-5 ' -chloro-
2 ',6 '-dime thy 1-
2 '-'chloro-6 ' -methyl-
Substituents
R2 R3 Rii Re Rg
— CH3 — CHg . . . . . .
-CH3 -Cl
-CH3 .. -CH3 ..
-CH3 . . -Cl
— CH3 . . > . — CH3 . .
— CH3 . . . . — Cl . .
-Cl . . . . -Cl
-CH30 .. .. -Cl
— CH3 . . . . . . — CH3
-Cl . . . . . . -CH3
Lamprey
LD100
(ppm)
3.0
0.7
3.0
0.5
1.0
0.5
0.3
0.7
>10.0
0.7
Trout
LD25
(ppm)
5.0
1.0
7.0
0.7
3.0
0.9
0.9
1.0
>10.0
1.0
-------
The above data indicate that an ortho-hydroxy phenolic substitu-
ent in the carboxylic acid moiety of salicylanilide is required for biological
activity. In addition, maximum selectivity and lethality were obtained by the
presence of a nitro group and a halogen in the same molecule.
4. Agricultural Chemicals
The problem of pollution by nitroaromatic agricultural chemicals
in lakes and streams poses a serious threat to many forms of aquatic life. These
chemicals, introduced into surface waters by runoff, application over water,
misuse, or accident, can easily become incorporated into the aquatic food chain
and ultimately affect all levels of life.
The effect of several nitroaromatic herbicides on freshwater
crustaceans was studied recently by Sanders (1970). Six test species were chosen
which represent important links in the food chain for fish. Bioassays were con-
ducted under static conditions without aeration of the water. A comparison of
the relative subacute toxicity of trifluralin (2,6-dinitro-N,N-di-n-propyl-a,
a, a-trifluoro-p_-toluidine) to crustaceans and bluegill sunfish is pre-
sented in Table 130. A large variation in the degree of toxicity to various
crustaceans clearly exists for trifluralin. Nevertheless, trifluralin was one
of the most toxic of the 16 herbicides tested in this study and the most toxic
of all those tested against bluegill sunfish.
A time-response bioassay using scud as a test species was con-
ducted on three nitroaromatic herbicides; dinoseb, trifluralin, and balan
(N-butyl-N-ethyl-a,a,ct-trifluoro-2,6-dinitro-£-toluidine). The various TL -
values for these compounds are presented in Table 131. The decreasing TL
values for both trifluralin and balan with respect to time indicate that they
may act as cumulative poisons.
'449
-------
Table 130. Estimated 48 Hour TL Values of Trifluralin to Six Species of
Freshwater Crustaceans and One Species of Fish (Sanders, 1970)
Organism and Temperature
48 hr TL50
(mg/D
Waterflea (Daphnia magna) 21°C
Seed shrimp (Cypridopsis vidua) 21°C
Scud (Grammarus fasciatus) 15.5°C
Sowbug (Asellus brevicaudus) 15.5°C
Glass shrimp (Palaemonetes hadiakensis) 21°C
Crayfish (Orconectes nails) 15.5°C
Bluegill (Lepomis macrochirus) 24°C
0..56
0.25
1.8
2.0
1.2
50.0
0.019*
Median tolerance limit-concentration in water which produces a 50 percent
mortality.
3Value reported by Cope (1966)
Table 131. Estimated TL Values and Confidence Limits (P = .05) for Several
Herbicides to Scud (Sanders, 1970)
Herbicide
P,P'-DDT
Reference
at 48 hours
(mg/1)
Values and Confidence Limits (mg/1)
24 hours
48 hours
96 hours
Dinoseb
Trifluralin
Balan
0.0056
0.0032
0.0022
2.8(1.1-4.9) 2.5(1.2-4.7)
3.2(1.9-17) 1.8(1.6-12)
8.2(2.4-28) 4.0(2.7-5.8)
1.8(0.72-6.1)
1.0(0.30-3.6)
1.1(0.61-1.9)
450
-------
3 3
The herbicide dinitramine (N , N -diethyl-2,4-dinitro-6-tri-
fluoromethyl-m-phenylenediamine) has recently been tested for toxicity to several
species of freshwater fish (Olson et^ al. , 1975). The results from 96 hour static
toxicity tests are presented in Table 132. The authors noted that dinitramine
Table 132. Toxicity of Dinitramine (99 + %) to Nine Species of Freshwater Fish
in Soft Water3 at 12° (Olson et al., 1975)
Average
Species Weight (g)
Coho salmon 0.8
(Oncorhynchus kisutch)
Steelhead trout 0.3
(Salmo gairdneri)
Brown trout 0. 7
(Salvelinus trutta)
Lake trout 0.6
(Salvelinus namaycush)
Carp '1.0
(Cvprinus carprio)
Fathead minnow 0.8
(Pimephales promelas)
Channel catfish 0.8
(Ictalurus punctatus)
Bluegill 1.4
(Lepomis macrochirus)
Yellow perch 0.8
(Perca flavescens)
LC _ and 95% Confidence
Interval (mg/1) at
24 hr .
>1.51
1.20
1.06-1.36
1.27
1.05-1.53
1.15
0.986-1.34
>2.00
2.63
b
2.99
2.20-4.06
2.88
1.96-4.24
1.00
0.870-1.15
96 hr
0.600
b
0.590
0.510-0.682
0.590
0.510-0.682
0.920
0.776-1.09
1.18
1.02-1.36
1.44
1.07-1.93
1.37
1.04-1.81
1.52
1.14-2.02
1.00
0.870-1.15
pH 7.2-7.6; total hardness = 40-44 mg/1 as CaCO.,
*
Insufficient data for computation of. confidence intervals
residues were much more persistent in fish than'residues of TFM and, because
of its low solubility in water, it was rapidly partitioned across the gills and
absorbed into the circulation.
451
-------
Alabaster (1969) has reported the results of fish toxicity
studies using the herbicides trifluralin and dinoseb, and also the fungicide
dinocap (dinitrocaprylphenol). The test results are summarized in Table 133,
Table 133. Toxicity of Trifluralin, Dinoseb, and Dinocap to Harlequin Fish
(Rasbora Heteromorpha) (Alabaster, 1969)
Compound
Dinoseb
Trifluralin
Dinocap
Approximate
Composition (%)
9.0
46
25
Median Lethal
Concentration (ppm)
24 hr
3.4
•i.o
0.6
0.39
48 hr
3.0
0.6
-
0.27
Estimated
Threshold
(ppm)
2.7
0.35
-
0.13
Water
Type
tap
soft
tap
soft
Fabacher and Chambers (1974) have recently demonstrated that
resistance to herbicides is possible in insecticide-resistant fish. Drainage
canal mosquito fish exposed to dinoseb or trifluralin displayed LC values of
0.96 and 4.10 ppm, respectively, whereas farm pond mosquito fish had LC values
of 0.87 and 2.00 for the same compounds. These results showed a 2.05 fold re-
sistance to trifluralin in the drainage canal fish and is a unique example
of fish resistance to herbicides.
Approximate toxicity ranges for DNOC, dinoseb, and dinocap to
\
freshwater fish have been established by the British government (Mawdesley-
Thomas, 1971). The range within which aqueous concentrations are lethal to
fish were given as above 1.0 to 10 ppm for DNOC, above 0.1 to 1.0 ppm for
dinoseb, and above 0.01 to 0.1 ppm for dinocap. These tests, however, were
of short duration (24 and 48 hours) and do not provide very reliable data.
452
-------
It has been suggested by many investigators that aquatic bioassays be conduc-
ted over a minimum period of 96 hours.
The fungicide 2,6-dichloro-4-nitroaniline (DCNA) was evaluated
for toxicity using both fish and wildlife (Knott and Scott, 1968). The LC
(50% mortality) values for fish were derived from 48 hour and 96 hour bioassays,
while mortality in bobwhite quail and mallard ducks was determined by subacute
toxicity studies. Their results are presented in Table 134.
Table 134. Mortality in Fish and Wildlife by DCNA, DDT, and Diphenamid (Knott
and Scott, 1968)
Species
Bobwhite quail
Mallard ducks
Rainbow trout
o
Bluegill sunfish
Goldfish3
Diphenamid
>9,000
15,000
8.6
>32.0
34.0
• LC . (ppm active
p,p'-DDT
486
525
0.0028
0.0022
0.0025
ingredient)
DCNA
2,438
8,850
1.6
37.0
>32.0
p,P'-DDT
486
525
0.0032
0.0020
0.0049
Q
96 hour mortality
5. Trinitrotoluene
A comprehensive review of the toxicity of munitions-related
compounds was made by Dacre and Rosenblatt (1974) and includes toxicity of the
nitrotoluene derivatives to aquatic organisms. In this review, they cite tox-
icity data for TNT and related compounds to 5-8 cm "minnows" at 23-24°C (Table 135),
453
-------
Table 135. Acute Toxicity of Several Nitroaromatic Compounds to Fish (Dacre
and Rosenblatt, 1974)
Compound
Trinitrotoluene
m-Dinitrophenol
Dinitro-o- cresol
o-Mononitrotoluene
m-Mononi tro to luene
£-Mononitro toluene
6 Hour Minimum
Hardness, 12.5 ppm CaCO«
4.0-5.0
0.5-1.0
1.5-2.0
18-20
14-18
20-22
Lethal Dose (mg/1)
Hardness, 150 ppm CaCO
4.0-5.0
35-38
3.0-4.0
35-40
25-30
45-50
These comparative data show that TNT has a greater acute toxicity (measured as
mortality) than the mononitrotoluenes but still is considerably less toxic than
dinitrophenol. These findings are consistent with toxicity data obtained from
mammalian studies, which indicated that increasing the number of nitro groups
raised the toxicity of nitrobenzene derivatives, but that these non-phenolic
chemicals were still less toxic than dinitrophenols (see Section III-D-1).
These authors also noted that dinitrotoluene (isomer unknown)
had 24, 48, and 96 hour median tolerance levels in bluegills of 50, 27, and
16 mg/1 respectively.
Nay (1974) has recently shown that bluegills exposed to TNT
displayed a median tolerance limit of 2.6 mg/1 in 96 hour static bioassay tests,
indicating a stream standard of 0.02 mg/1.
An extensive evaluation of the toxicity of TNT wastes on blue-
gills was conducted by Pedersen (1970). Calculations were made of the LCsn
(median lethal concentration) of alpha-TNT for bluegills in specific time periods
and under varying conditions of water hardness and temperature. These results
are summarized in Figure 76.
Figure 77 depicts the percent mortality after 96 hours versus
TNT concentration in four different bioassays. Statistical analysis revealed
that water hardness did not alter toxicity but that temperature did.
454
-------
Ul
100
Survival Time, minutes
500 1000
o
c
c
u
3000 5000 7000
!'
i
•• i i
i
i •
... , .- .__.
1
.. ....
' ' ; "
! • !
i • '
i
J
! ! ""
i :
I
! ' i
j._:-_j:.- -.
Figure 76. Relative Susceptibility of Bluegills to TNT at Different Water Hardness* and Temperature
(Pederso.n, 1970)
*Hard water, 180 ppm as CaCO.; soft water, 60 ppm as CaCO .
-------
o
c
0
99.9-
99.8'
99
98^
95
90-^
80-
70-
60-
50-
40-
30-
20-
10-
5-
2
1
0.5'
0.2
0.1
Alpha-TNT Concentrations, mg/l
I 3 4 5 » 8 10
Harai^S0
I jil.T
f-HSoft,;25°
_ .1 ..:!...:: .... li.'L 1.
Figure 77. Mortality Curves at Four Days (Pederson, 1970)
456
-------
F. Toxicity - Plants
Only limited amounts of information are available concerning the
toxicity of nitroaromatic compounds to higher plants. The chemical groups
which have received some attention are nitrophenols, nitrobenzenes, and nitro-
toluenes. Again, nitrophenols have been most extensively investigated perhaps
because of the interest of researchers in these compounds for their ability to
specifically uncouple oxidative phosphorylation from electron transfer. Further-
more, a number of nitrophenolic compounds are used as pest control agents and
understanding the mechanism of their action has been important from the point
of view of developing new and specific pest control agents.
. 1. Nitrophenols
An extensive study of the effect of o- and p- nitro, 2,4-dinitro
and, 2,4,6-trinitrophenol has been carried out by Simon and Blackman (1953).
Phytoxicity was studied using three different assay systems: (1) Inhibition of
the growth of the duck weed, Lemna minor, (2) Inhibition of the respiration of
vacuum infiltrated leaf disc of Brassica alba, and (3) Mortality of seedlings
of Brassica aIba. The concentration of each substance required to halve the
growth rate, respiration, and mortality, respectively, were determined. Since
the degree of ionization affects the toxicity of nitrophenols, in the measure-
ments above, pH values below pKa (where there is little ionization) were used.
The results of this study are presented in Table 136. As can be seen, nitro-
phenols at low concentrations inhibited growth of Lemna minor, and respir-
ation of the leaf discs of Brassica alba. The seedlings of Brassica alba were
affected only at high concentrations of the toxicants; however, the results are
inconclusive since nitrophenols were sprayed on the seedlings and there may well
457
-------
Table 136. Relative Toxicity of Mono- and Dinitro-substituted Phenols on
Plants (Simon and Blackman, 1953)
Concentrations Required For
Compound
tested
o-nitro-
phenol
(pK = 7.3)
£-nitro-
phenol
(pK = 7.2)
2,4-dinitro-
phenol
(pK = 4.0)
2,4,6-tri-
nitrophenol
(pK = 0.8)
50% reduction of
growth
Lemna
4.5 x
6.8 x
8.0 x
2.4 x
7.6 x
rate of
minor
M
-4
10 (5.4)**
-5
10 (5.2)
10~6 (5.2)
_4*
10 o* (5.1)
10 (5.1)
50% reduction of
respiration rate
of infiltrated leaf
disks of Brassica
alba
M
_T
1.6 x 10 ^ (3.0)
1.2 x 10 (3.0)
-4
2.5 x 10 (3.0)
1.3 x 10~5 (3.0)
-
50% mortality
seedlings of
Brassica alba
M
> 2.2 x 10 7
> 3.6 x 10
-2
1.2 x 10~
> 9 x 10
1.5 x 10~2
-
of
(3.0)
(3.0)
(3.0)
(3.0)
(3.0)
* Results with two batches of picric acid.
** The pH at which toxicity was measured is given in parentheses after each figure.
458
-------
have been significant losses of the chemicals. Of interest was the observation
that the relative toxicity of nitrophenols in all three test systems was similar.
For example, p-nitrophenol was always more toxic than the o-isomer. The utility
of the toxicity data in terms of the environmental effects is hard to assess
because of the unusually low pH values used in the studies. At lower pH, it
is questionable if the physiological state of the cell is normal and consequently
if response to the toxicant is normal. Since the toxicity of nitrophenols de-
creases as the pH is raised above the pK, at physiological pH values the toxicity
will perhaps be much lower than that determined by Simon and Blackman (1953).
Their results could at best be indicatiye of the upper limit of the toxicity
of nitrophenols.
Seedling emergence in Vic la faba was effectively inhibited by
p-nitrophenol (0.1%) but not by the o-isomer (Amer and Ali, 1968). Both the
isomers, however, were able to cause appreciable injury when sprayed on the
plants. Among the other seeds tested, p_-nitrophenol was found to inhibit
germination of Tritricum vulgare, Gossypium barbadense, Pisum sativum, and
£-nitrophenol inhibited T_. yulgare and G. barbadense. .Similar to the obser-
vations of Simon and Blackman (1953) , these results also suggest that the £-
isomer is relatively more toxic than the o-isomer.
A number of researchers have studied the effect of DNP on up-
take and respiration of exogenously supplied substrates in plants. In sun-
flower hypocotyls, Reinhold and Eilam (1964) measured the exogenous respiration
14
by supplying the hypocotyls with C0_ evolved. 2,4-Dinitrophenol (DNP) was
reported to drastically reduce the exogenous respiration while the overall
CO- production (CO evolved from endogenous + exogenous substrate) was actually
459
-------
-4
increased (Table 137). DNP (1.4 x 10 M) also raised the Respiratory Quotient
for the tissue from 1.0 to 1.2. Since the specific activity of the CO- evolved
decreased in the presence of DNP, it was suggested that DNP reduced the contri-
14
bution of exogenous C-labelled substrate in the (XL given off. Humphreys and
Dugger (1959), working with corn roots, noted that DNP inhibited the uptake of
exogenous substrates. Such an effect could be attributed to the disorganization
of the cell membranes by DNP (Reinhold and Eilam, 1964).
Table 137. Effect of 2,4-Dinitrophenol (pH 5.8) on Exogenous Respiration by
Segments of Sunflower Hypocotyl (Reinhold and Eilam, 1964)
Exogenous Substrate
DNP Concen.
(M)
Percent Change Over Control
Overall
CO- evolved
14
CO- evolved Specific
activity^
of CO,,
14
14
C-Glutamic acid
2 x 10 3 M
C-Glucose
5 x 10 3 M
1.6 x 10
3.2 x 10
2 x 10~4
-4
+37
+19
+71
-27
-31
-44
-47
-67
*
Specific activity indicates the amount of exogenous substrate respired relative to the
total substrate.
From the studies described above, it is clear that nitrophenols
are toxic to plants. They interfere with the uptake and respiration of sub-
strates, inhibit plant growth, and cause mortality in seedlings at higher con-
centrations. Similar to the observations with other organisms, the toxicity in
plants increased in the series mononitrophenol, dinitrophenol, but decreases with
460
-------
a third nitro group. Among mononitro-phenols, the jj-isomers are generally more
toxic than the o-isomers in plants.
2. Nitrotoluenes
The phytotoxicity of some products of TNT manufacture (mono-,
di-, and trinitrotoluenes, and one transformation product, 4-amino-2-nitro-
toluene) has been investigated by Schott and Worthley, 1974 (Table 138). In
the aquatic flowering plant Lemna perpusilla, both 2,4-dinitro- and 2,4,6-
trinitrotoluene depressed growth or killed colonies at concentrations of 1 ppm
or above; 4-amino-2-nitrotoluene was inhibitory to the test organism only at
concentrations of 10 ppm or higher. On the other hand, ^-nitrotoluene showed
no harmful effect in the test organisms except when the concentration reached
100 ppm. The response of the organism to the nitrotoluenes in acidic (6.3 pH)
and basic medium (8.3 pH) was similar except in the case of 4-amino-2-nitro-
toluene which was slightly more toxic in alkaline than in acidic medium. Upon
comparison of the response of Lemna perpusilla to a known plant toxicant, 2,4-
dichlorophenoxyacetic acid, it was noted that dinitro- and trinitro-substituted
toluenes were approximately one tenth as toxic as the herbicide.
A field study to evaluate the effect of discharges from Radford
Army TNT plant (located on the New River, Virginia) on submerged and emersed
aquatic vegetation has been carried out by Mitchell (1973). The author sur-
veyed the number of species of aquatic flowering plants, mosses, and "stemmed"
algae of the phylum Charophyta, and determined the relative biomass upstream
(designated as reference) and downstream from the plant discharge. A change in
plant population (species distribution or in total biomass) was attributed to
the introduction of TNT waste. Ten stations were chosen at the river bank to
461
-------
Table 138. Summary of Lemna perpusilla Colony Growth When Exposed to Various
Nitrotoluenes
Compound
.o_-Nitro toluene
oj-Nitro toluene
4-Amino-2-nitrotoluene
4-Amino-2-nitrotoluene
2 , 4-Dinitrotoluene
2 , 4-Dinitrotoluene
2,4, 6-Trinitrotoluene
2,4, 6-Trinitrotoluene
2 , 4-Dichlorophenoxy-
acetic acid
(Positive Control)
(Schott
Test
PH
6.3
8.5
6.3
8.5
6.3
8.5
6.3
8.5
6.3
and Worthley,
1974)
Plant
*
responses
Concentrations (ppm)
100 50
D
0 0
D X
X X
D
D D
D
D
-
10
0
0
0
X
D
D
D
D
D
5 1 0.5
- 0 -
- 0 -
- 0 -
- 0 -
- X X
D X -
D X 0
D X -
- D -
0.1 0.01 0.001
0 -
_
00
0 -
00
0 -
X 0 0
D = Death; X = Decrease in growth rate; 0 = no effect; - = not tested.
evaluate the individual effects of various discharges; only stations 5P and
higher numbers were located downstream from the TNT manufacturing site. In-
terpretation of the results to determine the effect of TNT waste alone is com-
plicated because these stations were also receiving effluents from an oleum
plant and a power plant located upstream from the TNT plant (see Figure 78).
The changes noted at station 5P may actually be collective rather than due to
the TNT waste alone. At best, rough conclusions concerning the harmful effect
of TNT waste can be drawn considering station 4P as the reference. If a species
is present at station 4P but cannot be detected at stations 5P or beyond, it
462
-------
P — PLANTS
A — ALGAE
B — BOTTOM FAUNA
F- FISH
NEUTRALIZED
ACID EFFLUENT
BURNING
GROUND
RJJNOFFx
OLeUM
PLAMT
\ EFFLUENT
\
\ -duLAa*
Figure 78. The Effect of Waste Discharges from Radford Army Ammunition Plant on the
Biota of the New River, Virginia - Location of Sampling Points
(Mitchell, 1973) (Reprinted with permission from the Water Resources Research
Center, Virginia Polytechnic Institute and State University.)
-------
could safely be attributed to the introduction of TNT waste. The results in-
dicated a decrease in the number of species by about one third at the point of
TNT plant effluent (Figure 79). The species which were characteristically
absent in the area of waste discharge are shown in Table 139.
40
30
10
1 23 4 5 6 7 8 9 10
station no.
Figure 79. Histogram Representing the Number of Species of Aquatic
Plants Present Below the High-Water Mark in Ten Samples
Along the New River, June 1971 (Mitchell, 1973)
(Reprinted with permission from the Water Resources
Research Center, Virginia Polytechnic Institute and
State University.)
464
-------
Table 139. Plant Genera and Species Affected by the Introduction of TNT Plant
Effluent in New River (Virginia) (Radford Army Ammunition Plant)
June, 1971 (Mitchell, 1973)
Plant Genera
or Species
Compositae .
Bidens frondosa L.
Rudbeckia laciniata L.
Cornaceae
Cornus amomum L.
Cruciferae
Arabis laevigata
(Muhl.) Poir.
Cyperaceae -
Eleocharis obtusa
(Willd.) Schultes
Gramineae
Echinochloa crysgalli
(L. ) Beauv.
Panicum sp .
(not flowering)
Hypericaceae
Hypericum mutilum L.
Labiatae
Mentha arvenis L.
Onagraceae
Ludwigia palustris
(L.) Ell.
Rosaceae
Agrimonia gryposepala
Wallr.
