vvERA,
Jr-.tM States
Environmental
Agencv
Regulations ana 5'anaarcs
Catena ano Standards Divi
Wasmngton 3C 20460
"980
Ambient
Water Quality
Criteria for
Nitrophenols
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AMBIENT WATER QUALITY CRITERIA FOR
NITROPHENOLS
Prepared 8y
U.S. ENVIRONMENTAL PROTECTION AGENCY
Office of Water Regulations and Standards
Criteria and Standards Division
Washington, D.C.
Office of Research and Development
Environmental Criteria and Assessment Office
Cincinnati, Ohio
Carcinogen Assessment Group
Washington, O.C.
Environmental Research Laboratories
Corvalis, Oregon
Duluth, Minnesota
Gulf Breeze, Florida
Narragansett, Rhode Is'and
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DISCLAIMER
This report has been reviewed by the Environmental Criteria and
Assessment Office, U.S. Environmental Protection Agency, and approved
for publication. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
AVAILABILITY NOTICE
This document is available to the public through the National
Technical Information Service, (NTIS), Springfield, Virginia 22161.
11
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FOREWORD
Section 304 (a)(l) of the Clean Water Act of 1977 (P.L. 95-217),
requires the Administrator of the Environmental Protection Agency to
publish criteria for water quality accurately reflecting the latest
scientific knowledge on the kind and extent of all identifiable effects
on health and welfare which may be expected from the presence of
pollutants in any body of water, including ground water. Proposed water
quality criteria for the 65 toxic pollutants listed under section 307
(a)(l) of the Clean Water Act were developed and a notice of their
availability was published for public comment on March 15, 1979 (44 FR
15926), July 25, 1979 (44 FR 43660), and October 1, 1979 (44 FR 56628).
This document is a revision of those proposed criteria based upon a
consideration of comments received from other Federal Agencies, State
agencies, special interest groups, and individual scientists. The
criteria contained in this document replace any previously published EPA
criteria for the 65 pollutants. This criterion document is also
published in satisifaction of paragraph 11 of the Settlement Agreement
in Natural Resources Defense Counci 1, et. al. ys. Train, 8 ERC 2120
(D.O.C. 1976), modified, 12 ERC 1833 (D.O.C. 1979).
The term "water quality criteria" is used in two sections of the
Clean Water Act, section 304 (a)(l) and section 303 (c)(2). The term has
a different program impact in each section. In section 304, the term
represents a non-regulatory, scientific assessment of ecological ef-
fects. The criteria presented in this publication are such scientific
assessments. Such water quality criteria associated with specific
stream uses when adopted as State water quality standards under section
303 become enforceable maximum acceptable levels of a pollutant in
ambient waters. The water quality criteria adopted in the State water
quality standards could have the same numerical limits as the criteria
developed under section 304. However, in many situations States may want
to adjust water quality criteria developed under section 304 to reflect
local environmental conditions and human exposure patterns before
incorporation into water quality standards. It is not until their
adoption as part of the State water quality standards that the criteria
become regulatory.
Guidelines to assist the States in the modification of criteria
presented in this document, in the development of water quality
standards, and in other water-related programs of this Agency, are being
developed by EPA.
STEVEN SCHATZOW
Deputy Assistant Administrator
Office of Water Regulations and Standards
111
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ACKNOWLEDGEMENTS
Aquatic Life Toxicology:
William A. Brungs, ERL-Narragansett
U.S. Environmental Protection Agency
John H. Gentile, ERL-Narragansett
U.S. Environmental Protection Agency
Mammalian Toxicology and Human Health Effects:
Van Kozak (author)
University of Wisconsin
Steven 0. Lutkenhoff (doc. mgr.)
ECAO-Cin
U.S. Environmental Protection Agency
Donna Sivulka (doc. mgr.) ECAO-Cin
U.S. Environmental Protection Agency
Kirby I. Campbell, HERL
U.S. Environmental Protection Agency
Ted Ericksen, HERL
U.S. Environmental Protection Agency
Sherwin Kevy
Children's Hospital Medical Center
V.M.S. Ramanujam
University of Texas Medical Branch
Alan B. Rubin, CSD
U.S. Environmental Protection Agency
John Autian
University of Tennessee
C. Stuart Baxter
University of Cincinnati
Karen Blackburn, HERL
U.S. Environmental Protection Agency
Patrick Durkin
Syracuse Research Corporation
Karl Gabriel
Medical College of Pennsylvania
Jeanne Manson
University of Cincinnati
Joseph P. Santodonato
Syracuse Research Corporation
Technical Support Services Staff: D.J. Reisman, M.A. Garlough, B.L. Zwayer
P.A. Daunt, K.S. Edwards, T.A. Scandura, A.T. Pressley, C.A. Cooper,
M.M. Denessen.