Solanaceae
Solanum carolinense L.
Urticaceae
Laportea canadensis
(L.) Wedd.
Station*
4P
Upstream from TNT plant
(Reference)
*
5P
Down
-
6P
stre
-
7P
am f
-
8P
rom
-
9P
TNT
*
10P
plant
-t
Key:
- indicates absence of taxa
+ indicates presence of taxa
* see map, Figure 78
465
-------
As the river left the discharge site, a number of these species reappeared,
and there was a gradual increase in the total number of taxa. By the time
the river approached sampling site 10P the number of species had returned to
more or less that of the reference. In this study, the authors also determined
the relative biomass at the sampling stations. However, the data is of little
use to assess the toxicity of TNT waste because the biomass, by the time the
river reached station 5, had already been severely affected by discharge from
other effluents upstream from the TNT waste discharge site.
In view of the fact that (1) nitro-substituted toluenes are
toxic to duckweed, an important member of the food web and that (2) the waste
from the TNT plant causes considerable changes in the species distribution in
aquatic vegetation, it can be said with certainty that nitro-substituted tol-
uenes will have an adverse effect on the aquatic and surrounding environment.
3. Nitrobenzenes
The toxicity of nitro-substituted benzenes to higher plants
has received very little attention. However, the effect of m-dinitrobenzene
on photosynthesis in Chlorella and spinach chloroplast has been examined. Among
the two phases of fluorescence induction (photochemical and thermal) , the
-4
thermal phase was reported to be inhibited at concentrations lower than 10 M.
The authors attributed the inhibition to an irreversible binding of the toxi-
cant to the reaction centers. At higher dinitrobenzene concentrations, the
photochemical phase appeared to have been inhibited. The data suggest that
m-dinitrobenzene may adversely affect plants by interfering with their
photosynthetic process.
466
-------
G. Toxicity - Microorganisms
A considerable amount of work has been done to determine the toxicity
and mechanisms of action of nitro-substituted aromatic compounds. Unfortunately,
in almost all of the reported studies, the concentrations of the toxicants used
are much higher than, and therefore do not simulate, field concentrations. One
of the main reasons for this appears to be the limited sensitivity of the para-
meter chosen to study the response of the organism. The types of microorganisms
which have been used in studying the toxicity of ni'troaromatics include yeast,
molds, and unicellular algae. Some studies have dealt with the effect of
nitroaromatic compounds on the activity and performance of activated sludge.
Nitrophenolic compounds (particularly 2,4-dinitrophenol) have been studied much
more extensively than any other group of nitroaromatics, perhaps because of the
interest of researchers in these compounds due to their specific uncoupling
effect. In this section, effort has been made to summarize all the literature
concerning the effect of nitroaromatics on microorganisms. The available in-
formation is organized by the type of organisms which have been used in the
toxicity studies, e.g., bacteria, yeast, unicellular algae, etc.
1. Effects on Bacteria
Studies dealing with the effect of nitroaromatic compounds on
bacteria are summarized in Table 140. As can be noted, a majority of the studies
falling in this section deal with the effect of nitrophenols, particularly
2,4-dinitrophenol, a long-known uncoupler of oxidative phosphorylation. The
compound effectively disconnects the oxidative phosphorylation energy trans-
ferring sequence from the electron transferring sequence, and thus deprives the
cell of the available energy. The susceptibility of bacteria to the toxic action
467
-------
Table 140. Summary of the Studies Dealing with Toxicity of Nitroaromatics to Bacteria
CO
Reference
Allwood and
Hugo, 1971
Kerridge,
1960
Aboud and
Burger, 1972
Eauerle and
Bennett, 1960
Rieder and
Bukatsch,
1956
Cowles and
Klotz, 1948
Test
Chemical Concentration
Studied Employed
2,4-Dinitro- 0.1 mM
phenol
" 1 mM
0.125 mM
" 25-250 ppm
2,4-Dinitro- 10~3 - 10~6 M
phenol
Several mono- , 1 x 10 , -
di-, and tri- 14 x 10 M_
nitro substi-
tuted phenols;
amino substi-
tuted nitro-
phenols
Test
Organism
Staphylococcus
aureus
Salmonella
typhimurium
Escherichia
coli-K12
Pseudomonas
aeruginosa
isolated from
spoiled
emulsion oil
Luminous bac-
teria: Photo-
bacterium phos-
phoreum
Escherichia
coli, Bacillus
mesentericus
Cell Function
Studied
1. Leakage of cations
and amino acids
2. Cell inactivation
1. Regeneration of
flagella
2. Protein and nucleic
acid synthesis
6-Galactosidase
synthesis
Oxidation of Ci,, C5,
C8> C10- C12. C13.
GI\, C16 saturated
aliphatic fatty acids;
cell growth
Glowing of bacteria
Cell multiplication
Analytical Method
Cations by flame photometer,
amino acids by ninhydrin method;
cell inactivation by pour plate
method
Motility estimation and flagellar
staining; total nucleic acid from
extinction at 260 my; DNA by di-
phenylamine method; protein
colorimetrically with Folin reagent
Assay of the enzyme, total protein
synthesis by measuring ll*C-amino
acid incorporation
Oxygen uptake by Warburg mano-
metric technique, growth by
measuring cell numbers
Measure light intensity
Visible growth
-------
Table 140. Summary of the Studies Dealing with Toxicity of Nitroaromatics to Bacteria (Cont'd)
VO
Reference
Cooper and
Mason, 1927,
1928
Demerec et
al. , 1951
Mohn, 1971
Clarke, 1971
Test
Chemical
Studied
2,4,6-Trinitro-
phenol (picric
acid)
2,4,6-Trinitro-
phenol
Pentachloro-
nitrobenzene
Pentachloro-
nitrobenzene
Concentration
Employed
200 - 3500 mg/
liter
100 - 180 mg/
liter
2 mg/ml
10 - 15 mg/
bacterial seeded
plate
Test
Organism
E. coli
P. fluorescens
S. marcescens
B. mesentericus
etc.
E. coli
E. coli 343,
(a non-induci-
ble, galactose
negative mutant)
E. coli B/r
ochre auxo-
tropic mutant
WWP-2
Cell Function
Studied Analytical Method
Growth
Mutagenic action
Forward mutation to Spot test on galactose minimal
galactose prototrophy medium
Reversion to tryp- Scoring the colonies of
tophan independence revertants
-------
of nitroaromatics has been ascertained by studying the effect on cell growth
and multiplication, and the effects on sensitive cell functions, such as
permeability, enzyme synthesis, oxidative ability, etc. For certain chemicals,
the mutagenic potential in bacteria has also been evaluated.
a. Growth Inhibition
One of the earlier studies dealing with the bactericidal
activity of certain nitrophenols is that of Cooper and Mason (1927, 1928) .
They studied the influence of picric acid on a number of bacteria, including
JL. coli, Pseudomonas jiluorescens and many others. Although picric acid was
inhibitory to all the bacteria tested, the effective concentration differed
from one organism to another (ranging from 125 mg/£ to 3330 mg/£, depending
upon the organism and the period of exposure).
Cowles and Klotz (1948) have investigated the toxic action
of 2,4-dinitrophenol in microorganisms in some detail. In this study, cultures
of Escherichja coli and Bacillus mesentericus were used as the test organisms,
and for media both complex broth solution and simple chemically defined media
were used. The bacteriostatic value was taken as the lowest concentration of
the test chemical which prevented visible growth for 4 days. The bacteriostatic
values with the two bacteria on either medium were found to be essentially sim-
ilar, and therefore, only the data obtained with JS. coli grown in yeast ex-
tract broth are given in Table 141. The bacteriostatic concentrations of the
compounds tested were found to be in the milllmolar range, which is far above
reasonable environmental concentrations. It is unlikely, therefore, that at
environmentally significant concentrations the nitroaromatic compounds will have
any bacteriostatic effect. The data further reveals that the toxicity of
470
-------
Table 141. Bacteriostatic Activity of Certain Nitrophenols (Cowles and
Klotz, 1948)
Test Compound
pKa Constant
Bacteriostatic Concentration*
(Molar x 10~3)
m-nitrophenol
p-nitrophenol
2-amino,'4-nitrophenol
2 ,5-dinitrophenol
2-amino ,4 ,6 ,-dinitrophenol
2 ,4-dinitrophenol
2 , 6-dinitrophenol
2,4, 6- trinitrophenol
8.3
7.1
7.0
5.1
4.4
4.0
3.6
0.8
2.8
2.5
8.0
1.0
11.0
5.6
11.0
14.0
* Lowest concentration which prevented growth for 4 days. The data given
are for culture pH 7.5.
nitrophenols, in the concentration range examined, decreases with an increase in
the number of nitro-groups on the molecule. In other words, the order of toxicity
is mono->di->tri-, except that the 2,5-substituted dinitrophenol was a
stronger inhibitor than any of the mono-nitro substituted phenols tested.
When the bacteriostatic action of various nitrophenols was examined in culture
medium at various pH conditions (5.5 - 8.5), it was shown that the effec-
tiveness of nitrophenols increased with a fall in pH. These observations led
the authors to conclude nitrophenols owe their activity to the undissociated
form.
Fujita (1966) applied a structure-activity correlation to
the data of Cowles and Klotz (1948) in order to assess the physiological activity
of nitrosubstituted phenols which exist partly as neutral molecule and partly
in the ionized form at physiological pH. The bacteriostatic activity
471
-------
was correlated with the chemical structure using the Hammet a constant and a
substituent constant TT for lipohydrophilic character of the molecule. The
analysis of the data revealed that perhaps both the electronic as well as
the lipohydrophilic character of the substituent were important in determining
the toxicity of nitrophenolic compounds. More definite conclusions could not
be drawn because the data for a wide variety of phenols were not available.
The influence of o-nitrobenzoic acid on reproduction and
growth of microorganisms has been studied by Durham and his coworkers.
Montgomery and Durham (1970) reported strong inhibition (about 94%) of the
growth of Pseudomonas fluorescens by £-nitrobenzoic acid (120 mM). The results
were unchanged whether the growth medium contained succinate or protocatechuate,
as carbon source, in the latter case the induced synthesis of protocatechuate
oxygenase being a requisite. Kirkland and Durham (1963) noted the inhibition
of growth by £-nitrobenzoic acid of Flavobacterium using p_-nitrobenzoic acid
as the sole source of carbon. In the above studies, the concentration of the
toxicant used was very high. Although growth inhibition was observed at these
concentrations, it is debatable if a significant degree of growth inhibition
will occur at low environmentally conceivable concentrations.
b. Effect on Cell Permeability
Allwood and Hugo (1971) reported an adverse effect of
2,4-dinitrophenol (2,4-DNP) on the permeability of the bacterium, Staphylococcus
aureus. They found that addition of concentrations of dinitrophenol as low as
0.1 mM to the suspension of cells caused a marked increase on K loss from the
cells. The efflux increased with an increase in concentration of the toxicant.
The minimum concentration of 2,4-DNP which was effective in causing loss of
472
-------
K also resulted in 20% loss of viability. No amino acid leakage was observed
after treatment with dinitrophenol under similar conditions. The loss of Na
Was also unaffected by the presence of dinitrophenol. However, Na loss was very
rapid and difficult to measure accurately, and therefore, these results are in-
conclusive.
c. Effect on Protein Synthesis
Since flagella from Salmonella typhimurium have been shown
to consist of a single well-characterized protein (Kauffmann, 1954; Ambler
and Rees, 1959), regenerating flagella have provided a suitable system to many
researchers for studying the effect of toxicants on protein synthesis.
Kerridge (1960) used this system to investigate the effect of 2,4-dinitrophenol.
A culture of Salmonella typhimurium was made non-flagellate by mechanical re-
moval of their flagella, and used in the regeneration studies. Although
_3
2,4-DNP (10 mM) had no effect on the motility of the normal flagellated
bacterium, the toxicant completely inhibited regeneration of flagella by
mechanically deflagellated J3. typhimurium (Table 142). Direct measurement of
the cellular protein and nucleic acid levels in the treated cell further con-
firmed that 2,4-DNP was an effective inhibitor of protein synthesis
The inhibitory effect of o-nitrobenzoic acid on microbial
protein synthesis has been noted by Montgomery and Durham (1970). They found
that the synthesis of inducible enzymes, protocatechuate oxygenase in Pseudo-
monas fluorescens, and of 8-galactosidase in E. coli, was delayed by £-nitro-
benzoic acid. The inhibitor at a concentration of 13 mM caused nearly 68% and
78% inhibition of protocatechuate oxygenase and 3-galactosidase, respectively. At
this concentration of ^-nitrobenzoic acid, no effect on cell viability or on the
473
-------
Table 142. Effect of 2,4-Dinitrophenol on the Regeneration of Flagella, and
on Protein and Nucleic Acid Synthesis by Salmonella typhimurium
(Kerridge, 1960)
Concn. of
2,4-DNP tested
(M)
Regeneration of Flagella
"Motile Flagella/
Bacteria Bacterium
(%) (mean)
% Inhibition
Total
Nucleic
Acid
of Synthesis
DNA Protein
5
1
2
-
x 10~4
_3
x 10
-3
x 10
90
50
1
1
3.6
2.1 80
100
100
-
70
100
100
-
54
94
100
activity of the existing enzyme was observed. At 10 fold lower concentrations
of o-nitrobenzoic acid, the synthesis of protocatechuate oxygenase was unaffected;
the effect of the lower toxicant concentration on 3-galactosidase synthesis was
not studied. The sensitivity of the inducible enzymes to o-nitrobenzoic acid
suggests that the compound interferes with the synthesis of protein. This
conclusion gains support from the finding that £-nitrobenzoic acid also in-
hibits the incorporation of radioactive amino acids into protein (TCA in-
soluble fraction) (Montgomery and Durham, 1970).
The inhibition of protein synthesis (i.e., flagella, in-
ducible enzymes, etc.) by toxic chemicals could have many environmental con-
sequences. For instance, the non-flagellated cells will fail to respond to
the stimulus provided by a chemical substance (chemotactic response) which is
474
-------
introduced in the environment. A chemotactic response is generally regarded
as a prerequisite for microbial attack on a chemical. Therefore, it may be
reasonable to conclude that the presence of an inhibitor of flagella synthesis
will affect the biodegradability of chemical substances in the environment.
Alexander (1973) has stated that enzymes participating in the initial phases
of decomposition of a number of synthetic and natural products require in-
duction. From this, it appears that toxicants which interfere with protein
synthesis (i.e., formation of inducible enzymes) will also have an impact on
biodegradability of chemical substances. However, from the data available, it
cannot be said if 2,4-dinitrophenol or £-nitrobenzoic acid will have such an
effect under field conditions and at low environmentally significant concen-
trations.
d. Influence on Oxidative Enzyme Systems
An extensive investigation concerning the effect of 2,4-
dinitrophenol on the oxidation of saturated aliphatic fatty acids (C. - C10)
H J.O
by Pseudomonas sp. was carried out by Bauerle and Bennett (1960). A pure
culture of Pseudomonas aeruginosa isolated from a spoiled emulsion oil was the
test organism. The results of the manometric studies indicated that the oxygen
uptake with nearly all fatty acids increased slightly in the presence of 2,4-
dinitrophenol (Table 143). However, similar concentrations of 2,4-DNP resulted
in inhibition of growth (growth studies were done only with capric acid). The
authors concluded that even though DNP stimulates oxygen uptake, the compound
has an inhibitory effect upon multiplication of the organism. It should be
added here that most compounds which act by uncoupling oxidative phosphorylation
from respiration are known to act in this manner.
475
-------
Table 143. Effect of 2,4-Dinitrophenol on the Oxidation of Saturated Aliphatic
Fatty Acids by Pseudomonas aeruginosa (Adapted from Bauerle and
Bennett, 1960)
Substrate
None (endogenous)
Butyric acid
Caproic acid
Caprylic acid
Capric acid
Laurie acid
Tridecanoic acid
Myristic acid
Palmitic acid
% Inhibit ion (-)
25ppm
+12
+
+
+
+
9
9
3
1
0
+15
+
0
4
or Stimulation(+) in 6 Hours
at DNP Concentrations
lOOppm
+14
- 3
+14
. + 8
+11
+ 4
+21
+ 5
- 2
e. Mutagenic Effects
So far, only a few nitroaromatic compounds have been tested
for mutagenic potential. The criterion used by Clarke (1971) in determining
mutagenicity of pentachloronitrobenzene (PCNB) was reversion of tryptophan
independence in the E_. coli B/r ochre auxotrophic mutant WWP-2. In this test,
PCNB was found to be detectably mutagenic in the her (excision repair de-
ficient) derivative of the strain. In the her strain (excision repair com-
petent), the compound was not measurably mutagenic. PCNB, however, could not
be shown to be mutagenic in another strain of E. coli with a different system
(Mohn, 1971) (i.e., forward mutation to galactose prototrophy in a phenotypic
galactose negative strain of E. coli). The concentration of PCNB was 2 rag/ml
in this test. Clarke (1971) used 10-15 mg PCNB/plate in his test. Since exact
toxicant concentration in the latter case is not known, comparison of the
results from the two sources is not justified.
476
-------
Demeree et^ aj.. (1951) have noted the mutagenic activity
of picric acid (2,4,6-trinitrophenol). These authors found that the number of
mutations exhibited by E. coli exposed to 100-180 mg/J, of picric acid exceeded
the controls by 10 fold.
f. Miscellaneous Effects
Rieder and Bukatsch (1956) reported the effect of 2,4-
dinitrophenol on the glowing of luminous bacteria, a process which has been
proven to be energy dependent. Treatment of a glowing culture of Photobacterium
3 -4
phosphoreuro with 2,4-dinitrophenol (10~ - 10 M) caused a rapid decrease in
the intensity of light. Lower concentrations of the toxicant (10 - 10 M)
on the other hand resulted in an increase in light intensity over control.
The results of this investigation provide further support to the fact that
2,4-DNP exerts its toxicity by interfering with the energy metabolism of the
cell.
2. Effect on Yeast and Fungi
The nitroaromatic compounds which have been extensively studied
for their effect: on yeast and fungi are 2,4-dinitrophenol and mr-dinitrobenzene.
Their toxic action on fungi and molds is evident from the fact that these com-
pounds are well known fungistatic agents.
The effect of 2,4-DNP on nitrogen (NH ) assimilation and res-
piration in the fungus Scopulariopsis brevicaulis has been examined by Macmillan
(1956). Although 2,4-DNP failed to inhibit the endogenous oxygen uptake of the
cells (in fact, it was stimulated), the compound effectively blocked the in-
creased respiration due to ammonia, as well as the assimilation of ammonia
(Table 144). The results are consistent with the view that DNP uncouples
477
-------
table 144. Effect of 2,4-Dinitrophenol on Nitrogen Assimilation and Respiration
in the Fungus Scopulariopsis brevicaulis (Mactnillan, 1956)
a. Respiration
Additions;
None (Control) _o
+DNP (2.5 x 10 M)
Control + NH_ 3
+DNP X2.5 x 10 M)
b. Nitrogen Assimilation
Additions:
Control (Energy source, 1%
glucose) _o
+DNP (2.5 x 10 M)
Oxygen consumed in 150 min.
(y£02/g dry wt.)
27
47
36
30
Assimilated in 150 min.
(mgN/g dry wt.)
16.5
4
478
-------
oxidation from phosphorylation and thus the energy required for assimilation
is not available. An additional interesting effect of DNP in the organism was
that in the treated cells, the amino acids synthesized were different. For
example, in the presence of DNP, the ammonia assimilated in alanine increased;
the increase was accompanied with a decrease in glutamine and certain other
amino acids.
Stanek and Drahonovsky (1964) reported stimulation of endogenous
as well as glucose-linked respiration by nitrated phenols (2,4-dinltrophenol,
2-nitro-3,4,6-trichlorophenol and 2,3-dichloro-4-nitrophenol) in the conidia
of Neurospora sitophila (Table 145). In agreement with the stimulation of
Table 145. Influence of Nitrophenols on the Metabolic Quotient, Oxygen
Consumption and Carbon Dioxide Evolution by Conidia of 1J.
Sitophila (Stanek and Drahonovsky, 1964)
Compound
Concn.
(M)
Oxygen
Consumption
E/mg/h
C02
Released
4/mg/h
Metabolic
Quotient*
Endogenous
Control (range from
three experiments)
2,4-Dinitrophenol
2-Nitro-3,4,6-
chlorophenol
2,3-Dichloro-4-
nitrophenol
Substrate - Glucose
10
10
-4
-4
10
-4
24.8-26.8
32.8
21.8
11.1
18.0-21.4
28.2
20.2
17.2
0.71-0.74
0.86
0.93
1.54
Control (range from
three experiments)
2 , 4-Dinitrophenol
2-Nitro-3,4,6-
chlorophenol
2,3-Dichloro-4-
nitrophenol
58.2-71.0
-4
10 I 84.5
10 78.7
10"4 22.4
18.0-21.4
95.5
80.0
51.7
0.98-1.02
1.13
1.02
2.30
* co2/o2
479
-------
respiration, was the finding that the total level of carbohydrates decreased
in the treated spores. Phenol derivatives, in addition, were also effective
in suppressing the germination capacity of the spores; the concentration for
50% suppression being 17, 8 and 30 mg/g dry weight of spores, respectively,
for 2,4-dinitro-, 2-nitro-3,4,6-trichloro-, and 2,3-dichloro-4-nitrophenol.
The inhibition of germination was accompanied with changes in permeability of
the cell membrane since the treated cell lost substantial quantities of cellu-
lar carbohydrates, low molecular weight phosphorus compounds (probably the deg-
radation products of nucleic acid, etc.) and nucleic acid bases, into the
incubation medium (Stanek and Drahonovsky, 1964). The chloronitrophenols,
2-nitro,3,4,6-trichlorophenol and 2,3-dichloro-4-nitrophenol possessed a
stronger activity in almost all respects than the typical phosphorylation
inhibitor, 2,4-dinitrophenol. Alteration of membrane permeability by nitro-
phenol has also been noted in bacteria.
-4
Nitrophenolic compounds (10 M) were also reported to inhibit
growth of bakers yeast Saccharomyces cerevisiae (Dedonder and Van Sumere, 1971).
The test results revealed that 2,4-dinitrophenol was more inhibitory than p_-
nitrophenol; growth inhibition being 60% for dinitrophenol and 22% for £-
nitrophenol. One tenth mM DNP was found to inhibit acetate and pyruvate oxi-
dation and assimilation in the yeast almost completely at pH 4.8 (or less)
(Stoppani, 1949;1951; Stoppani and Ramos, 1964). At a higher pH, the inhi-
bition decreased drastically; for example, with acetate as a substrate, the
inhibition by 0.1 mM DNP was 5% at pH 6.7, and none at pH 7.3. Glucose oxidation
in this organism was not affected by DNP. Studies with radio-labelled acetate
14
indicated significant changes in C-distribution in the soluble fraction in
480
-------
the presence of 2,4-DNP. For instance, labeling of tricarboxylic acids
diminished by 85% and similar, though much smaller, changes were observed
in aspartic acid and certain dicarboxylic acids. Their findings are con-
sistent with the conclusion that 2,4-DNP interfered with further oxidation
of acetate by the Krebs Cycle.
Among nitrobenzene compounds, the metabolic effect of m-di-
nitrobenzene has been investigated to some extent. This compound is a potent
inhibitor for molds; concentrations as low as 2.5 x 10 M produce complete
statis of Aspergillus niger growth (Higgins, 1960). The metabolic observa-
tions indicated that growth inhibition was the result of the depression in
the amino acid synthesis during the early growth phase (Higgins, 1960). At
growth depressing levels of nh-dinitrobenzene, no effect on oxygen utilization
or glucose/oxygen ratio was evident.
3. Effect on Protozoa
Only a few studies dealing with the influence of nitroaromatic
compounds on protozoa have been reported in the literature. Conner (1957)
studied the effect of 2,4-dinitrophenol on the growth of ciliated protozoan
Tetrahymena piriformis. The findings revealed that 2,4-DNP in the concen-
-4
tration range 0.5 - 1 x 10 M was an effective inhibitor of growth of this
organism. The ability of 2,4-DNP to act as an uncoupler of oxidative phos-
phorylation and to activate adenosine triphosphatase was considered to be
the mechanism underlying the growth inhibition.
Picric acid was reported to be extremely toxic to an unspecified
amoebae if applied to the surface at a concentration of 1% (Pollack, 1927).
Hajra (1959) noted that picric acid at a concentration of 10 mg/liter was half
481
-------
lethal for Acanthamoeba sp. , but was without effect in Neglaria gruberi.
Bringman and Kuehn (1959) found the 96 hour toxicity threshold to be 900 mg/fc
in the protozoan Microregina heterostoma. These studies revealed that indeed
both di- and trinitro-substituted phenols are toxic to amoebae, but that the
toxic concentration varies from species to species.
4. Effect on Unicellular Algae
In a preliminary study, Moberg et_ al. (1968) were able to show
-4
that 2,4-dinitrophenol at a concentration of 3 x 10 M inhibited the growth
of Chlorella pyrenoidosa in a synchronous culture. Dedonder and Van Sumere
(1971) confirmed the findings of Moberg (1968), but extended the investigation
to include the effect of nitrophenols on the respiration of Chlorella. At
concentrations which inhibited growth, the nitrophenols caused a stimulation
of the respiration of the algae (Table 146). This stimulation of respiration
is consistent with the established uncoupling action of nitrophenols. In
agreement with the fact that the effectiveness of nitrophenols increases with
a fall in pH (see Section III-G-1-a), it was found that stimulation was
greater in magnitude at pH 5.6 than at 7.2.
Table 146. Effect of Nitrophenolic Compounds on the Growth and Respiration of
Chlorella vulgaris (Dedonder and Van Sumere, 1971)
Test Compound
£-Nitrophenol
ii
2 , 4-Dinitrophenol
ii
Concn.
-5
5 x 10 M
1 x 10~4 M
5 x 10~5 M
-4
1 x 10 M
% Growth
Inhibition After
80 Hours
50
80
70
100
% Stimulation
Respiration, pH,
of
7.2
(5 Hours Incubation)
+30
+80
0
20
482
-------
The effect of 2,4-dinitrophenol on the uptake and metabolism of
2,4-dichlorophenoxyacetic acid (2,4-D), a synthetic auxin, has been reported by
Swets and Wedding (1964). Although the purpose of the authors in undertaking
such a study was to trace the sequence of reactions of 2,4-D in the cell, the
results of the investigation could also be valuable in assessing the hazards
associated with the interaction of the two contaminants in the environment.
For example, one contaminant may affect the toxicity, bioaccumulation and/or
persistence of another contaminant in the environment. Increasing concentrations
-4
of 2,4-DNP (up to 10 M) were reported to first cause a progressive inhibition
-4
of 2,4-D uptake, but as 2,4-DNP concentration increased (up to 3.2 x 10 M)
an increase in uptake was found (Table 147). In the absence of light or air,
2,4-DNP-linked uptake of 2,4-D was nearly doubled. The environmental con-
sequence of the interaction may be speculated to be an increased accumulation
of 2,4-D in the algae in the presence of 2,4-DNP. No information is available
concerning the toxicity and persistence of 2,4-D in the presence of low con-
centrations of 2,4-DNP.
Table 147. Effect of 2,4-Dinitrophenol on the Uptake of 2,4-Dichlorophenoxyacetic
Acid by Chlorella pyrenoidosa (Swets and Wedding, 1966)
2,4-DNP Concentration 2,4-D Uptake *
(M) (p moles/g dry wt)
None (control)
1 x 10~4
-4
3.2 x 10
3.2 x 10~3
0.10
0.03
0.13
0.10
* 10~4 M 2,4-D in the solution.
483
-------
The only report concerning the influence of nitroanilines on
algae is that of Villeret (1960). In this study, the effect of o-, m-, and
£-nitroaniline on respiratory gas exchange of algae Chlorella vulgaris and
Scenedesmus quadricauda was studied. The chemicals were found to stimulate
the respiration of Chlorella in the decreasing order of _o-, £-, m-nitroaniline.
Similar effects were not noted in Scenedesmus. The increase in respiration
in Chlorella could perhaps be due to the action of nitroaniline as an un-
coupling agent similar to that of 2,4-dinitrophenol, and may not be accompanied
with increased cell multiplication. The experimental data supporting or dis-
puting this conclusion are not available.
An algae survey in the vicinity of Radford Army Ammunition Plant
to determine the variations in the distribution of algae due to TNT plant effluent
was carried out by Wodehouse et^ ail. (1973). The authors noted the algae com-
munity appeared similar from station to station with only a few exceptions (for
location of stations, see Figure 78). Furthermore, the distribution of taxa
within major divisions of algae showed little qualitative change in response
to TNT waste. For instance, at station 6A (located immediately upstream from
the TNT plant), 53 taxa of algae were recorded, and at station 7A (located
immediately downstream from the plant), 49 taxa were recorded. The results
are, however, complexed by the fact that these stations also received other
waste from industrial plants located upstream from the TNT plant. It is possible
under these circumstances that one is dealing with abnormal algae population
to start with, and therefore, the utility of the data in terms of the effect
of TNT waste may be questionable.
484
-------
5. Influence of Nitroaromatics on the Microbiological Systems
Concerned With Waste Treatment
Most of the degradation in the environment and in waste treat-
ment processes is accomplished by microorganisms. Interference with the
activity of these microorganisms could result in the loss of the effectiveness
, of the treatment process. The following section is, therefore, devoted to the
studies dealing with the effect of nitroaromatics on the activity of the micro-
organisms involved in purification of streams and reservoirs and waste treat-
ment processes.
Under natural conditions, certain organic compounds, when present
at low concentrations, greatly retard the processes of self-purification of
reservoirs, and inhibit nitrification and the decomposition of other organic com-
pounds (Rogovskaya, 1951; Stasiak, 1967). For example, Rogovskaya (1951) noted
that 0.5 - 1.0 mg/1 trinitrotoluene only slightly affected stream self-purifi-
cation processes, whereas beyond 1.0 mg/1 marked damaging effects on self-
purification processes occurred. These results agreed with those of Ruchoft
ejt ad. (1945a) who found that concentrations of a-TNT as low as 1.17 mg/1 re-
tarded the BOD reaction.
In order to determine the effects of trinitrotoluene on sewage
treatment, Enzinger (1970) set up two Bush laboratory scale bio-oxidation units.
TNT was continuously pumped into the TNT test unit and the concentration was
gradually increased from 12 ppm to 29 ppm; the second unit fed only synthetic
sewage served as control. The monitoring of effluent COD and total carbon
analysis from the two units indicated that generally the TNT test unit was less
efficient in the oxidation of organic material than the control unit. That
TNT retarded the biological activity was shown from the fact that oxygen uptake
485
-------
rates for the TNT test unit were lower than the control unit. Warburg respiro-
metric studies undertaken to determine the effect of different levels of TNT on
samples from the TNT test unit and the control unit supported the theory that TNT
has adverse effects on the oxidation of organic material (Figure 80). TNT
affected the respiration rates of the microorganisms from the control unit more
severely than those from the TNT test unit. This suggests that the micro-
organisms in the TNT test unit may have been acclimated to TNT.
Contrary to the findings of Enzinger (1970), Bogatyrev (1973)
noted no effect of TNT on the activated sludge process at concentrations of
< 50 mg/£. Only a partial suppression of nitrification was observed in the
presence of TNT. Mono- as well as dinitrotoluenes were also without effect
on the activated sludge process.
The gross chemical composition and biochemical characteristics
of activated sludge grown on a given substrate remain remarkably unaltered
under normal circumstances. However, a change in the composition of the medium
or perhaps even addition of toxic substances could cause a modification in the
characteristics of activated sludge. This approach was used by Vaicum and
Eminovici (.1974) to assess toxic effects of trinitrophenol (picric acid) on
activated sludge systems. A laboratory activated sludge unit was run on a
i
nutrient feed which contained sodium acetate, urea, and phosphate. The toxi-
cant was included in the influent following a 14 day equilibration period.
The variation in respiration rate, enzyme activities (dehydrogenase and
catalase) and gross activated sludge composition (glucides and total pro-
tein) were measured over an extended period of time (from 14-75 days). At
the feeding level of 50 mg/1 trinitrophenol, all the parameters except the
486
-------
500 —
400—I
300-
o
LU
s
v>
Z
o
u
o1
200-
100 —
120 180 240
TIME ( min. )
(60)
LEGEND:
X TNT ACCLIMATED
o NOT ACCLIMATED
( ) TNT CONCENTRATION P. P. M.
300 360
Figure 80. Respiration Rate of TNT Acclimated and Nonacclimated Microorganisms
at Various Concentrations of TNT (Enzinger, 1970)
(Synthetic sewage as the carbon source)
487
-------
concentration of glucides showed a significant decrease after the first 24
hours. After 10 days, however, the COD removal efficiency and the sludge
total protein content returned to the normal level (Figure 81). Upon the
feeding of 200 mg/1 trinitrophenol to the acetate fed laboratory unit, sig-
nificant irreversible changes in the measured parameters were produced. After
75 days, there were no signs of recovery. The authors suggested that the
effect was either due to an inhibitory effect of the substance or to the
disappearance of the fraction of responsible microorganisms. The findings
revealed the adverse effect of trinitrophenol on the health and activity of
activated sludge systems, and consequently, on the breakdown of numerous chem-
icals which pass through the waste water treatment plants.
D
I
Picric acid
50 mg l.~1
140—1
COD removal %
Glucides
Proteins
%gMLVSS
120-
100 —
80 —
60-
40 —
20 —
Dehydrogenase 7 TTC mg" ' MLVSS 3 ~ 1 h
Catalase
Catalase I. g"1 (MLVSS) s
-1
Oehydrogenase
V
COD
Proteins
Glucides
I r i T i i i i i
14X1 16 18 20 22 24 26 28 30
Time (days)
Figure 81.
Effect of Trinitrophenol on the Biochemical Characteristics of
Activated Sludge (Vaicum and Eminovici, 1974)
488
-------
Whereas nitrotoluenes and certain nitrophenols have been shown
to have an adverse effect on the microbiological systems concerned with waste
treatment, and to reduce the efficiency of waste water treatment, the situation
is very different with 2,4-dinitrophenol. Wilkinson (1951) has found that the
presence of dinitrophenol in waste water (concentration unstated) causes an
increase in population of bacteria. Shah et^ al. (1975) have employed the un-
coupling action (ability to disconnect energy transferring sequence from the
electron transfer sequence) of 2,4-dinitrophenol to stimulate microbial res-
piration and substrate removal in a model waste water treatment system. Experi-
ments in a continuous reactor system with glucose as the waste (to stimulate
effluents from sugar and starch industries) and Saccharomyces cerevisiae as the
degrading organism, indicated as much as 85% increase in the degrading efficiency
of glucose at 2,4-DNP concentration of 5 x 10~ II (Figure 82). Cell growth
on the other hand decreased in the presence of DNP; a decrease of 70% was
recorded at 5 x 10 jl DNP (Figure 83). The experimental data provide no
information regarding the fate of DNP itself. Since the study was restricted
to a model system, it cannot be said with certainty if DNP will have similar
effects on specific waste-water treatment systems in the field where one is
dealing with complex mixtures of chemical substances and with a variety of
degrading microorganisms. Furthermore, since DNP is shown to effectively
inhibit cell growth, it appears likely that upon prolonged incubation, the
oxygen consumption rates will fall and may even be lower than those in the
absence of DNP. Shah et^ al. (1975) terminated their study at the end of 24
hours and this limits the conclusion that can be drawn concerning the long
range effects of this nitroaromatic on waste water treatment.
489
-------
9 12 15
TIME (hr)
Figure 82. Concentration-Time Profiles of Carbon Dioxide in Exit Stream at
Different Bulk DNP Concentrations (Shah jejt _al. , 1975)
s
CM
0.020
0.000
0.0 1x10~8 1 x 10~7 1 x10~6 1 x 10~5 1 x 10~4
BULK DNP CONCENTRATION ( MOLAR )
Figure 83. Effect of Bulk DNP Concentration on Cell Growth (Shah e_t a_l. , 1975)
490
-------
\
6. Effect on Natural Microbial Populations
The effect of nitroaromatic compounds (other than pesticides)
on natural microbial populations of soil or aquatic systems has rarely been
studied. Katznelson and Stevenson (1956) have reported results of the study
in which they examined the effect of 2,4-dinitrophenol on the oxidative activity
of soil. Unlike the reported effect of 2,4-DNP on pure cultures of micro-
-4
organisms, the addition of 2,4-DNP (5 x 10 M) to the natural communities
of microorganisms in soil was found to completely inhibit oxygen uptake with
casamino acids as substrate. Similar DNP concentrations failed to exhibit any
appreciable effect on the respiration of unamended soil (Figure 84). However,
no significant increase in the number of microorganisms was observed in the
presence of 2,4-DNP during the period of oxidation, suggesting that the in-
hibitor interfered with cell multiplication. Picric acid was reported to be
without effect on the microorganisms in domestic sewage (Ruchhoft and Norris,
1946). For example, the five-day BOD of domestic sewage was not lowered sig-
nificantly by picric acid at 100 mg/J,, and was lowered only 36% at 1000 mg/£.
However, it must be emphasized that assessment of the toxicity of nitrophenols
from the oxygen consumption data can be misleading because of the fact that
nitrophenols generally stimulate oxygen consumption or leave it unaffected
while inhibiting energy generation. In view of this shortcoming of the assay
method, the actual toxic effect of picric acid on sewage microorganisms remains
uncertain.
491
-------
2200 -i
1800-
- 1400
a.
Q.
D
_l
<
O
1000-
600 -I
200-
i
4
iCASAMINO ACIDS
CAS. + DNP
DNP
UNAMENDED SOIL
6
HOURS
10
12
Figure 84. Inhibition of Casamino Acid Oxidation by 2,4-Dinitrophenol
(Katznelson and Stevenson, 1956)
492
-------
IV. Regulations and Standards
A. Current Regulation
Regulation and control over nitroaromatic chemicals is provided under
several different authorities. Because this group of compounds is involved in
a large number of different applications, product control at the federal level
is quite varied. Effluent control, on the other hand, is exercised under basi-
cally the same authority for all the nitroaromatic compounds.
Many of the nitroaromatics with agricultural applications are regu-
lated under the Federal Insecticide, Fungicide, and Rodenticide Act (7 U.S.C.
135-135k). This act would cover all of the nitroaromatic herbicides, fungicides,
and lamprey larvicides. The major law concerning pesticides is now the Federal
Environmental Pesticide Control Act of 1972, which has revised the Federal
Insecticide, Fungicide, and Rodenticide Act of 1947. Under this new act, nitro-
aromatic chemicals with pesticide applications are required to be registered
by the EPA.
Tolerances for pesticide chemical residues in or on new agricultural
commodities have been established under the Federal Food, Drug, and Cosmetic
Act (21 U.S.C. 346a). Specific tolerances for a number of nitroaromatic com-
pounds have been reported in 40 CFR 180. Among the substances listed are:
2,6-dichloro-4-nitroaniline (40CFR180.200)
2,3,5,6-tetrachloronitrobenzene (40CFR180.203)
trifluralin (40CFR180.207)
N-butyl-N-ethyl-a,a,a-trifluoro-2,6-dinitro-£-toluidine (40CRF180.208)
dinitro-£-toluidine (40CFR180.281)
pentachloronitrobenzene (40CFR180.291)
2,4-dinitro-6-octylphenyl crotonate and 2,6-dinitro-4-octylphenyl
crotonate (40CFR180.341)
4,6-dinitro-o-cresol and its sodium salt (40CFR180.344)
493
-------
The major federal law governing hazardous substances is the Federal
Hazardous Substances Act of 1960 (15 U.S.C. 1261-1273). However, this act
covers household products and toys but not the raw materials from which they
are manufactured. Therefore, it does not apply directly to most of the chemi-
cals of concern in this report.
Since several nitroaromatic compounds are used as constituents in
fragrances and hair dyes, these substances would be regulated under the Federal
Food, Drug, and Cosmetic Act of 1938 (21 U.S.C. 301 et seq.).
The major authority for controlling hazardous pollutants released
into the environment is provided by the Federal Water Pollution Control Act
(33 U.S.C. 446 et seq.) and the Clean Air Act. Both laws have been extensively
amended in recent years and have set standards for air and water quality. A
"sample" list of hazardous substances was issued as an advanced notice of pro-
posed rulemaking under authority of Section 311 and Section 501 of the Federal
Water Pollution Control Act as amended (33 U.S.C. 1251 et seq.) (Federal Regis-
ter, 39(164):30466-30471, Aug. 22, 1974). Among the substances listed are
dinitrobenzene, dinitrophenol, and nitrophenol.
The transportation of hazardous materials by rail and highway is
regulated by the Hazardous Materials Control Act of 1970. This law is adminis-
tered by the Hazardous Materials Regulation Board of the Department of Trans-
portation. Air Transportation regulations are issued by the Federal Aviation
Administration while the U.S. Coast Guard supervises inland and coastal water
shipments. Oceanborne shipping of hazardous materials is regulated by the
Federal Maritime Commission.
494
-------
In January, 1974, the Department of Transportation proposed extensive
changes in the rules governing the transportation of hazardous chemicals, espe-
cially by air. These changes, published in the Federal Register (January 24, 1974)
include the following nitroaromatic chemicals classified as being hazardous:
dinitrobenzene
dinitrochlorobenzene
dinitrocyclohexylphenol
dinitrophenol
nitrobenzene
nitrochlorobenzene
ortho-nitroaniline
para-nitroaniline
picric acid
trinitrobenzene
trinitrobenzoic acid
trinitroresorcinol
trinitrotoluene
A code for the manufacture, transportation, storage, and use of
explosive materials has been prepared by the National Fire Protection Associa-
tion. This code would apply to all nitroaromatic munitions compounds.
Employee safety from hazardous chemicals is controlled by the
Occupational Safety and Health Administration, established in 1971 by the
Department of Labor. OSHA requires all chemical manufacturers to fill out ,
Material Safety Data Sheets for each shipment containing chemical substances.
B. Consensus and Similar Standards
Limits have been established for maximum permissible exposure to
hazardous chemicals by several agenices. Threshold limit values (TLV's) for
chemicals in the workroom environment have been established by the American
Conference of Governmental Industrial Hygienists. These TLV's, which are
periodically revised and updated, include a number of nitroaromatic chemicals.
The values given in Table 148 refer to the maximum concentration which may be
present in the working environment and include the potential contribution to
overall exposure by contact with the skin.
495
-------
Table 148. Adopted Threshold Limit Values for Nitroaromatic Compounds (Data
from American Conference of Governmental Industrial Hygienists, 1974)
Substance
Dinitrobenzene (all isomers)
Dinitro-o-cresol
Dinitrotoluene
£-Nitroaniline
Nitrobenzene
£-Nitrochlorobenzene
4-N i t rob iph eny 1
Nitrotoluene
Picric Acid
Tetryl
Trinitrotoluene
3,5-Dinitrotoluamide
. 3
ppm mg/m
0.15 1
0.2
1.5
1 6
1 5
- 1
*
5 30
0.1
1.5
0.2 1.5
5
*Human carcinogen - no assigned TLV
Exposure limits for hazardous substances have also been set by the
Occupational Safety and Health Administration and the National Institute for
Occupational Safety and Health. Their list of standards, which has been pub-
lished in the Federal Register (October 19, 1972), includes the same compounds
as in Table 148, and for which the official exposure limits are the same as
above.
The Manufacturing Chemists Association has sponsored the publication
of Dangerous Properties of Industrial Materials, by Irving Sax, Reinhold Book
Corp., N.Y. , 1968. This compendium lists over 1,200 chemicals and their tox-
icity ratings, including many nitroaromatic compounds. Although the book does
not provide documentation for the classification of each chemical, it is a
useful guide to general toxicity information.
496
-------
C. Foreign Authority
Limits for toxic substances in drinking water have been established
in the U.S.S.R. (Stofen, 1973). Standards for a number of nitroaromatic chemi-
cals are included among these values and are given below:
Substance Limit in mg/1
Dinitrobenzene 0.5
Dinitrochlorobenzene 0.5
Dini t ronaph thalene 1.0
2,4-Dinitrophenol 0.03
Nitrochlorobenzene 0.05
ifr-Nitrophenol 0.06
jo-N it rophenol 0.06
£-Nitrophenol 0.02
Trinitrotoluene 0.5
497
-------
498
-------
V. Summary and Conclusions
A. Summary
Commercial chemicals which fall into the category of nitroaromatics
are numerous and have varying physical and chemical properties, production
quantities, uses, environmental fate, and biological effects. Because of the
large number of compounds studied in this report, a detailed assessment of any
particular chemical was not possible. However, the conclusions discussed in
this section will help develop priorities for the compounds that should receive
further study and research.
Approximately 250-300 compounds are listed as commercial nitroaro-
matic compounds (see Chemical Index, p. 525). However, most of those compounds
are produced in such small quantities that they are little more than laboratory
curiosities. Nevertheless, there are approximately 40 compounds that are
consumed in quantities greater than 0.5 million pounds per year, and perhaps
50-100 compounds that exceed 100,000 pounds per year. Even larger numbers of
compounds fall in the 0-100,000 pound per year range.
In order to consider the relative environmental contamination poten-
tial of the individual nitroaromatic compounds, a variety of factors, which
are reviewed in detail in the previous sections, must be considered. Table 149
summarizes the information that is available for most of the major commercial
products. Some explanation of the table is necessary.
The 93 compounds included in Table 149 were: (1) listed as commercial
products (produced in at least 1000 pounds or worth at least $1000, annually),
and (2) had some environmental fate and/or biological effects information.
499
-------
Table 149. Summary of Information on Nitroaromatic Chemicals
CO
U
E
OJ
£.
C.J
2-Amino-4-nitrophenol
4-Amino-4 ' -nitro-2 , 2 ' -
stilbenedisulfonic acid
2-Bromo-4,6-dinltro-
aniline
2-sec-Butyl-4,6-dinitro-
phenol
6-tert-Butyl-3-methyl-
2,4-dinitroanisole
5-tert-Butyl-2 ,4 ,6-
trinitro-m-xylene
l-Chloro-2,4-dinitro-
benzene
2-Chloro-4-nitroaniline
3
C C
E O CJ*
< -H PH
U C.
tn e BO
o> 3 n
aj w — '
1. C U
a o 3
J 119 (72)
>163 (70)
6,626
(68)
2,500-3,000
(75)
o «-*
,—. ^-. ra
ti- a. u u~i
•—• w e •
V. T3 •- £ II
o c o o «
U CQ O O
u . •- u
a 01 i-t m a
U. OJ 1! O ~<
-a co • T3
a) -H QJ o u
OJ OJ tl) -rJ C
a: a. c. w -r-«
0.05
0.05
0.05
1,00
1.00
(perfume)
1.00
(perfume)
0.05
0.05
c
o
1-1
Contaminat
Factor
(C x RF)
12
12
47
3,000
119
L63
331
L50
Biodegradability
>,
i_i
to
3
cn
_
c
LI
c3
O OJ
t-> a.
a.
ca a
>i CO
S3
Transport
u
Bioconcent
tion
ca a
CJ U
'3)11.
O -H
r- C
O OC
0 cc
cd z:
0
m
Q ••-*
J -- OC
co j:
--^ u ^
CS C3 OO
u a B
O ^--'
1,280 (Mice
4,490
25-60
339
500-1,593
Metabolic
Effects
+
u
Hematolog
Effects
—
_
+
«
.M
l-i
CO
E
-------
Table 149. Summary of Information on Nitroaromatic Chemicals (Cont'd)
to
u
•H
e
•«
<; T_, _^ M -a
u — eu c
•WO. E C3
cn e oo ^ «J
O 3 C J-» 3
M CD 1-1 l~t
CJ 4) OJ ~- C
ni fc a « -H
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
c
Contadllnat:
Factor
(C x RF)
25
395
3,000
5,500
37
30
9
17
Biodegradability
x
^
to
3
l/l
—
—
+
fij
r """
Days to
Disappearar
>64
>64
>64
Transport
Bioconcent
tlon
27
22
27
c
Ecological
Magnificat
307
227
224
0
i/i
3 ~"3
(fl .*
cfl en at)
v- a: s
O ^"^
555
288
420
Metabolic
Effects
_
—
—
[Hematologi
Effects
+
+
+
a-.
.*
<3
£
3J
OS
U>5Q (Mice) = 63 mg/kg I.V.
Monitored in river, drinking, and
waste water
Monitored in river and waste water
Monitored in waste water
-
Ul
o
-------
Table 149. Summary of Information on Nitroaromatic Chemicals (Cont'd)
cn
u
1
o-(4-Chloro-3-nitro-
benzoyl)benzoic acid
2-Chloro-4-nitrotoluene
2-Chloro-6-nitro toluene
4-Chloro-2-nitro toluene
4-Chloro- 3-nitrotoluene
2 ,6-Dichloro-4-nitro-
aniline
1 , 2-Dichloro-4-nitro-
benzene
1 , 4-Dichloro-2-nitro-
benzene
2 , 5-Dichloro-3-nitro-
benzoic acid
Largest Annual
Consumption (C)
During (19 )
(import quantities
sometimes used)
(thousands of pounds)
147 (67)
693 (63)
102 (66)
607 (66).
3,000-3,600
(75)
700-800 (75)
Release Factor (RF)
Pesticides and
perf umes=l .00; Explo-
sives^. 05; Chemical
intermediates=0.05
0.05
0.05
0.05
0.05
0.05
0.05
(same use as
fungicide)
0.05
0.05
0.05
Contamination
Factor
(C x RF)
7
35
5
30
180
40
Biodegradability
Summary
Days to
Disappearance
Transport
Bioconcentra-
tion
Ecological
Magnification
3,020
579
686
0
m
3 -M
CD ^
~t *J ^
co a oo
o ^ ^
418-
>5,000
643
1,210
3,500
Metabolic
Effects
+
Hematologic
Effects
+
-------
Table 149. Summary of Information on Nitroaromatic Chemicals (Cont'd)
CO
O
0
2 , 4-Dichlorophenyl-4-
nitrophenyl ether
(Nitrofen)
0,O-Diethyl-o,£-nitro-
phenylphosphoro thloate
(Ethyl parathion)
0,0-Dime thy 1-iJ, £-nitro-
phenylphosphorothioate
(Methyl parathion)
2,4-Dinitroaniline
£-(2,4-Dinitroanilino)-
phenol
2,4-Dinitroanisole
3" ,4-Dinitrobenzanilide
1,3-Dinitrobenzene
"* ii ""
c c
c o &
<; •** ^
01 a oo
0) => C
00 to -H
CO O -
j o a
^
CO C
tH o
-^ -a
c co o
CO 3
3 CO
01 1 1
4-1 E CO
jj -H en
O JJ 3
i" 1 °
• — • to "- '
15,259 (70)
48',890 (73)
>679 (72)
33 (64)
16 (69)
12,000 (72)
1
O — !
.— ^ «-. (3
ti. a, u m
^ u E -
41 O
1-. T3 ^- JS I)
j-i CO O QJ
(J . •- W
t- OJ II O -t-(
*o en • *o
0) -H 0) O 0)
01 cj E tl E
ca -11 3 M J-.
cu cy oj -H c
^ P-, D. W i-<
1.00
1.00
1.00
0.05
0.05
0.05
0.05
0.05
c
o
ca
c ^*
^t Ex.
'•e ^ a:
CO O
ij *J X
O ra U
0 b. — -
15,259
48,890
34
2
1
600
Biodegradability
S
3
to
+
, en
pa
>M
Transport
,
oconcent
on
ec *->
8
c
co co
CJ O
•H T-t
0 -H
-H C
O 60
w x:
o
m
O ^
^ x-v 00
to ^
ca ra oo
3,050
3.6-13
9-25
1,800
tabolic
fects
E UJ
*
+
—
00
o
^-i to
o *-»
1~> CJ
fO !U
e «w
X W
+
n
CO
E
a:
48 hr. TL (Bluegills) - 47 mg/1
m
Non-tumorigenic ; LD (Racs) =
100 mg/kg oral
Monitored in drinking water
O
u>
-------
Table 149. Summary of Information on Nitroaromatic Chemicals (Cont'd)
to
o
•H
E
X
0
3,5-Dinitrobenzoic acid
4,4' -Dinit robiphenyl
Dinitrobutylphenol ,
ammonium salt
Dinit rocaprylphenyl
crotonate
4,6-Dinitro-o-cresol
2 , 4-Dinitro-a-naphthol
2,4-Dinitrophenol
4,4' -Dinit rostilbene-
2,2'-disulfonic acid
3 , 5-Dinitrotoluamide
Largest Annual
Consumption (C)
During (19 )
(import quantities
sometimes used)
(thousands of pounds)
500 (75)
58 (67)
>218 (72)
1,000 (75)
9,858 (72)
Release Factor (RF)
Pesticides and
perf times3! .00; Explo-
sives=0.05; Chemical
intermediates1^. 05
0.05
0.05
1.00
1.00
1.00
0.05
0.05
0.05
0.05
Contamination
Factor
(C x RF)
25
58
218
50
493
Biodegradability
Summary
—
+ + +
±
Days to
Disappearance
Transport
Bioconcentra-
t ion
8
Ecological
Magnification
o
_] *-* 00
tn ^
rt tn oo
k. 05 ^
45
980-1,190
10-50
30
560
Metabolic
Effects
-t-
+
-
+
— •
Hematologic
Effects
—
~™"
—
—
—
at
V
U.
Produced neoplasms
48 hr. TL (Harlequin Fish) =
0.27 ppm
Non-tumorigenic; 48 hr. TL
m
(Rainbow Trout) = 210 mg/1;
monitored in cheiaical plant
•effluent
LD (Dog) = 30 mg/kg I.V.
Non-tumorigenic; LC . (Fish) =
mln
0.5-38 mg/1
••Jl
o
-------
Table 149. Summary of Information on Nitroaromatic Chemicals (Cont'd)
a
o
1
SI
CJ
2,4-(and 2,6-)Dinitro-
toluene
l-Fluoro-2 ,4-dinitro-
benzene
£-Fluoronitrobenzene
4-(Methylsulfonyl)-2,6-
dinitro-N,N-diphenyl-
anillne (Nitralin)
3'-Nitroacetanilide
3'-Nitroacetophenone
o-Nitroaniline
m-Nitroaniline
£-Nitroaniline
Largest Annual
Consumption (C)
During (19 )
(import quantities
sometimes used)
(thousands of pounds)
471,237 (73)
15 (70)
6,000 (75)
>192 (73)
14,000 (75)
Release Factor (RF)
Pesticides and
perfumes=1.00; Explo-
sives^. 05; Chemical
lntermediutes=0. 05
0.05
1.00
0.05
0.05
0.05
0.05
Contamination
Factor
(C x RF)
23,561
1
300
10
700
Biodegradability
>,
V-
D
W
+ +
+ +
+ +
+ + +
Days to
Disappearance
>64
>64
>64
Transport
Bioconcentra-
tion
12
8
6
c
o
~H U
ce Q
u u
OC tu
O -H
^-i C
O 00
£%
o • •
C5 *~-
J — 00
w ^
^ . *J -^
g « g,
o ^ -^
268-707
>2,000
3,250
535-3,520
535-900
1,410-3,249
Metabolic
Effects
—
—
—
—
Hematologic
Effects
+
+
+
+
+
01
J*
14
CO
I
Non-tumorigenic; 96 hr. TL
m
(Bluegills) « 16 mg/1; monitored
in drinking water and waste water
Skin sensitizer; tumor promoter
LD (Rats) = 50 mg/kg oral
LD (Rats) =- 250 mg/kg oral
Ul
o
Ul
-------
Table 149. Summary of Information on Nitroaromatic Chemicals (Cont'd)
' r- 1
c3
O
•H
1
u
4-Nitro-o-anisidine
(1-amino)
5-Nitro-o-anisidine
(1-amino)
o-Nitroanisole
£-Nitroanisole
£-Nitrobenzaldehyde
Nitrobenzene
m-Nit robenzenesul f onic
acid and sodium sale
m-Nitrobenzenesulfonyl
chloride
o-Nitrobenzoic acid
m-Nitrobenzoic acid
and sodium salt
est Annual
iimption (C)
ng (1.9 )
ort quantities
times used)
usands of pounds)
i- c u e e ^
!3 O 3 TH O 4-1
J 0 C -* in —
>345 (73)
>127 (67)
2,500-3,500
(75)
750-1,500 (75)
655,000 (74)
3,654 (70)
23 (63)
911 (69)
ase Factor (RF)
icldes and
umes=1.00; Explo-
s=0.05; Chemical
rmediates=0.05
—i m i-i > *J
4J "U (1) — < C
K a. c. en -H
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
aihlnation
or
RF)
c: u
O (U O
u fc ^
17
6
150
75
32,750
183
1
46
Biodegradability
>,
u
3
«
+
+ + +
+
Days to
Di sappearance
>64
>64
>64
8
>64
Transport
Bloconcentra-
tion
13
7
11
Ecological
Magnification
79
o
in
O ^
*1 ^* ofl
en &
r-i ij '
cj a so
v- a; e
704
640-664
1,820
Metabolic
Effects
—
—
—
Hematologic
Effects
+
+ .
+
+
en
^
tu
§
-------
Table 149. Summary of Information on Nitroaromatic Chemicals (Cont'd)
t-t
CO
CJ
•H
e
CU
j=
u
£-Nitrobenzoic acid
o-Nitrobiphenyl
A-Nitrod iphenylamine
1-Nitronaphthalene
3-Nitro-l , 5-naphthalene-
disulfonic acid
7-(and 8-)Nitronaphth-
(l,2-d)U,2,3)oxadia-
zole-5-sulfonic acid
o-Nitrophenol
£-Nitrophenol and
sodium salt
4 ' - (j>-Nitrophenyl)
acetophenone
Largest Annual
Consumption (C)
During (i9 )
(import quantities
sometimes used)
(thousands of pounds)
>460 (72)
6,290 (72)
223 (65)
551 (69)
10,000-15,000
(75)
60,000r
100,000 (75)
42 (70)
ase Factor (RF)
icides and
umes=1.00; Expio-
s=0.05; Chemical
rmediates=0.05
^H 05 U > U
v y a) -* c
a: a, &. tn t-
0.05
1.00
0.05
0.05
0.^5
0.05
0.05
0.05
0.05
amination
or
i RF)
C U
O TO U
U fc. -^
23
315
11
28
750
5,000
2
Biodegradability
Summary
+ + +
+
+ + +
Days to
Disappearance
4
>64
16
Transport
Bioconcentra-
tion
13
12
14
Ecological
Magnification
3
.43
o
in
O ^
J .— 00
tn ^
i-H U ^>
CD CO OC
" S.-S:
1,960
1,230
120
2,828
350-467
Metabolic
Effects
—
—
—
—
+
Hematologic
Effects
— '
+
+
+
CO
.*
U
<3
E
CU
01
Non-tumorigenic
Non-tumorigenic : 48 hr. TL
m
(Bluegills) = 46.3-51.6; monitored
in chemical plant lagoon
Non-tumorigenic; monitored in
parathion plant water effluent
O
—I
-------
Table 145. Summary of Information on Nitroaromatic Chemicals (Cont'd)
tc
y
oj
.n
o
2-Nitro-£-phenylene-
diamlne
4-Nitro-o-phenylene-
diamine
4-Nitrostilbene
2-Nitrotoluene
3-Nitro toluene
4-Nitro toluene
5-Nitro-o-toluenesulfonic
acid (1-SO.jH)
3-Nitro-j>-toluenesulfonic
acid (1-S03H)
Largest Annua]
Consumption (C)
During (19 )
(import quantities
sometimes used)
(thousands of pounds)
10,000-12,000
(75)
17,750 (68)
7,955
81 (68)
Release Factor (RF)
Pesticides and
perfumes=1.00; Explo-
aives=0.05; Chemical
intermedlates=0.05
1.00
(hair dye)
1.00
({lair dye)
0.05
0.05
0.05
0.05
0.05
Contamination
Factor
(C x RF)
600
888
398
4
Biodegradabillty
X
3
±
Days to
Disappearance
>64
>64
>64
Transport
Bioconcentra-
tion
23
28
26
Ecological
Magnification
7
9
0
in
Q *-*
,J ,-,00
01 j>:
to to oo
o ^ ^
891
1,072-2,282
2,144
Metabolic
Effects
—
—
—
Hematologic
Effects
+
^
+
to
I*
0}
a:
Carcinogenic
Carcinogenic
Carcinogenic; ^°iOQ (Mice) =
500 mg/kg I. P.
6 hr. LC . (Fish) - 18-40 mg/1;
min
monitored in TNT plant water
effluent
6 hr. LC . (Fish) = 14-30 mg/1
min
6 hr. LC' (Fish) - 20-25 mg/1;
min
monitored in TNT plant water
effluent
Ul
o
03
-------
Table 149. Summary of Information on Nitroaromatic Chemicals (Cont'd)
t-t
CO
a
E
0)
o
2-Ni t ro-j>- toluldine
(1-amino)
4-Nitro-o-toluidine
(1-amino)
5-Nitro-o-toluidine
(1-amino)
Nitroxylenes
£-Nitro-o-xylene
Pentachloronitrobenzene
2,3,5, 6-Te trachloro-
nitrobenzene
2,3,4,6-Tetranitroaniline
N , 2 , 4 , 6-Te tranitroaniline
(Tetryl)
l,2,4-Trichloro-5-
nitrobenzene
Largest Annual
Consumption (C)
During (19 )
(import quantities
sometimes used)
(thousands of pounds)
864 (67)
>334 (72)
353 (73)
545 (59)
>407 (72)
3,000 (72)
3,600 (73)
(discontinued)
Release Factor (RF)
Pesticides and
perf umes=l .00; Explo-
sives^. 05; Chemical
intermed iates=0.05
0.05
0.05
0.05
0.05
0.05
1.00
1.00
0.05
Contamination
Factor
(C x RF)
43
17
18
27
20
3,000
Biodegradability
Summary
Days to
Disappearance
—
Transport
Bioconcentra-
tion
Ecological
Magnification
25,520
4,074
1,555
o
in
a ___-—
— 1 AJ ^
CO CO 00
574
1,650-1,740
Metabolic
Effects
—
—
Hematologic
Effects
—
—
CO
t-
1
(U
S£.
Carcinogenic
Produced neoplasms
LD (Dogs) « 2,500 mg/kg S.C.
nun
Produced neoplasms
LD50 (Blackbirds) = 100 mg/kg oral
(Jl
o
VO
-------
Table 149. Summary of Information on Nitroaromatic Chemicals (Cont'd)
o)
u
t-i
§
ji
a,a,a-Trifluoro-2,6-
dinitro-N,N-dypropyl-j>-
toluidine (Trifluralin)
2,4, 6-Trini trophenol
2,4,6-Trinitrotoluene
2,4,6-Trinitroresorcinol
3
c c
Largest An
Consumptio
During (19
en
en c
— < O
i_l 0) UJ
C 01 O
a a
( impo r t q u
sometimes
(thousands
25,500 (72)
432,000 (73)
i
o —
S X •— O
^- U3 E •
D O
V- T3 •* -C U
o c o u «
u cd O cU
b« CJ II O -~H
•O 01 -13
cn cj e ll E
to — ' 3 « Vi
u
(£ SU 0. X -^
1.00
0.05
0.05
0.05
c
o
•H
Contaminat
Factor
; (c x RF)
i
25,500
21,600
3iodegradability
u
CO
3
±
+
u
c
Days to
Disappear*
Transport
ij
Bioconcent
tion
T<
CD (3
CJ U
-H i-i
DO u-i
O 11
O 00
U CD
w 2:
O
u-i
O — *
_3 ^-N 00
a a oo
£ 53
5,000
1 Metabolic
Effects
—
• —
u
1 Hematolog
Effects
—
+
tn
C!
a:
48 hr. TL (Trout) = 11 mg/1
Q)
Non-tumorigenic; 96 hr. TL
m
(Bluegills) =2.6 mg/1; monitored
in TNT plant effluent
Ol
(-"
o
-------
Commercial compounds having no data on fate or effects were selected if avail-
able information indicated that they were produced or imported (the sign, >, was
used when import data were used) in quantities exceeding 100,000 pounds per year.
Unfortunately, the chemical marketing literature is not comprehensive enough
to assure that all nitroaromatic compounds consumed in over 100,000 pounds are
included. However, numerous manufacturers were personally contacted in an effort
to identify all nitroaromatics consumed in over 500,000 pounds per year. Thus,
the list is fairly comprehensive for nitroaromatics in that consumption category.
Nitroaromatic compounds find applications as pesticides, perfumes,
explosives, and chemical intermediates. Each of these uses has a different
potential for release of the compound to the environment. The release factor,
which was calculated in Table 149, was used to convert the consumption figure
into a number related to the quantities likely to be released to the environ-
ment. Pesticides and perfumes were assigned a factor of 1.00, since they would
be used in such a way that 100% would be released to the environment or would
come into human contact. Chemical intermediates were given a small factor since
most of the chemicals will be converted to another material (0.05 is arbitrary,
but is probably an upper limit of the amount that might be released during pro-
duction, transport, and use). Even though a high release factor was used, there
are only about six non-pesticidal chemicals that are produced in large enough
quantities that the amount likely to be released to the environment would exceed
the quantity from a relatively low volume pesticide (^ 1 million pounds per year),
An attempt was made to include the biodegradation data in the
contamination factor calculation. However, after examining the information,
it did not appear that a quantitative value should be assigned. Thus, the
511
-------
summary uses only qualitative signs of biodegradability (see Table 44 for the
key). Also, much of the biodegradation work was done with different pure cultures
of microorganisms and correlation of the results to environmental fate is diffi-
cult. One study by Alexander and Lustigman (1966) did evaluate the biodegrad-
ability of a number of disubstituted nitroaromatics with a mixed population of
microorganisms. These results are also included in Table 149t (the days noted
correspond to the time necessary for the ultraviolet adsorbancy to return to
the value of the blank). In general, nitroaromatics appear to be fairly stable,
although, depending upon the other substituents, some chemicals do biodegrade.
On the other hand, some nitroaromatics (e.g. , chloronitrobenzenes) are extremely
persistent. With the exception of TNT, not enough information is available to
determine the environmental photodegradation potential for the nitroaromatics.
However, most of the chemicals absorb ultraviolet light at wavelengths greater
than 290 nm (wavelengths available in sunlight).
The bioconcentration and ecological magnification potential have
been calculated for a number of compounds included in Table 149. With the
exception of the highly chlorinated nitrobenzenes, which are mostly used as
pesticides, none of the compounds is likely to bioaccumulate in higher trophic
levels to the same extent as many of the organochlorine pesticides (for compari-
son, the bioconcentration of endrin was 2,953; ecological magnification for DDT
was 16,950). However, increases in the calculated concentration of several
hundred are not uncommon.
Many of the important commercial nitroaromatic compounds have been
detected in drinking water, river water, and waste water effluents (see remarks,
Table 149). Detecting a chemical in the environment is an important factor in
determining its potential hazard. However, lack of detection cannot be interpreted
512
-------
to indicate that a chemical is not an environmental contaminant unless a very
sensitive and specific analytical method is used with a well-designed sampling
scheme. This is rarely the case, as evidenced by the fact that no nitroaromatic
compounds have been detected in effluent or ambient air samples, even though
the high vapor pressure of some of the chemicals would suggest substantial
evaporation. Several non-commercial nitroaromatic compounds have been detec-
ted in water samples (see Table 150). Most of these chemicals are by-products
Table 150. Nitroaromatic Compounds Detected in River, Drinking, or Waste Waters
That Are Not Commercial Products
Chemical
Type of Sample
Concentration
4,6-Dinitro-2-aminophenol
2,4-Dinitrotoluene-5-sulfonic acid
2,4-Dinitrotoluene-3-sulfonic acid
3,5-Dinitrobenzenesulfonic acid
Trinitrobenzoic acid
3,4-Dinitrotoluene
Drinking water
TNT plant water effluent
TNT plant water effluent
TNT plant water effluent
TNT plant water effluent
Explosives (DNT) plant
water effluent
0.80 ppm
40 ppm
from TNT or DNT plants (with 4,6-dinitro-2-aminophenol, the source is unknown)
Other nitration processes may produce by-products which could be emitted to
the environment, but little information on identity or quantities lost is
available.
513
-------
Nitroaromatic compounds exhibit several distinct and important
biological effects. Nitrobenzene and its derivatives (dinitrobenzenes, nitro-
toluenes) primarily affect the hematologic system through the production of
methemoglobinemia, sulfhemoglobinemia, Heinz bodies, and red cell destruction.
2,4-Dinitrophenol and related structures (2-sec-buty1-4,6-dinitrophenol, 4,6-
dinitro-o-cresol) are unique in their ability to "uncouple" oxidative phos-
phorylation by suppressing the coupling of electron flow to synthesis of ATP.
This uncoupling effect produces a profound disturbance of metabolic function.
In addition, a number of nitroaromatic chemicals (most of which are not pro-
duced in large commercial quantities) are active tumor-producing agents in
animals.
Repeated exposures to nitroaromatic chemicals can typically result
in irreversible damage to the major organs responsible for foreign compound
detoxification (principally the liver and kidneys). Numerous fatalities have
occurred in humans as a result of occupational and accidental poisoning by
nitroaromatic compounds. Non-fatal exposure to these substances has produced
a plethora of cases involving cyanosis, anemia, CNS disturbance, cataracts,
liver disease, allergic reactions, and severe contact dermatitis. In most
instances of acute exposure, however, the resulting effects on the hematologic
system or metabolic function are rapidly reversible following removal from
exposure. In only a few cases (e.g., 4,6-dinitro-o_-cresol, m-dinitrobenzene)
does it appear that cumulative toxicity may occur from long-term, low-level
exposures.
In general, the dinitrophenol derivatives are far more acutely
toxic than the nitrobenzene compounds (rat oral LD < 75 mg/kg) and the
danger from single exposures may be very great. Most of the nitroaromatic
514
-------
compounds which have been tested are readily absorbed by oral, dermal, and
inhalational routes. Increasing substitution of the nitroaromatic nucleus,
as well as the addition of bulky substituents, tends to decrease both absorp-
tion and toxicity. In lower animals such as fish, the nitroaromatics are
comparatively quite highly toxic, with nitrophenol derivatives being particu-
larly active.
A number of nitroaromatic chemicals are active tumor-initiators,
tumor-promoters, or complete carcinogens in animals (Table 151). These com-
pounds appear to owe their tumorigenic activity to a highly reactive metabolic
intermediate, believed to be a nitroso or hydroxylamino derivative. These
same intermediates are formed during the metabolic conversion of the classical
aromatic amine carcinogens. Thus, it has been possible to demonstrate that
most nitro. analogs of aromatic amine carcinogens are likewise active tumor-
producing agents. Moreover, it is known that certain heterocyclic nitro
compounds and polychlorinated nitrobenzenes possess unique tumorigenic proper-
ties. It is important to note, however, that while many nitroaromatic com-
pounds produce tumors in animals, these neoplastic growths are not necessarily
malignant. The information presented in Table 151 distinguishes between com-
pounds that produced neoplasms (not necessarily malignant) and those that
induced malignant carcinomas.
It is exceedingly difficult to assess the overall toxic hazard posed
by many of the commercially significant nitroaromatic chemicals. The published
literature is sorely deficient in data regarding chronic exposures, mutagenic
and teratogenic effects, and possible carcinogenic bioactivation for many of
the high-volume nitroaromatics produced today. The priorities of past toxi-
cology research with the nitroaromatics have generally not emphasized those
compounds with highest production volume or greatest environmental contamina-
tion potential.
515
-------
Table 151. Tumor Production in Animals by Nitroaromatic Chemicals
Compound
Effect
2-Amino-4- (_p_-nitropheny 1) thiazole
l-Chloro-2,4-dinitronaphthalene
2,6-Dichloro-4-nitroaniline
l,2-Dichloro-3-nitronaphthalene
4,4'-Dinitrobiphenyl
2,5-Dinitrofluorene
2,7-Dinitrofluorene
l-Fluoro-2,4-dinitrobenzene
Hexanitrodiphenylamine
2-Hydrazino-4- (p_-nitrophenyl)thiazole
_p_-Nitrobiphenyl
2-Nitrofluorene
5-Nitro-2-furaldehyde semicarbazone
N-[4-(5-Nitro-2-furyl)-2-thiazolyl]acetamide
2-Nitronaphthalene
_p_-Nitroperbenzoic acid
4-Nitro-o-phenylenediamine
2-Nitro-jg-phenylenediamine
4-Nitroquinoline-N-oxide
£-Nitrostilbene
Pentachloronitrobenzene
2,3,4,5-, 2,3,4,6-, and 2,3,5,6-Tetrach'loronitrobenzene
carcinoma
carcinoma
neoplasm
neoplasm
neoplasm
carcinoma
carcinoma
tumor
promotion
neoplasm
carcinoma
carcinoma
carcinoma
carcinoma
carcinoma
neoplasm
neoplasm
carcinoma
carcinoma
carcinoma
carcinoma
carcinoma
neoplasm
516
-------
B. Conclusions
This report has focused on non-pesticidal nitroaromatic compounds.
Most of the commercial products are consumed as chemical intermediates and,
therefore, the major source of environmental contamination is from chemical
production or use plants. However, with the exception of TNT production and
use, information on the quantities of chemicals released is not available.
Many of the chemicals appear to be persistent and some may bioaccumulate
slightly. The toxicologic information is not adequate, considering the quanti-
ties of some compounds that are produced. Few of the commercially important
compounds have been tested for carcinogenic, mutagenic, or teratogenic effects.
Nevertheless, an attempt has been made to determine the relative environmental
hazard of the non-pesticidal nitroaromatic compounds (Table 152).
The compounds in Table 152 are listed in descending order by the
value of the contamination factor (0.05 x consumption) calculated in Table 149.
However, the order is not necessarily the same as for pollution potential,
although an exact ordering based upon the available information does not
seem justified. Nevertheless, some of the compounds appear to have high
priorities for further study. All the nitroaromatic compounds that have been
detected in drinking water should be closely examined and tested. Detection
in drinking water is the best evidence that the chemical is persistent, at
least long enough to be transported from the chemical plant to the drinking
water plant. Air monitoring studies, which unfortunately are not available,
would have been extremely useful in setting priorities.
The monochloronitrobenzene group is interesting in that the one
compound produced in the smallest quantity (l-chloro-3-nitrobenzene) ±s the
only one detected in drinking water. Many explanations are possible, including:
517
-------
Table 152. Nitroaromatic Compounds Which Have a High Potential for Being Environmental Pollutants
Chemical
-Nitrobenzene
Dinitrotoluene
2,4,6-Trinitrotoluene (TNT)
l-Chloro-4-nitrobenzene
£-Nitrophenol
l-Chloro-2-nitrobenzene
4-Nitrotoluene
o-Nitrophenol
£-Nitroaniline
Contamination
Factor
32750
23561
21600
5500
5000
3000
888
750
700
Biodegradable
7
Yes
Yes
No
Yes
No
1
7
7
Monitored in
Water Effluents
or
River Water
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Monitored
in
Drinking
Water
Yes
Yes
No
No
No
No
No
No
No
Oral LD5Q
(rats)
mg/kg
640-664
268-707
—
420
350-467
288
2144
2828
1410-3249
Fish Toxicity
(96 TLM;
mg/1)
—
16
2.6
—
—
—
20-50
(6 hr.
LCMIN>
46.3-51.6
(48 hr.
—
Tested for
Carcinogenicity
No
Yes-Neg.
Yes-Neg.
No
Yes-Neg.
No
No
Yea-Neg.
No
C3
-------
Table 152. Nitroaromatic Compounds Which Have a High Potential for Being Environmental Pollutants
(Cont'd)
Chemical
2-Nitrotoluene
1 , 3-Dinitrobenzene
4,4' -Dinitrostilbene-2 , 2 ' -disul-
fonic acid
5-Nitro-<3-toluenesulfonic acid
l-Chloro-3-nitrobenzene
l-Chloro-2 ,4-dinitrobenzene
1-Nitronaphthalene
c^-Nitroaniline
m-Nitrobenzenesulfonic acid
Contamination
Factor
600
600
493
398
395
331
315
300
183
Biodegradable
No
No
—
—
No
No
—
?
—
Monitored in •
Water Effluents
or
River Water
Yes
Yes
No
No
Yes
Yes
No
No
No
Monitored
in
Drinking
Water
No
Yes
No
No
Yes
No
No
No
No
Oral LD5Q
(rats)
mg/kg
891
—
— '
—
555
500-1593
120
535-3520
—
Fish Toxicity
(96 TLM;
mg/1)
18-40
(UW
—
—
—
—
—
—
—
—
Tested
for
Carcino-
genicity
No
No
No
No
No .
No
No
No
No
Ln
-------
Table 152. Nitroaromatic Compounds Which Have a High Potential for Being Environmental Pollutants
(Cont'd)
Chemical
1 , 2-Dichloro-4-nitrobenzene
6-tert-Butyl-2 , 4 , 6-trini tro-m-
xy'lene
2-Chloro-4-nitroaniline
c>-Nitroanisole
6-tert-Butyl-3-methyl-2 ,4-
dinitroanisole
£-Nitroanisole
2,4-Dinitrophenol
2-Bromo-4 ,6-dinitroaniline
m-Nitrobenzoic acid
2-Nitro-g-toluidine
Contamination
Factor
180
163
150
150
119
75
50
47
46
43
Biodegradable
—
—
—
No
—
No
7
—
7
—
Monitored in
Water Effluents
or
River Water
No
No
No
No
No
No
No
No
No
.No
Monitored
in
Drinking
Water
No
No
No
Yes*
No
Yes*
No
No
No
No
Oral LD50
(rats)
rag/kg
643
—
—
—
339
--
30
4490
1820
—
Fish Toxicity
(96 THj;
mg/1)
—
—
—
—
—
—
0.5-38
(L(W
—
—
—
Tested for
Carcinogenic! ty
No
No
No
No
No
No
Yes-Neg .
No
Yes-Neg.
No
Ul
M
O
* Nitroanisole has been removed from EPA's list of organic chemicals identified in drinking water.
-------
Table 152. Nitroaromatic Compounds Which Have a High Potential for Being Environmental Pollutants
(Cont'd)
Chemical
l,4-Dlchloro-2-nitrobenzene
4-Chloro-3-nitrobenzenesul-
fonamlde
4-Chloro-?2-nitro toluene
2 , 4-Dini t roaniline
2,6-Dichloro-4-nltroaniline
2-Chloro-5-nitrobenzenesul-
fonlc acid
7-(and 8-)Nitronaphth(i,2-d)
(1,2 , 3) oxadlazole-5-sulf onlc
acid
Nitroxylenes
4-Chloro-2-nitroaniline
Contamination
Factor
40
37
35
34
30
30
28
27
25
Biodegradable
—
—
—
— -
—
—
—
—
—
Monitored in
Water Effluents
or
River Water
No
No
No
No
No
No
No
No
No
Monitored
in
Drinking
Water
No
No
No
No
No
No
No
No
No
Oral LD5Q
(rats)
rag/kg
1210
—
—
1800
418-
>5000
—
—
• —
—
Fish Toxicity
(96TLM;
mg/1)
—
—
—
—
37
—
— '
—
—
Tested for
Carcinogenicity
No
No
No
.No
Yes;
produced
tumors
No
No
No
No
Ul
NJ
-------
1) the meta-isomer may be more stable in the environment (the fact that the
ortho-isomer was detected after traveling 1000 miles in the Mississippi River
does not support this explanation) and 2) the meta-isomer may be an undesirable
by-product and, therefore, only minimal efforts are exerted to recover the
chemical when produced. The chloronitrobenzene compounds in general appear to
be very persistent and, therefore, even the compounds produced in small quanti-
ties should be studied. l-Chloro-2,4-dinitrobenzene, in particular, should be
examined further because it is such a potent skin sensitizer.
p-Nitrophenol is another compound that is produced in large enough
quantities that sizable amounts might be released to the environment. However,
since £-nitrophenol is a major breakdown product of the parathions and nearly
50-60 million pounds of parathions are intentionally released to the environ-
ment each year, it seems unlikely that losses of j>-nitrophenol from product
and chemical intermediate use would be significant compared to the parathion-
derived source.
There is one compound, 4,6-dinitro-2-aminophenol, that has been
detected in drinking water but is not an important commercial product or an
obvious by-product of a large commercial product. It seems likely that the
precursor to the dinitroaminophenol probably contains three nitrogen substitu-
ents. This limits possibilities for its source considerably, with prime can-
didates being the dinitroaniline herbicides or TNT.
Although only a few of the compounds have been tested for carcino-
genic activity, one compound, 2,6-dichloro-4-nitroaniline, has been found to
be tumorigenic. The low production volume suggests that this compound may
pose an occupational or localized environmental problem, but is not likely to
be a widespread environmental contaminant. However, the compound is also used
as a fungicide which will result in considerable release to the environment
and potential human exposure to the chemical.
522
-------
1-Nitronaphthalene may be a significant environmental contaminant
since it is related by reduction to a-naphthylamine, a suspected carcinogen.
For this reason, study of the effluents from the one plant (DuPont) that pro-
duces the compound seems desirable.
In conclusion, it appears that a sizable number of nitroaromatic
compounds are produced in large enough quantities that significant quantities
can be released to the environment, even though most of the chemicals are used
as chemical intermediates. Detection of these chemicals in drinking water
supports this contention. Air monitoring and further water monitoring studies
to more accurately determine the extent of nitroaromatic environmental contami-
nation would seem desirable. Many of the chemicals are stable in the environ-
ment, and some seem to be extremely persistent. Most of the biological effects
that have been noted are reversible and have no-effect concentrations above
likely environmental concentrations. However, in vitro screening of these
compounds for mutagenic/carcinogenic effects using inexpensive methods, such
as devised by Ames e£ jal^ (1975), would appear to be very desirable.
523
-------
524
-------
Chemical Index
525
-------
8'-Acetamido-l-(4-acetamido-2-hydroxy-
5-nitrophenyl azo)-2-naphthol
Toms River • Toms River, NJ
N-Acetyl'-4,4'-dinitrodiphenylamine
(p. 88)
4
N -Acetyl-N'-(4-nitrophenyl)sulfa-
nilamide
Salsbury Labs Charles City, IA
2-(j)-Aminoanilino)-5-nitrobenzene-
sulfonic acid (p. 51, 54)
Mobay Chem. Bayonee, NJ
Charleston, SC
Toms River Toms River, NJ
2-Amino-5-chloro-l-nitrobenzene
(p. 26)
4-Amino-3-chloro-l-nitrobenzene
(p. 26)
2-Amino-3-chloro-5-nitrobenzonitrile
(p. 54)
2-Amino-4-chloro-5-nitrophenol
(p. 54, 88)
2-Amino-4-chloro-6-nitrophenol
(p. 54, 88)
Nyanza Ashland, MA
2-Amino-6-chloro-4-nitrophenol
(p. 54, 88)
6-Amino-4-chloro-5-nitrophenol
(p. 88)
4-Amino-.l,3-dimethyl-5-nitrobenzene
(5-Nitro-4-amino-l,3-dimethyl-
benzene) (p. 94)
2-Amino-4,6-dinitrophenol (4,6-
Dinitro-2-aminophenol; Picramic
acid) (p. 55, 88, 92, 96, 125,
128, 130, 471)
2-Amino-4,6-dinitrophenol, sodium salt
(Picramic acid, sodium salt; sodium
picramate) (p. 96)
Martin Marietta Sodyeco, NC
2-Amino-4,6-dinitrotoluene (6-Amino-
2,4-dinitrotoluene) (p. 177, 235)
4-Amino-2,6-dinitrotoluene (2,6-Dinitro-
4-aminotoluene) (p. 235).
2-Amino-N-ethyl-5-nitrobenzenesulfon-
anilide (p. 54)
GAF Rensselaer, NY
2-Amino-5-nitrobenzenesulfonic acid
(p. 51, 54, 74, 88)
GAF Linden, NJ
Toms River Toms River, NJ
2-Amino-5-nitrobenzenesulfinic acid,
sodium salt (p. 54)
2-Amino-5-nitrobenzenesulfonic acid,
ammonium salt (p. 54)
2-Amino-4-nitrobenzoic acid
Salsbury Labs Charles City, IA
2-Amino-5-nitrobenzoic acid (5-Nitro-
anthranilic acid) (p. 54)
Toms River Toms River, NJ
2-Amino-5-nitrobenzonitrile (p. 54)
2-Amino-6-nitrobenzothiazole (p. 54)
Inmont Hawthorne, NJ
2-Amino-5-nitrobenzotrifluoride
Olin Rochester, NY
1-Amino-N-nitronaphthalene (N-Nitro-
1-aminonaphthalene) (p. 94)
5-Amino-l-nitronaphthalene (p. 27)
2-Amino-5-nitro-N-(phenethylBenzene-
surf onamide (p. 54)
526
-------
2-Amino-4-nitrophenol (4-Nitro-2-amino-
phenol) (p. 4, 18, 26, 49, 54, 83, 88,
94, 156, 164, 223, 224, 225, 241, 358,
387, 471)
GAF Rensselaer, NY
2-Amino-4-nitrophenol, sodium salt
(p. 54)
2-Amino-5-nitrophenol (p. 18, 54, 88,
236, 239)
GAF Rensselaer, NY
4-Amino-2-nitrophenol (p. 18, 88, 163,
164, 224, 225, 241, 387)
Mobay Bayonne, NJ
Charleston, SC
4-Amino-3-nitrophenol (4-Amino-5-
nitrophenol) (p. 88)
Ashland Great Meadows, NJ
2-Amino-6-nitro-l-phenol-4-sulfonic
acid (p. 88)
4-Amino-6-ni t ro-1-pheno1-2-s uIfoni c
acid (p. 88)
6-Amino-4-nitro-l-phenol-2-sulfonic
acid (p. 88, 89)
2-Amino-(_p_-nitrophenylazo)naphthalene
American Color
and Chem. Lock Haven, PA
2-Amino-4- (p_-nitrophenyl) thiazole
(p. 415)
4-Amino-4'-ni tro-2,2'-s tilbenedisul-
fonic acid (p. 4, 52, 54, 89)
GAF Rensselaer, NY
Toms River Toms River, NJ
4-Amino-4'-nitro-2,2'-s tilbenedisul-
fonic acid, disodium salt ..
Allied Buffalo, NY
2-Amino-4-nitrotoluene (see 5-Nitro-
o-toluidine)
4-Amino-2-nitrotoluene (see 3-Nitro-
pj-toluidine)
Ammonium picrate (see 2,4,6-Trinitro-
phenol, ammonium salt)
2-sec-Amyl-4,6-dinitrophenol (p. 358)
2-(o-Anisidino)-5-nitrobenzenesulfonic
acid
Toms River Toms River, NJ
Balan (see N-Butyl-N-ethyl-a,a,a-tri-
fluoro-2,6-dinitro-p_-toluidine
Benefin (see N-Butyl-N-ethyl-a,a,a-
trifluoro-2,6-dinitro-p_-toluidine)
Benzyl _p_-nitrophenyl ether
GAF Linden, NJ
BNP (see 2,6-Di-tert-butyl-4-nitrophenol)
2-Bromo-6-chloro-4-nitroaniline (p. 89)
American Color
and Chem. Lock Haven, PA
Martin Marietta Sodyeco, NC
4-Bromo-2-chloro-6-nitrophenol (p. 444)
2-Bromo-4,6-dinitroaniline (p. 4, 19,
50, 53, 54, 59, 63, 83, 352)
American Color
and Chem. Lock Haven, PA
Marin Marietta Sodyeco, NC
Toms River Toms River, NJ
3-Bromo-2,4-dinitrophenol (p. 444)
3-Bromo-4,6-dinitrophenol (p. 444)
2-Bromo-4'-nitroacetophenone
GAF Rensselaer, NY
l-Bromo-2-nitrobenzene (o-Bromonitro-
benzene) (p. 54, 122)
2-Bromo-4-nitrobenzoic acid (2-Bromo-
4-nitrobenzoate) (p. 142, 143, 151,
152, 182)
527
-------
2-Bromo-4-nitrophenol (p. 440, 442)
2-Bromo-4-nit.rophenol, sodium salt
(p. 440)
3-Bromo-4-nitrophenol (p. 440, 442)
4-Bromo-2-nitrophenol (p. 442)
5-Brojno-2-nitrophenol (p. 442)
2'-Bromo-3-nitrosalicylanilide (p. 447)
3'-Bromo-3-nitrosalicylanilide (p. 447)
4'-Bromo-3-nitrosalicylanilide (p. 447)
4'-Bromo-5-nitrosalicylanilide (p. 447)
a-B r omo-j>-ni t ro t oluene
RSA Ardsley, NY
3-Bromo-2,4,6-trinitrophenol (p. 444)
N-sec-Butyl-4-tert-butyl-2,6~dinitro-
aniline (p. 42)
4'-tert-Butyl-2',6'-dimethyl-3'.5'-
dinitroacetophenone (musk ketone)
(p. 54)
Givaudan Clifton, NJ
2-sec-Butyl-4,6-dinitrophenol (DNBP,
Dinoseb, Dinitrobutylphenol; 4,6-
Dinitro-o-sec-butylphenol) (p. 4, 41,
42, 54, 15, 59, 63, 82, 89, 92, 183,
210, 211, 212, 213, 242, 243, 301,
302, 303, 323, 327, 329, 346, 349,
350, 360, 361, 375, 377, 378, 385,
386, 406, 408, 409, 449, 450, 452)
Blue Spruce Edison, NJ
Dow Chem. Midland, MI
Vicksburg Vicksburg, MS
2-sec-Butyl-4,6-dinitrophenol,
ammonium salt (p. 89)
FMC Middleport, NY
2-sec-Butyl-4,6-dinitrophenol,
alkanolamine salt (p. 89, 308)
Dow Chem. Midland, MI
2-sec-Butyl-4,6-dinitrophenol,
isopropanolamine salt (p. 89, 301)
Blue Spruce Edison, NJ
2-sec-Butyl-4,6-dinitrophenol,
triethanolamine salt (p. 89, 92,
301)
Blue Spruce Edison, NJ
FMC Middleport, NY
2-sec-Butyl-4,6-dinitrophenyl acetate
(p. 371, 400)
2-tert-Butyl-4,6-dinitrophenyl acetate
(p. 346, 371)
2-sec-Butyl-4,6-dinitrophenyl-3,3-
dimethylacrylate (Morocide, binapcryl)
(p. 89, 304, 347, 371)
FMC Middleport, NY
2-sec-Buty1-4,6-dinitrophenyl isopropyl
carbonate (Dinobuton) (p. 349, 350)
Union Carbide Institute, WV
S. Charleston, WV
2-sec-Butyl-4,6-dinitrophenyl-3-
methyl-2-butenoate (see 2-sec-Butyl-
4,6-dinitrophenyl-3,3-dimethyl
acrylate)
2-sec-Butyl-4,6-dinitrophenyl-3-methyl
crotpnate (see 2-sec-Butyl-4,6-
dinitropheny1-3,3-dimethylacrylate)
6-tert-Butyl-2,4-dinitrophenylmethane
sulfonate (p. 371)
N-Butyl-N-ethyl-a,a,a-trifluoro-2,6-
dinitro-£-toluidine (Benefin; Balan)
(p. 89, 449, 450, 493)
Eli Lilly Lafayette, IN
528
-------
6-_tert-Butyl-3-methyl-2,4-dinitro-
anisole (Musk ambrette) (p. 4, 54,
76, 89, 371)
Givaudan Clifton, NJ
l-tert-Butyl-3,4,5-trimethyl-2,6-
dinitrobenzene (Musk tibetene)
(p. 89)
Givaudan Clifton, NJ
2-tert-Butyl-5-methyl-4,6-dinitro-
phenyl acetate (Medinoterb acetate)
(p. 346, 371)
N-sec-Butyl-p-nitroaniline (p. 89)
5-tert-Butyl-2,4,6-trinitro-m-xylene
(Musk xylene) (p. 54, 89)
Givaudan Clifton, NJ
N,N'-Carbonylbis(4-methoxy-6-nitro-
metanilic acid)
GAF Rensselaer, NY
2-Chloro-l,4-dibutoxy-5-nitrobenzene
(p. 54)
2-Chloro-l,4-diethoxy-5-nitrobenzene
(p. 54)
Fairmount Newark, NJ
GAF Rensselaer, NY
2-Chloro-N,N-diethyl-4-nitroaniline
Dupont Deepwater, NJ
4-Chloro-N,N-dimethy1-3-nitrobenzene-
sulfonamide
GAF Rensselaer, NY
2-Chloro-4,6-dinitroaniline (p. 19, 54)
GAF Rensselaer, NY
l-Chloro-2,4-dinitrobenzene (2,4-
dinitrochlorobenzene; DNCB) (p. 3, 4,
26, 30, 48, 53, 54, 59, 62, 76, 82,
113, 124, 129, 135, 139, 182, 219,
252, 259, 260, 296, 317, 318, 334,
355, 389, 390, 395, 396, 397, 424,
495, 497)
Martin Marietta Sodyeco, NC
2-Chloro-3,5-dinitrobenzenesulfonic
acid (p. 89)
3-Chloro-4,6-dinitrobenzenesulfonic
acid
Toms River Toms River, NJ
2-Chloro-3,5-dinitrobenzoic acid
Ashland Great Meadows, NJ
4-Chloro-3,5-dinitrobenzoic acid
Ashland Great Meadows, NJ
GAF Linden, NJ
2-Chloro-3,5-dinitrobenzotrifluoride
(p. 89)
l-Chloro-2,4-dinitronaphthalene
(p. 415, 430)
2-Chloro-4,6-dinitrophenol (6-Chloro-
2,4-dinitrophenol) (p. 90, 333, 334,
358, 444)
4-Chloro-2,6-dinitrophenol (p. 90, 444)
5-Chloro-2,4-dinitrophenol (p. 333, 334)
N-(.2-Chloroethyl)-4-(2-chloro-4-nitro-
phenylazo)-N-ethylaniline
GAF Rensselaer, NY
3-Chloro-4-fluoronitrobenzene
Olin Rochester, NY
4-Chloro-N-isopropyl-3-nitrobenzene
sulfonamide
Toms River Toms River, NJ
4-Chloro-N-methyl-3-nitrobenzenesul-
fonamide
Toms River Toms River, NJ
2'-Chloro-6'-methyl-3-nitrosalicylanilide
(p. 448)
4-Chloro-3-(methylsulfonyl)nitrobenzene
Toms River Toms River, NJ
529
-------
4-Chloro-3-nitroacetanilide
Frank Enterprises Columbus, OH
4-Chloro-5-nitro-2-aminophenol (see
2-Amino-4-chloro-5-nitrophenol)
2-Chloro-4-nitroaniline (p. 4, 50, 53,
54, 59, 63, 74, 76, 82, 83, 351)
Chemetron Huntington, WV
Dupont Deepwater, NJ
2-Chloro-5-nitroaniline (p. 19,' 54)
Olin Rochester, NY
4-Chloro-2-nitroaniline (2-Nitro-4-
chloroaniline) (p. 5, 19, 50, 53,
54, 74, 76, 83, 90, 122, 351)
American Color
and Chem. Lock Haven, PA
Mobay Bayonne, NJ
Charleston, SC
Dupont Deepwater, NJ
4-Chloro-3-nitroaniline (3-Nitro-4-
chloroaniline) (p. 4, 19, 54, 351)
Frank Enterprises Columbus, OH
Olin Rochester, NY
4-Chloro-2-nitroanisole (p. 83)
4-Chloro-3-nitroanisole (p. 54)
5-Chloro-2-nitroanisole (p. 19, 54)
2-Chloro-4-nitrobenzamide
Salsbury Labs Charles City, IA
4'-Chlcro-3-nitrobenzanilide (p. 446)
l-Chloro-2-nitrobenzene (o-Chloro-
nitrobenzene; 2-Chloronitrobenzene)
(p. 3, 5, 20, 24, 26, 48, 53, 54,
58, 61, 68, 71, 73, 74, 77, 80,
104, 122, 123, 124, 128, 129, 135,
139, 140, 182, 188, 191, 193, 225,
226, 227, 261, 280, 294, 295, 356,
389, 390, 401, 495, 497
American Color
and Chem. Lock Haven, PA
Dupont Deepwater, NJ
Monsanto Sauget, IL
l-Chloro-3-nitrobenzene (3-Chloro-
nitrobenzene; m-Chloronitrobenzene)
(p. 3, 5, 48, "53, 59, 62, 68, 71,
73, 82, 122, 124, 128, 129, 191,
193, 225, 226, 227, 356, 495, 497)
GAF Linden, NJ
l-Chloro-4-nitrobenzene (p-Chloro-
nitrobenzene; 4-Chloronitrobenzene)
(p. 3, 5, 20, 26, 33, 48, 53, 57,
58, 61, 68, 71, 73, 77, 78, 79, 80,
98, 101, 104, 122, 124, 129, 135,
139, 182, 191, 193, 225, 226, 227,
257, 258, 261, 280, 282, 294, 295,
356, 357, 380, 381, 389, 390, 495,
496, 497)
Dupont Deepwater, NJ
Monsanto Sauget, IL
2-Chloro-5-nitrobenzenesulfinic acid
Toms River Toms River, NJ
4-Chloro-3-nitrobenzenesulfonamide
(p. 5, 52, 60, 64, 83)
GAF Rensselaer, NY
Inmont Hawthorne, NJ
Nyanza Ashland, MA
Salsbury Labs Charles City, IA
Toms River Toms River, NJ
4-Chloro-3-nitrobenzenesulfonanilide
Toms River Toms River, NJ
2-Chloro-4-nitrobenzenesulfonic acid
(p. 5)
2-Chloro-5-nitrobenzenesulfonic acid
(p. 5, 51, 60, 64, 84)
Dupont Deepwater, NJ
Toms River Toms River, NJ
Nyanza Ashland, MA
2-Chloro-5-nitrobenzenesulfonic acid,
sodium salt (p. 6, 20, 51, 60, 64,
84)
Dupont Deepwater, NJ
4-Chloro-3-nitrobenzenesulfonic acid
(p. 5, 51, 90)
GAF Rensselaer, NY
Toms River Toms River, NJ
530
-------
4-Chloro-3-nitrobenzenesulfonic acid,
potassium salt
Allied Chem. Buffalo, NY
4-Chloro-3-nitrobenzenesulfonyl chloride
(p. 6, 52, 90)
2-Chloro-4-nitrobenzoic acid (2-Chloro-
4-nitrobenzoate) (p. 6, 55, 90, 142,
143, 151, 152, 182)
Bofors Indust. Linden, NJ
RSA Corp. Ardsley, NY
Salsbury Labs Charles City, IA
2-Chloro-5-nitrobenzoic acid (p. 90)
Toms River Toms River, NJ
4-Chloro-3-nitrobenzoic acid (p. 55)
Ashland Great Meadows, NJ
5-Chloro-2-nitrobenzoic acid (p. 90)
2-Chloro-5-nitrobenzotrifluoride (p. 90)
4-Chloro-5-nitrobenzotrifluoride
(4-Chloro-a,a,a-trifluoro-3-nitro-
toluene) (p. 55, 90)
GAF Rensselaer, NY
Olin Rochester, NY
2-Chloro-4-nitrobenzoyl chloride
Bofors Linden, NJ
o-(4-Chloro-3-nitrobenzoyl)benzoic
~ acid (p. 6, 51, 90)
American Color
and Chem. Lock Haven, PA
GAF Linden, NJ
2-Chloro-4-nitrophenol (p. 55, 155,
158, 182, 227, 334, 358, 442)
2-Chloro-5-nitrophenol (p. 227, 440)
3-Chloro-2-nitrophenol (p. 227, 442)
3-Chloro-4-nitrophenol (p. 227, 358,
442)
4-Chloro-2-nitrophenol (p. 26, 76, 83,
155, 158, 182, 442)
4-Chloro-3-nitrophenol (p. 227)
5-Chloro-2-nitrophenol (p. 442)
4-Chloro-6-nitro-l-phenol-2-sulfonic
acid (p. 90)
6-Chloro-2-nitro-l-phenol-4-sulfonic
acid (p. 90)
0-2-Chloro-4-nitrophenyl-0,0-diethyl
phosphorothioate
American Cyanamid Linden, NJ
2-Chloro-5-nitrophenyl methyl sulfone
Toms River Toms River, NJ
4-Chloro-3-nitrophenyl methyl sulfone
Toms River Toms River, NJ
2-Chloro-3-nitropyridine
Olin Rochester, NY
2-Chloro-5-nitropyridine
Olin Rochester, NY
2'-Chloro-3-nitrosalicylanilide (p. 447)
2'-Chloro-5-nitrosalicylanilide (p. 447)
3'-Chloro-3-nitrosalicylanilide (p. 447)
3'-Chloro-5-nitrosalicylanilide (p. 447)
4'-Chloro-3-nitrosalicylanilide (p. 446,
447)
4'-Chloro-5-nitrosalicylanilide (p. 447)
a-Chloro-m-nitrotoluene
Eastman Kodak Rochester, NY
2-Chloro-4-nitrotoluene (p. 6, 90, 368)
Dupont Deepwater, NJ
531
-------
2-Chloro-6-nitrotoluene (6-Chloro-
2-nitrotoluene) (p. 6, 27, 76, 91,
360)
Dupont Deepwater, NJ
4-Chloro-2-nitrotoluene (p. 6, 27, 49,
60, 64, 76, 83)
American Color
and Chem. Lock Haven, PA
Synalloy Spartanburg, SC
(Blackman Uhler)
4-Chloro-3-nitrotoluene (p. 6, 49, 55,
91)
Synalloy Spartanburg, SC
(Blackman Uhler)
l-Chloro-2,4,6-trinitrobenzene (2,4,6-
Trinitrochlorobenzene; Picryl
chloride) (p. 30, 97, 115, 398, 399)
Northrop Asheville, NC
2-Cyano-4-nitroanisole (2-Methoxy-5-
nitrobenzonitrile) (p. 39)
DCNA (see 2,6-Dichloro-4-nitroaniline)
Diaminohexanitrobiphenyl
Northrop Asheville, NC
Diaminonitrotoluene (Nitrodiamino-
toluene) (p. 177)
Diaminotrinitrobenzene (p. 91)
Northrop Asheville, NC
Diazodinitrophenol (2-Diazo-4,6-
dinitrophenol) (p. 91)
Hercules Kenvil, NJ
2,6-Dibromo-4-nitroaniline (p. 91)
Martin Marietta Sodyeco, NC
2,6-Dibromo-4-nitrophenol (p. 236, 333,
334, 442)
Sherwin-Williams St. Bernard, OH
4,6-Dibromo-2-nitrophenol (p. 442)
2,6-Di-tert-butyl-4-nitrophenol (BNP)
(p. 213, 214)
2,6-Dibutyl-4-nitrophenol (p. 359)
2'>5-Dichloro-3-tert-butyl-4'-
nitrosalicylanilide (p. 264, 265)
l,2-Dichloro-4,5-dinitrobenzene (p. 354)
2,5-Dichloro-4,6-dinitrophenol (p. 444)
2,5-Dichloro-4-nitroaniline (p. 19)
2,6-Dichloro-4-nitroaniline (DCNA;
l-Amino-2,6-dichloro-4-nitrobenzene)
(p. 6, 19, 50, 53, 55, 60, 64, 74,
76, 84, 120, 236, 237, 264, 338,
348, 351, 376, 377, 388, 389, 415,
453, 493)
GAF Rensselaer, NY
Kewanee Louisville, KY
(Harshaw)
Upjohn North Haven, CT
l,2-Dichloro-4-nitrobenzene (3,4-
Dichloro-1-nitrobenzene) (p. 3, 7,
26, 59, 63, 82, 122, 193, 354)
Blue Spruce Edison, NJ
l,3-Dichloro-4-nitrobenzene (p. 44, 91,
122)
RSA Ardsley, NY
l,4-Dichloro-2-nitrobenzene (2,5-
Dichloro-1-nitrobenzene) (p. 3, 7,
26, 48, 60, 64, 74, 83, 91, 122,
193, 354)
Dupont Deepwater, NJ
Mobay Bayonne, NJ
2,3-Dichloronitrobenzene (p. 122, 193)
2,4-Dichloro-l-nitrobenzene (see 1,3-
Dichloro-4-nitrobenzene)
2,5-Dichloro-3-nitrobenzoic acid
(p. 7, 91, 371)
GAF Linden, NJ
532
-------
Dichloronitrobenzoic acid, isomeric
mixture (p. 91)
GAP Linden, NJ
2,5-Dichloro-3-nitrobenzoic acid,
ammonium salt
GAF Linden, NJ
2,5-Dichloro-3-nitrobenzoic acid,
iminodi-2,2'-ethanol salt
GAF Linden, NJ
2,5-Dichloro-6-nitrobenzoic acid,
sodium salt
1,2-Dichloro-3-nitronaphthalene
(p. 429, 430, 432)
2,3-Dichloro-4-nitrophenol (p. 479, 480)
2,4-Dichloro-6-nitrophenol (4,6-
Dichloro-2-nitrophenol) (p. 91, 442)
2,5-Dichloro-4-nitrophenol (p. 440, 442)
2,5-Dichloro-4-nitrophenol, sodium salt
(p. 440)
2,6-Dichloro-4-nitrophenol (p. 158,
182, 358, 442)
4,5-Dichloro-2-nitrophenol (p. 442)
2',5'-Dichloro-3-nitrosalicylanilide
(p. 448)
2',5-Dichloro-4'-nitrosalicylanilide
(Niclosamide) (p. 91, 446)
Mobay Kansas City, MO
2',5-Dichloro-4'-nitrosalicylanilide,
2-aminoethanol salt"
Mobay Kansas City, MO
2,4-Dichlorophenyl-4-nitrophenyl ether
(2,4-Dichlorophenyl-j>-nitrophenyl
ether; Nitrofen) (p. 7, 42, 91,
347, 371)
Rohm and Haas Philadelphia, PA
N3,N3-Diethyl-2,4-dinitro-6-trifluoro-
methyl-m-phenylenediamine (Dinitramine)
(p. 120, 451)
0,0-Diethy 1-0- (p_-nitrophenyl phosphoro-
thioate) (Parathion, ethyl parathion)
(p. 7, 49, 53, 57, 62, 77, 80, 81,
119, 120, 121, 247, 302)
Monsanto Anniston, AL
Stauffer Mt. Pleasant, TN
1,5-Difluoro-2,4-dinitrobenzene
Pierce Rockford, IL
2,4-Difluoronitrobenzene
Olin Rochester, NY
2,5-Difluoronitrobenzene
Olin Rochester, NY
4,5-Difluoro-2-nitrophenol (p. 442)
4,6-Difluoro-2-nitrophenol (p. 442)
2,6-Diiodo-4-nitrophenol (p. 91, 358,
359)
l,4-Dimethoxy-2-nitrobenzene (p. 55)
2,5-Dimethoxy-4'-nitrostilbene
Upj ohn Kalamazoo, MI
0,0-Dimethyl-0-(3-methyl-4-nitrophenol)
phosphorothioate (p. 42)
N,N-Dimethyl-m-nitroaniline (p. 92)
N,N-Dimethyl-o-nitroaniline (p. 92)
0,0-Dimethyl-0-(p_-nitrophenyl)phosphoro-
thioate (Methyl parathion) (p. 7, 49,
53, 57, 58, 62, 76, 80, 81, 120, 121)
Hercules Plaquemine, LA
Kerr-McGee Hamilton, MS
Monsanto Anniston, AL
Stauffer Mt. Pleasant, TN
Vicksburg Vicksburg, MS
2',3'-Dimethyl-3-nitrosalicylanilide
(p. 448)
533
-------
2',4'-Dimethyl-3-nitrosalicylanilide
(p. 448)
2', 6'-Dimethyl-3-nitrosalicylanilide
(p. 448)
N ,N-Dimethyl-3-nitro-_p_- toluenesulfon-
amide
GAF Rensselaer, NY
2,4'-Dimethy1-3,3',5,5'-tetranitro-
ONN-azoxybenzene (p. 44)
2',4-Dimethyl-3,3',S.S'-tetranitro-ONN-
azoxybenzene (p. 44)
2,4-Dinitroacetanilide (p. 55)
2 ,4-Dinitro-6-s_e£-amylphenol (p. 41)
2,4-Dinitroaniline (p. 7, 26, 50, 55, 60,
64, 74, 76, 82, 83, 92, 113, 351)
American Color
and Chem. Lock Haven, PA
Marin Marietta Sodyeco, NC
£-(2,4-Dinitroanilino)phenol (p. 92)
£-(2,4-Dinitroanilino)phenol (p. 7, 49,
92)
GAF Rensselaer, NY
2,4-Dinitroanisole (p. 8, 92, 333,
334, 371, 415)
Am. Hoechst Somerville, NJ
Chemtronics Swannanoa, NC
4,6-Dinitroanthranil (p. 44)
3,3'-Dinitroazoxybenzene (p. 224)
3,5-Dinitrobenzamide
Salsbury Labs Charles City, LA
3',4-Dinitrobenzanilide (p. 8, 50, 92)
Toms River Toms River, NJ
1,2-Dinitrobenzene (o-Dinitrobenzene)
(p. 22, 122, 134, 136, 182, 191, 495,
496, 497)
1,3-Dinitrobenzene (m-Dinitrobenzene;
2,4-Dinitrobenzene; 2,6-Dinitrobenzene)
(p. 8, 22, 27, 30, 33, 55, 59, 62, 76,
81, 114, 122, 124, 134, 136, 137, 138,
182, 191, 223, 224, 225, 245, 293, 334,
339, 340, 354, 377, 437, 466, 477, 481,
494, 495, 496, 497)
DUpont Wilmington, DE
jD-Dinitrobenzene (1,4-Dinitrobenzene)
(p. 22, 134, 136, 137, 138, 182, 191,
354, 495, 496, 497)
2,4-Dinitrobenzenesulfonic acid (p. 92)
Eastman Kodak Rochester, NY
Toms River Toms River, NJ
2,4-Dinitrobenzenesulfonic acid, sodium
salt (p. 92)
Frank Columbus, OH
Hease State College, PA
3,5-Dinitrobenzenesulfonic acid (p. 128)
2,2'-Dinitrobenzidine (p. 92)
3,3'-Dinitrobenzidine (p. 92)
Northrop Asheville, NC
2,4-Dinitrobenzoic acid (p. 142, 151,
182, 275)
2,5-Dinitrobenzoic acid (p. 142, 151, I
182) ,
3,4-Dinitrobenzoic acid (p. 142, 151,
182)
3,5-Dinitrobenzoic acid (3,5-Dinitro-
benzoate) (p. 55, 60, 64, 84, 151,
182)
Ashland Great Meadows, NJ
Bofors Linden, NJ
Salsbury Labs Charles City, IA
3,5-Dinitrobenzoic acid, sodium salt
(3,5-Dinitro-Na-benzoate) (p. 143,
150)
534
-------
3,5-Dinitrobenzoyl chloride (p. 55, 92)
Bofors Linden, NJ
Eastman Kodak Rochester, NY
Guardian Hauppauge, NY
(Eastern)
2,4'-Dinitrobiphenyl
Northrop Asheville, NC
4,4'-Dinitrobiphenyl (p. 8, 428)
Northrop Asheville, NC
Dinitrobutylphenol, ammonium salt
(4,6-Dinitro-o-sec-butylphenol,
ammonium salt) (p. 8, 52, 74, 92)
Dow Chem. Midland, MI
4,6-Dinitro-o-sec-butylphenol, tri-
ethanolamine salt (see 2-sec-Butyl-
4,6-dinitrophenol, triethanolamine
salt)
2,4-Dinitro-o-cresol (p. 93, 128, 158)
2,6-Dinitro-£-cresol (p. 329, 363)
4,6-Dinitro-o-cresol (DNOC; Dinitro-
£-cresol; 2,4-Dinitro-6-methylphenol)
(p. 8, 41, 55, 93, 129, 156, 162, 164,
165, 182, 198, 199, 200, 201, 202,
203, 204, 205, 206, 207, 208, 209,
219, 220, 270, 272, 299, 300, 301,
304, 306, 308, 321, 323, 327, 328,
329, 331, 333, 334, 346, 349, 361,
362, 378, 385, 386, 406, 416, 443,
452, 454, 493, 496)
Blue Spruce Edison, NJ
4,6-Dinitro-£-cresol, sodium salt
(p. 93, 301, 302, 303, 493)
Blue Spruce Edison, NJ
4,6-Dinitro-o-cyclohexyl phenol
(2-Cyclohexy1-4,6-dinitrophenol)
(p. 41, 93, 212, 213, 327, 329,
349, 350, 363, 376, 385, 495)
2,4-Dinitrodiazobenzene (p. 93)
3,4-Dinitro-dimethylaniline (isomer
unknown) (p. 416)
4,4'-Dinitrodiphenylamine (p. 92, 93)
2,6-Dinitro-N,N-dipropyl cumidine (p. 371)
2,6-Dinitro-N ,N-dipropyl-p_-toluidine
Eli Lilly Lafayette, IN
2,5-Dinitrofluorene (p. 416, 428)
2,7-Dinitrofluorene (p. 411, 416, 428)
2,7-Dinitrofluoren-9-one
Mackenzie
Chem. Works Central Islip, NY
2,4-Dinitro-5-fluoroaniline
Pierce Rockford, IL
Dinitrofluorobenzene
3',5'-Dinitro-2'-hydroxyacetanilide
Toms River Toms River, NJ
2,4-Dinitro-6-hydroxylaminotoluene
(p. 177)
2,6-Dinitro-4-hydroxyalminotoluene
Cp. 177, 235)
l-(3,5-Dinitro-2-hydroxyphenylazo)-
2-naphthol
Toms River Toms River, NJ
2,6-Dinitro-4-isopropylphenol (p. 365)
1,5-Dinitronaphthalene (p. 27, 497)
2,4-Dinitro-a-napthol (p. 8, 207,
208, 209, 210, 334, 372)
Carroll Wood River, RI
2,4-Dinitro-6-octylphenyl crotonate
(Karathane; Dinocap; 2-Capry1-4,6-
dinitrophenyl crotonate; Dinitrocapryl
phenylcrotonate; 4,6-Dinitro-2-(l-
methyl heptyl)phenyl crotonate)
Cp. 8, 92, 93, 94, 210, 211, 304, 347
350, 363, 372, 378, 452, 493)
Rohm & Haas Bristol, PA
Philadelphia, PA
535
-------
2,4-Dinitrophenetole (p. 333, 334, 372)
Dinitro-o-propylphenol (p. 386)
2,3-Dinitrophenol (p. 329, 495)
2,4-Dinitrpphenol (DNP) (p. 8, 26, 49,
53, 55, 59, 63, 76, 82, 83, 155, 156,
159, 162, 163, 164, 183, 191, 207,
208, 209, 210, 223, 224, 241, 242,
245, 259, 262, 263, 264, 266, 267,
268, 269, 270, 271, 272, 273, 274,
275, 276, 298, 299, 306, 307, 308,
323, 324, 325, 326, 327, 329, 331,
333, 334, 335, 336, 337, 338, 346,
349, 350, 363, 364, 365, 375, 378,
379, 385, 386, 387, 399, 403, 404,
405, 406, 407, 416, 444, 454, 457,
458, 459, 460, 467, 468, 470, 471,
472, 473, 474, 475, 476, 477, 478,
479, 480, 481, 482, 483, 484, 489,
490, 491, 492, 494, 495, 497)
Martin Marietta Sodyeco, NC
2,5-Dinitrophenol (p. 155, 162, 163,
164, 183, 191, 259, 329, 471, 495)
2,6-Dinitrophenol (p. 55, 155, 162,
163, 164, 165, 183, 191, 329, 333,
334, 471, 495)
3,4-Dinitrophenol (p. 329, 495)
3,5-Dinitrophenol (p. 191, 329, 495)
2,6-Dinitro-l-phenol-4-sulfonic acid
(P- 93)
2,4-Dinitrophenoxyethanol
Hummel S. Plainfield, NJ
(2 ,4-Dinitrophenyl)hydrazine
Guardian Hauppauge, NY
(Eastern)
2,4-Dinitro-6-phenylphenol (p. 365)
Dinitrophenyllysine hydrochloride
(p. 426)
2,4-Dinitroresorcinol (p. 93, 157,
167, 183, 372)
3,5-Dinitrosalicylic acid
Eastman Kodak Rochester, NY
Salsbury Labs Charles City, IA
4,4'-Dinitrostilbene (p. 27)
4,4'-Dinitrostilbene-2,2'-disulfonic
acid (p. 9, 27, 52, 55, 59, 62, 82)
American Cyanamid Bound Brook, NJ
Ciba-Geigy Mclntosh, AL
GAP Rensselaer, NY
Toms River Toms River, NJ
2,4-Dinitrothymol (p. 334)
3,5-Dinitrotoluamide (Dinitolmide)
(p. 9, 297, 372, 379, 399, 496)
Dow Chem. Gainesville, GA
Midland, MI
2,3-Dinitrotoluene (p. 368, 496)
2,5-Dinitrotoluene (p. 19, 368, 496)
2,4-Dinitrotoluene (p. 9, 16, 19, 20,
29, 33, 36, 48, 55, 66, 68, 69, 76,
105, 113, 114, 128, 129, 169, 171,
172, 174, 183, 259, 316, 334, 340,
344, 368, 393, 394, 461, 462, 496)
Air Products
and Chemicals Pensacola, FL
Dupont Deepwater, NJ
Rubicon Geismar, LA
2,4-(and 2,6)-Dinitrotoluenes (p. 9, 16,
20, 48, 53, 58, 61, 66, 80, 87, 103,
104, 107, 108, 109, 128, 129, 174,
369, 379, 496)
Dupont Deepwater, NJ
Mobay Cedar Bayou, TX
New Martinsville, WV
2,6-Dinitrotoluene (p. 341, 369, 496)
536
-------
3,4-Dinitrotoluene (p. 129, 369, 496)
3,5-Dinitrotoluene (p. 19, 496)
Dinitrotoluene Oil (Dinitrotoluenes)
(p. 16, 20, 280, 496)
2,4-Dinitrotoluene-3-sulfonic acid
(p. 128)
2,4-Dinitrotoluene-5-sulfonic acid
(p. 128)
3,5-Dinitro-p_-toluenesulfonic acid
GAP Rensselaer, NY
3,5-Dinitro-p-toluidine (p. 372)
Dinitrotrichlorobenzene (p. 355)
2,4'-Dinitro-4-trifluoromethyldiphenyl
ether (p. 55, 93)
Ciba-Geigy Mclntosh, AL
2,4-Dinitro-l,3,5-trimethylbenzene
(2,4-Dinitromesitylene) (p. 334, 342)
4,6-Dinitro-l,3-xylene (p. 341)
Dinocap (see 2,4-Dinitro-6-octylphenyl
crotonate)
Dinoseb (see 2-sec-Butyl-4,6-dinitro-
phenol)
5,5-Dithiobis-(2-nitrobenzoic acid)
Pierce Rockford, IL
DNCB (see l-Chloro-2,4-dinitrobenzene)
DNFB (see l-Fluoro-2,4-dinitrobenzene)
DNOC (see 4,6-Dinitro-£-cresol)
DNP (see 2,4-Dinitrophenol)
4-Ethoxy-3-nitroacetanilide
American Color
and Chem. Lock Haven, PA
4-Ethyl-2,6-dinitrophenol (p. 365)
N,N'-Ethylenebis(3-nitrobenzenesulfona-
mide)
Salsbury Labs Charles City, IA
Ethyl-m-nitrobenzoate (p. 38)
Ethyl-p_-nitrobenzoate (p. 33, 38)
l-Fluoro-2,4-dinitrobenzene (DNFB;
2,4-Dinitrofluorobenzene) (p. 9, 219,
260, 355, 399, 424, 425, 426)
Eastman Kodak Rochester, NY
Olin Rochester, NY
Pierce Chem. Rockford, IL
2-Fluoro-3,5-dinitro-benzotrifluoride
(p. 89)
4-Fluoro-2,6-dinitrophenol (p. 444)
2-Fluoro-5-nitroaniline
Olin Rochester, NY
4-Fluoro-2-nitroaniline
Olin Rochester, NY
4-Fluoro-3-nitroaniline
Olin Rochester, NY
m-Fluoronitrobenzene (p. 122)
Olin Rochester, NY
£-Fluoronitrobenzene (p. 122)
~ Olin Rochester, NY
j>-Fluoronitrobenzene (p. 9, 122, 355)
Olin Rochester, NY
2-Fluoro-4-nitrobenzoic acid (2-Fluoro-
4-nitrobenzoate) (p. 151, 152, 182)
3-Fluoro-4-nitrobenzoic acid (3-Fluoro-
4-nitrobenzoate) (p. 142, 143, 182)
4-Fluoro-3-nitrobenzoic acid
Olin Rochester, NY
537
-------
2-Fluoro-4-nitrophenol (p. 442)
3-Fluoro-2-nitrophenol (p. 442)
3-Fluoro-4-nitrophenol (p. 442)
4-Fluoro-2-nitrophenol (p. 442)
5-Fluoro-2-nltrophenol (p. 442)
6-Fluoro-2-nitrophenol (p. 442)
2'-Fluoro-3-nitrosalicylanilide (p. 447)
3'-Fluoro-3-nitrosalicylanilide (p. 447)
4'-Fluoro-3-nitrosalicylanilide (p. 447)
4'-Fluoro-5-nitrosalicylanilide (p. 447)
2-Fluoro-4-nitrotoluene
Olin Rochester, NY
2-Fluoro-5-nitrotoluene
Olin Rochester, NY
4-Fluoro-2-nitrotoluene
Olin Rochester, NY
Fluoro-2,4,6-trinitrobenzene (2,4,6-
Trinitrofluorobenzene) (p. 30)
L-6-Glutamyl-p_-nitroanilide
Beckman . Carlsbad, CA
1,2,3,4,5,6-Hexachloro-7-nitro-
naphthalene (p. 416)
Hexanitroazobenzene
Northrop Asheville, NC
Hexanitrodiphenylamine (p. 93, 434,
435)
Hexanitrodiphenyl sulfone
Northrop Asheville, NC
Hexanitrostilbene (2,2*,4,4',6,6'-
Hexanitrostilbene) (p. 9., 416)
Northrop Asheville, NC
2-Hydrazino-4- (p_-nitrophenyl) thiazole
(p. 417)
4-Hydroxylamino-2-nitrophenol (p. 163)
6'-Hydroxy-5'-[(2-hydroxy-5-nitrophenyl)
azo]-m-acetotoluidide
Toms River Toms River, NJ
N-[7-Hydroxy-8-([2-hydroxy-5-nitrophenyl]
azo)-l-naphthy1]acetamide
Toms River Toms River, NJ
4-Hydroxy-7 (p_-nitrobenzamido)-2-
naphthalene sulfonic acid
GAF Rensselaer, NY
4-Hydroxy-3-nitrobenzenearsonic acid
Salsbury Labs Charles City, IA
4-Hydroxy-3-nitrobenzenearsonic acid,
monosodium salt
Salsbury Labs Charles City, IA
4-Hydroxy-3-nitrobenzenesulfonic acid
(p. 93)
2-Hydroxy-4-nitrobenzoic acid (p. 182)
3-Hydroxy-4-nitrobenzoic acid (p. 142,
182)
2-Hydroxy-5-nitrometanilic acid
Toms River Toms River, NJ
3-Hydroxy-3'-nitro-2-naphthanilide
(p. 93)
Pfister Ridgefield, NJ
l-(2-Hydroxy-4-nitrophenylazo)-2-
naphthol
Toms River Toms River, NJ
N-(2-Hydroxy-5-nitropheny1)glycerine
(p. 93)
2-Iodo-3-nitrobenzoic acid (p. 93)
2-Iodo-3-nitrophenol (p. 442)
538
-------
2'-Iodo-3-nitrosalicylanilide (p. 447)
3'-Iodo-3-nitrosalicylanilide (p. 447)
4'-Iodo-3-nitrosalicylanilide (p. 447)
4'-Ipdo-5-nitrosalicylanilide (p. 447)
2- (p_-Iodopheny 1) - 3- (p_-nitropheny 1) - 5-
phenyltetrazoliam chloride
Aldrich Milwaukee, WI
Isopropyl-£-nitrobenzoate (p. 33)
2-Iodo-4-nitrobenzoic acid (2-Iodo-
4-nitrobenzoate) (p. 142, 152, 182)
Karathane (see 2,4-Dinitro-6-octylphenyl
crotonate)
Lead 2,4-dinitroresorcinate (p. 93)
Lead nitroresorcinol, mono
Tyler Tamaqua, PA
2'-Methoxy-5'-chloro-3-nitrosalicyl-
anilide (p. 448)
6-Methoxy-8-nitroquinoline
Sterling Drug Rensselaer, NY
2-(a-Methylbenzyl)-4,6-dinitrophenol
(p. 365)
2'-Methyl-3'-chloro-3-nitrosalicyl-
anilide (p. 448)
2'-Methyl-5'-chloro-3-nitrosalicyl-
anilide (p. 448)
l-Methyl-3,4-dimethoxynitrobenzene
Orbis Products Newark, NJ
4,4'-Methylenebis(N,N-dimethyl-3-
nitroaniline)
GAP Rensselaer, NY
N-Methyl-4'-nitroacetanilide
GAF Linden, NJ
N-Methyl-£-nitroaniline
GAF Linden, NJ
2-Me thy1-5-ni troaniline
Pfister Ridgefield, NJ
3-Methyl-2-nitrobenzoic acid (2-Nitro-
m-toluic acid) (p. 56, 182)
Salsbury Labs Charles City, IA
3-Methyl-4-nitrobenzoic acid
Bofors Linden, NJ
Salsbury Labs Charles City, IA
3-Methyl-6-nitrobenzoic acid
Salsbury Labs Charles City, IA
2-Methyl-4-nitrophenol (p. 155, 183)
N-Methyl-N-nitro-2,4,6-trinitroaniline
(p. 14, 29, 96, 110, 113, 114, 115,
118, 130, 291, 292, 314, 315, 352,
383, 397, 398, 417, 496)
Hummel S. Plainfield, NJ
4-(MethyIsulfonyl)-2,6-dinitro-N,N-
dipropylaniline (p. 9, 352)
Shell Denver, CO
2-(MethyIsulfonyl)-4-nitroaniline
(p. 55)
Toms River Toms River, NJ
Musk ambrette (see 6-tert-Butyl-3-
methy1-2,4-dinitroanis ole)
Nigrosine (mixture containing nitrobenzene,
nitrophenol, or nitrocresols) (p. 297,
399)
3'-Nitroacetanilide (p. 50, 94)
GAF Rensselaer, NY
Toms River Toms River, NJ
4'-Nitroacetanilide (p. 50)
GAF . Rensselaer, NY
Toms River Toms River, NJ
3'-Nitroacetanilide (p. 10)
GAF Rensselaer, NY
539
-------
3'-Nitroacetophenone (m-Nitroaceto-
phenone) (p. 10, 2457 372)
Syntex Newport, TN
A'-Nitroacetophenone (p_-Nitroaceto-
phenone) (p. 253)
3-Nitro-4-aminoanisole (see 2-Nitro-
p_-anisidine)
m-Nitroaniline (3-Nitroaniline) (p. 10,
27, 50, 55, 76, 122, 179, 180, 181,
182, 185, 191, 197, 223, 224, 225,
280, 352, 401, 484)
o-Nitroaniline (2-Nitroaniline; 2-Nitro-
~ phenylamine) (p. 10, 55, 59, 63, 77,
80, 82, 95, 122, 179, 180, 181, 182,
185, 191, 353, 484, 495)
Monsanto Sauget, IL
_g-Nitroaniline (4-Nitroaniline) (p. 10,
26, 33, 35, 38, 50, 53, 55, 58, 62,
74, 77, 78, 80, 81, 98, 104, 106, 107,
136, 178, 179, 180, 181, 182, 185,
191, 197, 217, 218, 236, 238, 239,
280, 305, 311, 312, 353, 379, 401,
484, 495, 496)
American Color
and Chem. Lock Haven, PA
Monsanto Sauget, IL
Universal Oil
Products McCook, IL
4-Nitroaniline-3-sulfonic acid (5-
Amino-2-nitrobenzenesulfonic acid;
6-Nitrometanilic acid) (p. 94)
2-Nitro-_p_- anisidine (4-Amino-3-ni tro-
anisole; 3-Nitro-4-aminosole) (p. 19,
20, 55)
Dupont Deepwater, NJ
4-Nitro-o-anisidine (2-Amino-5-nitro-
anisole; 4-Nitro-2-anisidine) (p. 10,
19, 49, 55, 74, 94)
Dupont Deepwater, NJ
4-Nitro-3-anisidine (p. 94)
5-Nitro-^-anisidine (2-Amino~4-
nitroanisole) (p. 10, 19, 49, 53,
55, 94, 372)
American Cyanamid Marietta, OH
Synalloy
(Blackman Uhler) Spartanburg, SC
o-Nitroanisole (2-Nitroanisole)
(p. 11, 20, 26, 55, 59, 63, 77, 83,
372)
Dupont Deepwater, NJ
Monsanto St. Louis, MO
£-Nitroanisole (4-Nitroanisole)
(p. 11, 20, 33, 38, 39, 59, 63, 77,
83, 372, 379)
Dupont Deepwater, NJ
nr-Nitrobenzaldehyde (p. 28, 55, 245, 401)
Aldrich Milwaukee, WI
o-Nitrobenzaldehyde (p. 28, 31, 94)
Aldrich Milwaukee, WI
RSA Ardsley, NY
£-Nitrobenzaldehyde (p. 11, 234, 245,
343, 373, 401)
Sterling Drug Rensselaer, NY
£-Nitrobenzamide (p. 373)
4'-Nitrobenzanilide
GAF Rensselaer, NY
Nitrobenzene (Mononitrobenzene)
(p. 3, 11, 20, 21, 22, 23, 24, 27, 33,
34, 35, 37, 38, 40, 48, 53, 58, 61,
65, 66, 67, 74, 77, 78, 79, 80, 85,
86, 87, 104, 105, 106, 107, 123, 124,
. 125, 128, 129, 134, 136, 138, 182,
185, 188, 191, 192, 193, 214, 215,
216, 218, 221, 222, 223, 245, 246,
253, 254, 255, 278, 280, 282, 294,
309, 310, 311, 318, 319, 320, 339,
341, 343, 344, 345, 355, 356, 380,
390, 391, 392, 393, 399, 401, 411,
435, 436, 454, 495, 496)
Allied Chem. Moundsville, WV
American Cyanamid Bound Brook, NJ
Willow Island, W
540
-------
Nitrobenzene (Cont'd)
Dupont Beaumont, TX
Gibbstown, NJ
First
Mississippi Pascagoula, MS.
Mobay New Martinsville, WV
Monsanto Sauget, IL
Rubicon
Chem. Geismar, LA
£-Nitrobenzenesulfonamide (p. 249, 250,
373)
3'-Nitrobenzenesulfonanilide
GAF Rensselaer, NY
m-Nitrobenzenesulfonic acid (p. 51, 59,
63, 74, 82)
Toms River Toms River, NJ
m-Nitrobenzenesulfonic acid, potassium
salt (p. 63)
American Cyanamid Bound Brook, NJ
m-Nitrobenzenesulfonic acid, sodium
salt (p. 51, 55, 59, 63, 74, 82)
GAF Linden, NJ
USM
(Crown Metro) Greenville, SC
m-Nitrobenzenesulfonyl chloride (p. 52)
GAF Rensselaer, NY
ch-Nitrobenzenesulfonyl chloride (p. 94)
£-Nitrobenzenesulfonyl chloride
Eastman Kodak Rochester, NY
2-Nitrobenzidine (p. 94)
3-Nitrobenzidine (p. 94)
6-Nitrobenzimidazole
Fairmount Newark, NJ
6-Nitrobenzimidazole nitrate
Fairmount Newark, NJ
6-Nitrobenzimidazole, sodium salt
Fairmount Newark, NJ
m-Nitrobenzoic acid (m-Nitrobenzoate)
(p. 11, 28, 38, 51, 55, 60, 63, 83,
141, 142, 143, 144, 145, 146, 147,
148, 150, 182, 191, 196, 245, 373)
Bofors Linden, NJ
Salsbury Labs Charles City, IA
Sterling Drug Cincinnati, OH
£-Nitrobenzoic acid (o-Nitrobenzoate)
(p. 11, 31, 55, 141, 142, 143, 144,
145, 146, 147, 148, 149, 150, 151,
182, 191, 196, 231, 373, 472, 473,
474, 475)
Bofors Linden, NJ
Salsbury Labs Charles City, IA
£-Nitrobenzoic acid (PNBA; £-Nitrobenzoate;
4-Nitrobenzoate) (p. 11, 20, 27, 33, 38,
55, 77, 95, 141, 142, 143, 144, 145, 146,
147, 148, 149, 150, 151, 152, 158, 182,
191, 231, 234, 245, 246, 249, 250, 251,
275, 373, 472)
Bofors Linden, NJ
Dupont Deepwater, NJ
m- and £-Nitrobenzoic acids (p. 11, 17, 51)
£-Nitrobenzoic acid, ethyl ester
Bofors Linden, NJ
m-Nitrobenzoic acid, sodium salt
(Sodium-m-nitrobenzoate) (p. 11, 51,
60, 63, 83, 143, 145)
Salsbury Labs Charles City, IA
£-Nitrobenzoic acid, sodium salt
Co-Nitro-Na-benzoate) (p. 145)
£-Nitrobenzoic acid, sodium salt
(£-Nitro-Na-benzoate) (p. 143, 145)
m-Nitrobenzonitrile (p. 38)
£-Nitrobenzonitrile (p. 33, 37, 38, 39)
m-Nitrobenzptrifluoride
Olin Rochester, NY
6-Nitro-2-benzoxazolinone
GAF Rensselaer, NY
541
-------
2-(m-Nitrobenzoyl)-£-acetanisidide
GAF Linden, NJ
ro-Nitrobenzoyl chloride (p. 55)
Aceto Carlstadt, NJ
Bofors Linden, NJ
o-Nitrobenzoyl chloride
Bofors Linden, NJ
£-Nitrobenzoyl chloride (p. 55)
Occidental
Petroleum Niagara Falls, NY
(Hooker)
£-Nitrobenzyl alcohol (p. 231)
£-Nitrobenzyl alcohol (p. 234, 245)
Eastman Kodak Rochester, NY
£-Nitrobenzyl bromide (p. 95)
RSA Ardsley, NY
Stauffer Edison, NJ
4- (_p_-Nitrob enzy 1) pyridine
Eastman Kodak Rochester, NY
c>-Nitrobiphenyl (2-Nitrobiphenyl)
(p. 12, 373, 381)
jD-Nitrobiphenyl (4-Nitrobiphenyl)
(p. 246, 373, 382, 411, 417, 427,
429, 496)
4'-Nitro-4-biphenylcarboxylic acid
Toms River Toms River, NJ
4-Nitrocatechol (p. 161, 221, 222)
3-Nitro-4-chloroacetanilide
Frank Enterprises Columbus, OH
2-Nitro-4-chloroaniline (see 4-Chloro-
2-nitroaniline)
3-Nitro-4-chloroaniline (see 4-Chloro-
3-nitroaniline)
2-Nitro-5-chloroanisole (see 5-Chloro-
2-nitroanisole)
£-Nitrocresol (p. 246)
2-Nitro-£-cresol (p. 12, 77, 129, 366)
Sherwin-Williams Chicago, IL
4-Nitro-m-cresol (p. 56, 366)
6-Nitro-m-cresol
4-Nitro-6-cyclohexylphenol (p. 387)
2-Nitro-£-cymene
Eastman Kodak Rochester, NY
5-Nitro-l-diazo-2-naphthol-4-sulfonic acid
(p. 55)
6-Nitro-l-diazo-2-naphthol-4-sulfonic acid
(p. 95)
4-Nitro-N,N-diethylaniline (p. 35)
2-Nitrodiphenylamine
American Cyanamid Marietta, OH
4-Nitrodiphenylamine (p. 56)
Monsanto Sauget, IL
2-Nitrodiphenylamine-4-sulfonanilide
Salsbury Labs Charles City, IA
4-Nitrodiphenylamine-2-sulfonic acid
(p. 95)
Nitrododecylbenzene
Monsanto Sauget, IL
4-Nitro-N-ethylaniline (p. 35)
2-Nitrofluorene (p. 246, 411, 417, 428,
430)
6-Nitro-2-furaldehyde semicarbazone
Cp. 417)
5-Nitrofuran (p. 411)
N-[4-(5-Nitro-2-furyl)-2-thiazolyl]-
acetamide (p. 418)
542
-------
p_-Nitrohippuric acid (p. 231)
Nitrohydroquinone (p. 161)
5-Nitroimidazole (p. All)
5-Nitroisophthalic acid
Ashland Great Meadows, NJ
Bofors Linden, NJ
GAF Linden, NJ
1-Nitronaphthalene (a-Nitronaphthalene)
(p. 12. 27, 59, 63, 82, 246, 373)
Dupont Wilmington, DE
2-Nitronaphthalene (g-Nitronaphthalene)
(p. 246, 256, 374, 411, 418, 429)
3-Nitro-l,5-naphthalenedisulfonic acid
(Nitro casella acid) (p. 12, 52)
GAF Linden, NJ
Toms River Toms River, NJ
l-Nitro-3,6,8-naphthalene trisulfonic
acid (p. 27)
4-Nitronaphthalic anhydride
GAF Linden, NJ
7-(and 8-)Nitronapth(l,2-d)(l,2,3)oxa-
diazole-5-sulfonic acid (p. 12, 52,
60, 64, 84)
GAF Rensselaer, NY
Mobay Bayonne, NJ
Charleston, SC
Toms River Toms River, NJ
3-Nitro-2-naphthylamine (p. 429, 430,
431)
£-Nitroperbenzoic acid (p. 418, 433)
3-Nitro-p-phenetidine (3-Nitro-p_-
ethoxyaniline) (p. 56)
4-Nitro-o-phenetidine (4-Nitro-£-
ethoxyaniline) (p. 56)
5-Nitro-p_-phenetidine (5-Nitro-o-
ethoxyaniline) (p. 56)
5-Nitro-p_-phenetidine (5-Nitro-p_-
ethoxyaniline) (p. 56)
m-Nitrophenol (3-Nitrophenol) (p. 56,
73, 95, 154, 155, 156, 158, 159, 160,
161, 183, 191, 221, 222, 223, 240,
245, 259, 275, 277, 366, 386, 387,
401, 444, 471, 497)
o-Nitrophenol (2-Nitrophenol) (p. 12,
35, 56, 58, 62, 73, 77, 81, 104,
129, 154, 155, 156, 158, 159, 160,
161, 183, 185, 189, 191, 193, 221,
222, 240, 258, 259, 275, 276, 366,
386, 387, 418, 437, 438, 439, 444,
457, 458, 459, 497)
Martin Marietta Sodyeco, NC
Monsanto Sauget, IL
£-Nitrophenol (4-Nitrophenol)
(p. 12, 20, 26, 33, 35, 38, 49, 53,
56, 57, 58, 61, 73, 74, 78, 79, 80,
101, 112, 119, 120, 125, 129, 130,
154, 155, 156, 158, 159, 160, 161,
183, 191, 192, 193, 207, 208, 209,
210, 215, 216, 221, 222, 223, 240,
245, 274, 275, 277, 313, 331, 366,
367, 382, 386, 387, 401, 418, 443,
444, 457, 458, 459, 471, 480, 482,
494, 497)
Dupont Deepwater, NJ
Martin Marietta Sodyeco, NC
Monsanto Anniston, AL
Sauget, IL
Northern Fine Franklin, NJ
G.D. Searle Norwood, OH
(Will Ross)
jD-Nitrophenol, sodium salt (sodium
£-Nitrophenate) (p. 20, 49, 53, 58,
61, 80)
Dupont Deepwater, NJ
Northern Fine Franklin, NJ
6-Nitro-l-phenol-2,4-disulfonic acid
(p. 95)
543
-------
£-Nitrophenyl acetate
Eastman Kodak Rochester, NY
(£-Nitrophenyl)acetic acid (£-Nitro-a-
toluic acid)
Stauffer Chem. Edison, NJ
4'-(p_-Nitrophenyl) acetophenone
(P. 12, 52, 95)
GAF Linden, NJ
2-Nitrophenylamine (see o-Nitroaniline)
4-Nitrophenylarsonic acid (p. 95)
Salsbury Charles City, IA
2- (c>-Nitrophenylazo)-j»-cresol
Toms River Toms River, NJ
2-Nitro-p_-phenylenediamine (2-NPPD;
m-Nitro-£-phenylenediamine) (p. 12,
18, 56, 95, 401, 402, 403, 433)
Ashland Great Meadows, NJ
Martin Marietta Sodyeco, NC
Olin Rochester, NY
4-Nitro-j3-phenylenediamine (4-NOPD;
Nitro-j3-phenylenediamine) (p. 13,
18, 56, 94, 95, 401, 402, 433)
Ashland Great Meadows, NJ
Fairmount Chem. Newark, NJ
4-Nitro-m-phenylenediamine (p. 56, 95)
5-Nitro-m-phenylenediamine (p. 95)
j>-Nitrophenyl ethyl ether (p. 26)
N-(p_-Nitrophenyl) glycine (p. 95)
(j[v-Nitrophenyl)hydrazine
Eastman Kodak Rochester, NY
m-Nitrophenylhydroxylamine (p. 95)
£-Nitrophenyl isocyanate
Eastman Kodak Rochester, NY
_p-Nitrophenylmercapturic acid (p. 221)
2-(4-Nitrophenyl)-(2H)-naphtho(l,2-d)-
triazole-6,8-disulfonic acid
Toms River Toms River, NJ
2-(p_-Nitrophenyl)-l-octadecyl-5-
benzimidazolesulfonic acid
GAF Linden, NJ
l-(m-Nitrophenyl)-5-oxo-2-pyrazoline-3-
carboxylic acid (p. 56)
Mobay Bayonne, NJ
jg-Nitrophenylphosphate, disodium salt
(p. 56)
Aldrich Milwaukee, WI
jj-Nitrophenyl phosphate, sodium salt
Regis Morton Grove, IL
3-(2-Nitrophenyl)propenoic acid
(p. 28, 31)
^-Nitrophenylsulfenyl chloride
Pierce Rockford, IL
Bis(4-Nitrophenyl)sulfide
American Cyanamid Willow Island, WV
£-Nitrophenyl-a,a,a-trifluoro-2-nitro-
2~tolyl ether
Nor-Am Chicago, IL
3-Nitrophthalic acid
Eastman Kodak Rochester, NY
3-Nitrophthalic anhydride
Eastman Kodak Rochester, NY
4-Nitrophthalimide
Martin Marietta Sodyeco, NC
j>-Nitrophenylphosphate
Pierce Rockford, IL
4-Nitropyridine-N-oxide
Aldrich Milwaukee, WI
4-Nitropyrogallol (p. 96)
Nitroquinol (p. 221)
544
-------
4-Nitroquinoline-N-oxide (p. 411, 414,
419)
5-Nitrosalleylaldehyde
Eastman Kodak Rochester, NY
Nitrosalicylanilides (p. 406, 446)
3-Nitrosalicylanilide (p. 446)
3-(and 5-)Nitrosalicylic (p. 195,
196)
GAF Rensselaer, NY
jD-Nitrosodium phenolate (see j>-Nitro-
phenol, sodium salt)
4-Nitroso-2-nitrophenol (p. 163)
4-Nitrostilbene (p. 13, 411, 418)
GAF Rensselaer, NY
4-Nitro-4'-(5-sulfo-2H-naphthol(l,2d)-
triazol-2-yl)-2,2'-stilbenedisulfonic
acid
Toms River Toms River, NJ
4-Nitro-2-sulfotoluene (see 5-Nitro-
o-toluenesulfonic acid)
m-Nitrotoluene (3-Nitrotoluene)
~ (p. 13, 16, 33, 77, 96, 129, 185, 191,
193, 277, 342, 369, 393, 394, 399,
454, 496)
First Mississippi Pascagoula, MS
^-Nitrotoluene (2-Nitrotoluene) (p. 13,
~ 16, 20, 24, 27, 31, 33, 36, 59, 62,
66, 69, 77, 81, 125, 129, 185, 191,
193, 231, 277, 280, 341, 370, 393,
394, 399, 454, 461, 462, 496)
Dupont Deepwater, NJ
First Mississippi Pascagoula, MS
£-Nitrotoluene (4-Nitrotoluene) (p. 13,
16, 20, 27, 33, 38, 48, 56, 58, 62,
66, 69, 77, 81, 118, 125, 129, 169,
171, 183, 185, 189, 191, 231, 234,
245, 253, 277, 280, 339, 342, 343,
370, 393, 394, 399, 401, 454, 496)
p-Nitrotoluene (Cont'd)
Dupont Deepwater, NJ
First Mississippi Pascagoula, MS
Nitrotoluenes, mixed (p. 280, 496)
Dupont Deepwater, NJ
5-Nitro-£-toluenesulfonanilide
GAF Linden, NJ
3-Nitro-p_-toluenesulfonic acid (p. 13,
51, 96)
Nyanza Ashland, MA
Toms River Toms River, NJ
4-Nitrotoluene-2-sulfonic acid (4-
Nitro-o-toluenesulfonic acid) (p. 13)
Dupont Deepwater, NJ
5-Nitro-o-toluenesulfonic acid (4-
Nitro-2-sulfotoluene) (p. 52, 59,
62, 81, 82, 96)
American Cyanamid Bound Brook, NJ
Dupont Deepwater, NJ
GAF Rensselaer, NY
Toms River Toms River, NJ
4'-Nitro-p_-toluenesulfono-o-toluidide
GAF Rensselaer, NY
5-Nitro-o-toluenesulfonyl chloride
GAF Linden, NJ
3-Nitro-o-toluic acid (2-Methyl-3-nitro-
benzoic acid) (p. 56)
3-Nitro-p_-toluic acid (4-Methyl-3-
nitrpbenzoic acid) (p. 56)
2-N;i.tro-p_-toluidine (4-Amino-3-nitro-
toluene) (p. 27, 50, 53, 56, 60, 63,
74, 77, 83)
Sherwin-Williams Chicago, IL
3-Nitro-p_-toluidine (4-Amino-2-nitro-
toluene) (p. 13, 28, 374, 461, 462)
4-Nitro-£-toluidine (2-Amino-5-nitro-
toluene) (p. 13, 50, 56, 74)
GAF Rensselaer, NY
545
-------
5-Nitro-o-toluidine (2-Amino-4-nitro-
toluene) (p. 14, 50, 53, 56, 368, 374)
Dupont Deepwater, NJ
Pfister Chem. Ridgefield, NJ
Synalloy Spartanburg, SC
(Blackman Uhler)
5-Nitro-2-£-toluidinobenzenesulfonic
acid
Toms River Toms River, NJ
2-Nitro-4-trifluoromethylchlorobenzene
Pfister Chem. Ridgefield, NJ
7-Nitro-2-trifluoromethylphenothiazine
Olin Rochester, NY
3-Nitro-a,a,a-trifluorotoluene (a,a,a-
Trifluoro-m-nitrotoluene) (p. 370)
Olin Rochester, NY
£-Nitro-^o-xylene (p. 56)
Nitroxylenes (p. 14, 48)
4-Nitro-2,6-xylenol (p. 374)
4-NOPD (see 4-Nitro-o-phenylenediamine)
2-NPPD (see 2-Nitro-p_-phenylenediamine)
Parathion (see 0,0-Diethy1-0-[£-nitro-
phenyl]-phosphorothioate)
Pentachloronitrobenzene (PCNB) (p. 3,
14, 42, 56, 96, .119, 120, 186, 187,
190, 192, 193, 230, 231, 232, 233,
297, 345, 348, 357, 382, 399, 401,
410, 419, 420, 421, 422, 423, 469,
476, 493)
Olin Mclntosh, AL
1,1,3,3,5-Pentamethy1-4,6-dinitroindan
(p. 96)
Givaudan Clifton, NJ
Picramic acid (see 2-Amino-4,6-dinitro-
phenol)
Picramic acid, sodium salt (see 2-Amino-
4,6-dinitrophenol, sodium salt)
Picramide (p. 434, 435)
Picric acid (see 2,4,6-Trinitrophendl)
Picric acid, ammonium salt (see 2,4,6-
Trinitrophenol, ammonium salt)
Picric acid, sodium salt (see 2,4,6-
Trinitrophenol, sodium salt)
Picrolonic acid (3-Methyl-4-nitro-l-
(p_-nitrophenyl)-5-pyrazolone)
RSA Aresley, NY
Picryl chloride (see l-Chloro-2,4,6-
trinitrobenzene)
PNBA (see £-Nitrobenzoic acid)
Sodium-m-nitrobenzenesulfonate
GAF Linden, NJ
Monsanto St. Louis, MO
Salsbury Labs Charles City, IA
Sodium picramate (see 2-Amino-4,6-
dinitrophenol, sodium salt)
Tetrachloronitroanisole (p. 119)
Tetrachloronitrobenzene (1,2,4,5-Tetra-
chloro-3-nitrobenzene) (p. 3, 14, 96,
119, 120)
Aceto Flushing, NY
2,3,4,5-Tetrachloronitrobenzene
(p. 193, 228, 229, 230, 247, 419,
420, 421)
2,3,4,6-Tetrachloronitrobenzene
(p. 193, 419, 420, 421)
2,3,5,6-Tetrachloronitrobenzene
(p. 193, 228, 229, 247, 419, 420,
421, 422, 493)
Sterling Cincinnati, OH
546
-------
2,3,5,6-Tetrachloro-4-nitrophenol
(p. 442)
2,3,4,6-Tetranitroaniline (p. 14, 345,
353)
Hummel Chem. S. Plainfield, NJ
2,2',6,6'-Tetranitro-4,4'-azoxytoluerie
(p. 44, 177, 235)
4,4',6,6'-Tetranitro-2,2'-azoxytoluene
(p. 44, 177)
1,2,4,6-Tetranitrobenzene (p. 30)
Tetranitrofluoren-9-one
Mackenzie Central Islip, NY
Tetranitroxylene (p. 374)
Tetryl (see N-Methyl-N-nitro-2,4,5-tri-
nitroaniline)
2,2'-Thiobis(5-nitrobenzenesulfonic
acid)
GAF Rensselaer, NY
D-Threo-l-(p_-nitrophenyl)-2-amino-l,3-
propanediol
Warner-Lambert Holland, MI
Triaminotrinitrobenzene
Northrop Asheville, NC
2,3,5-Tri-0-benzyl-l-0-£-nitrobenzoyl-
D-arabinofuranose
Pfanstiehl Waukegan, IL
l,2,4-Trichloro-5-nitrobenzene
(p. 15, 357)
Alliance Newark, NJ
Pfister Ridgefield, NJ
2,3,4-Trichloronitrobenzene (p. 193)
2,4,5-Trichloronitrobenzene (p. 122,
193)
2,3,6-Trichloro-4-nitrophenol (p. 442)
3,4,6-Trichloro-2-nitrophenol (p. 440,
442, 479, 480)
3,4,6-Trichloro-2-nitrophenol, sodium
salt (p. 440)
a,ot,a-Trif luoro-2,6-dinitro-N,N-dipropyl-
£-toluidine (Trifluralin) (p. 15, 34,
42, 58, 62, 81, 247, 248, 348, 372,
449, 450, 452, 493)
Eli Lilly Lafayette, IN
2-Trifluoromethyl-4-nitrophenol (p. 442,
443)
3-Trifluoromethyl-2-nitrophenol (p. 442)
3-Trifluoromethyl-4-nitrophenol (p. 96,
120, 367, 440, 441, 442, 443, 445,
446, 451)
3-Trifluoromethyl-4-nitrophenql, sodium
salt (p. 440)
4-Trifluoromethyl-2-nitrophenol (p. 442)
Trifluralin (see a,a,a-Trifluoro-2,6-
dinitro-N,N-dipropyl-p_-toluidine)
2,4,6-Trinitroaniline (p. 96)
2,4,6-Trinitroanisole (p. 30, 374, 396)
2,4,6-Trinitrobenzaldehyde (p. 44)
1,2,3-Trinitrobenzene (p. 22, 495)
1,3,5-Trinitrobenzene (2,4,5-Trinitro-
benzene) (p. 22, 29, 44, 45, 96, 115,
124, 134, 136, 137, 138, 182, 339,
340, 383, 495)
2,4,6-Trinitrobenzenesulfonic acid (p. 15)
Pierce Rockford, IL
2,4,6-Trinitrobenzoic acid (p. 96, 129,
142, 151, 182, 275, 495)
2,4,6-Trinitrobenzoic acid, sodium salt
(p. 143, 150)
547
-------
2,4,6-Trinitrobenzonitrile (p. 44)
2,4,6-Trinitrobenzyl alcohol (p. 235)
2,4,6-Trinitro-m-cresol (p. 156, 165,
182, 329, 367)
2,4,7-Trinitrofluoren-9-one
Eastman Kodak Rochester, NY
MacKenzie Chem. Central Islip, NY
2,4,6-Trinitrophenetole (p. 398)
2,4,6-Trinitrophenol (Picric acid)
(p. 14, 29, 77, 82, 97, 113, 155, 156,
162, 163, 164, 183, 274, 275, 298,
367, 382, 396, 399, 434, 435, 457,
458, 469, 470, 471, 477, 481, 486,
488, 491, 495, 496)
Martin Marietta Sodyeco, NC
2,4,6-Trinitrophenol, ammonium salt
(Ammonium picrate; Picric acid,
ammonium salt) (p. 89, 169, 396)
2,4,6-Trinitrophenol, sodium salt
(Sodium picrate; Picric acid, sodium
salt)
Hummel S. Plainfield, NJ
Northrop Asheville, NC
Trinitrophenolate (Lead picrate) (p. 94)
2,4,6-Trinitrophenyl ether (p. 30)
2,4,6-Trinitroresorcinol (Styphnic acid)
(p. 15, 110, 157, 165, 167, 183, 495)
Northrop Asheville, NC
Olin East Alton, IL
Trinitroresorcinol, lead salt (Styphnic
acid, lead salt) (p. 94, 97)
Remington Arms Bridgeport, CT
2,4,6-Trinitrotoluene (TNT; a-TNT)
(p. 15, 16, 19, 28, 29, 33, 43, 45,
56, 58, 61, 70, 72, 73, 80, 81, 102,
103, 106, 108, 109, 110, 111, 113,
114, 115, 116, 117, 118, 126, 129,
130, 168, 169, 170, 171, 172, 173)
2,4,6-Trinitrotoluene (Cont'd)
(p. 174, 175, 176, 177, 178, 183,
185, 186, 189, 235, 286, 287,
288, 289, 290, 291, 292, 314, 315,
316, 317, 340, 344, 345, 370, 383,
384, 393, 394, 396, 453, 454, 455,
456, 461, 462, 464, 465, 466, 484,
485, 486, 487, 495, 496, 497)
Trinitrotoluene (2,3,5-; 2,4,5-; 2,3,4-)
(p. 19, 28, 70, 72, 495, 496)
2,4,6-Trinitro-l,3-xylene (p. 342)
548
-------
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TECHNICAL REPORT DATA
(Please read Inutuctions on the reverse before completing)
i. REPORT NO.
EPA-560/2-76-010
2.
3. RECIPIENT'S ACCESSIOI»NO.
4. TITLE AND SUBTITLE
Investigation of Selected Potential Environmental
Contaminants: Nitroaromatics
5. REPORT DATE
June 1976
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Philip H. Howard, Joseph Santodonato, Jitendra Saxena,
Judith E. Mailing, Dorothy Greninger
8. PERFORMING ORGANIZATION REPORT NO
TP 76-573
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Center for Chemical Hazard Assessment
Syracuse Research Corporation
Merrill Lane
Syracuse, New York 13210
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
EPA 68-01-2999
12. SPONSORING AGENCY NAME AND ADDRESS
Office of Toxic Substances
U.S. Environmental Protection Agency
Washington, D.C. 20460
13. TYPE OF REPORT AND PERIOD COVERED
Final Technical Report
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This report considers the large number of chemicals which contain at least
one nitro substituent .on an aromatic ring. Approximately 250-300 chemicals are
listed as commercial nitroaromatic compounds. However, only about 40 compounds
are produced or consumed annually in quantities over 500,000 pounds and perhaps
another 50-100 compounds exceed 100,000 pounds. Nitroaromatic compounds are used
as pesticides, perfumes, explosives, and chemical intermediates. This report
focuses upon the non-pesticidal nitroaromatics. Because of the large number of
compounds considered in this report, comprehensive information on individual com-
pounds could not be developed. However, adequate information is available to
provide priorities for further study and research. Production volume, uses, en-
vironmental fate, monitoring, and biological effects were considered. In general,
nitroaromatic compounds appear to be fairly persistent and exhibit either hemato-
logic or metabolic effects at high levels of exposure. Most of the large-volume
nitroaromatics have not been screened for carcinogenic, mutagenic, or teratogenic
effects.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
COSATI l-'iclcl/Group
18. DISTRIBUTION STATEMENT
Document is available to the public through
the National Technical Information Service,
Springfield, Virginia 22151
19. SECURITY CLASS (Tills Report}
21. NO. OF PAf.bS
624
20. SECURITY CLASS (Thispage)
22. PRICE
EPA Form 2220-1 (9-73)
601
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