Clerical Staff: C.A. Haynes, S.J. Faehr, L.A. Wade, D. Jones, B.J. Bordicks,
B.J. Quesnell, C. Russom, B. Gardiner.
1v
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TABLE OF CONTENTS
Page
Criteria Summary
Introduction A-l
Aquatic Life Toxicity B-l
Introduction B-l
Effects B-2
Acute Toxicity B-2
Chronic Toxicity 3-3
Plant Effects 3-3
Residues B-6
Miscellaneous B-6
Summary B-3
Criteria B-8
References B-19
Mononitrophenols C-l
Mamalian Toxicology and Human Health Effects C-l
Introduction C-l
Exposure C-2
Ingestion from Water C-2
Ingestion from Food C-6
Inhalation C-12
Dermal C-13
Pharmacokinetics C-13
Absorption and Distribution C-13
Metabolism C-14
Excretion C-16
Effects C-19
Acute, Subacute, and Chronic Toxicity C-19
Synergism and/or Antagonism C-24
Teratogenicity C-25
Mutagenicity C-25
Carcinogenicity C-26
Dinitrophenols C-27
Mammalian Toxicology and Human Health Effects C-27
Introduction C-27
Exposure C-30
Ingestion from Water C-30
Ingestion from Food C-31
Inhalation C-32
Dermal C-32
Pharmacokinetics C-33
Absorption C-33
Distribution C-33
Metabolism C-34
Excretion C-35
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-I'-ects C-26
Ac-Jte, Suoacjte, and Chronic Toxic'ty ;-36
Synergism and/or Antagcn'S~ ~.i;
Teratogenicity 2-~3
Mutagenicity C-4g
Carcinogenicity C-51
Trinitropnenols C-52
Mammalian Toxicology and Human Health Effects C-52
Introduction C-52
Exposure C-52
Ingestion from Water C-52
Ingestion from Food C-56
Inhalation C-56
Dermal C-56
Pharmacokinetics C-57
Absorption C-57
Distribution C-57
Metabolism C-58
Excretion C-53
Effects C-53
Acute, Subacute, and Chronic Toxicity C-53
Synergism and/or Antagonism C-62
Teratogenicity C-62
Mutagenicity C-62
Carcinogenicity C-63
Oinitrocresols C-64
Mammalian Toxicology and Human Health Effects C-54
Introduction C-6£
Exposure C-67
Ingestion from Water C-57
Ingestion from Food C-67
Inhalation C-68
Dermal C-63
Pharmacokinetics C-68
Absorption C-68
Distribution C-69
Metabolism C-73
Excretion C-75
Effects C-77
Acute, Subacute, and Chronic Toxicity C-77
Synergism and/or Antagonism C-86
Teratogenicity C-86
Mutagenicity C-36
Carcinogenicity C-37
Criteria Formulation C-88
Existing Guidelines and Standards C-8S
Current Levels of Exposure C-3S
Special Groups at Risk C-39
Basis and Derivation of Criterion C-90
References C-95
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CRITERIA :oc'jy-N
Aquatic Life
The available data for m'troonenols indicate that acute toxicity to
freshwater aouatic life occurs at concentrations ds low as 230 ug/l and
would occur at lower concentrations among species that are more sensitive
than those tested. No data are available concerning the chronic toxicity of
nitroohenols to sensitive freshwate- aoautic life but toxicity to one soe-
cies of alqae occurs at concentrations as low as 150 ug/1.
The available data for nitroohenols indicate that acute toxicity to
saltwater aouatic Tife occurs at concentrations as low as 4,350 ug/1 and
would occur at lower concentrations among soecies that are more sensitive
than those tested. No data are available concerning the chronic toxicity of
rvtroohenols to sensitive saltwater aouatic life.
Human Health
Due to the insufficiency in the available data for mono- and trintro-
ohenols, satisfactory criteria cannot be derived at this time, using the
present guidelines.
For the protection of human health from the toxic properties of dinitro-
ohenols and 2,4-dinitro-o-cresol ingested through water and contaminated
aiuatic organisms, the ambient water criteria are determined to be 70 ug/1
and 13.4 ug/1, respectively.
For the protection of human health from the toxic properties of dinitro-
ohenols and 2,4-dinitro-o-cresol ingested throuch contaminated aouatic or-
ganisms alone, the ambient water criteria are determined to be H.3 mg.M anc1
755 uo/1, respectively.
vi i
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wononitrophenol has three isomeric forms, distinguished by the position
of t^e m'tro grouo on the phenolic ring. Three isomeric forms are possible,
namely 2-nitrophenol, 3-nitrophenol, and 4-nitrophenol. The compounds are
also commonly referred to as o-nitrophenol, m-nitrophenol, and p-nitro-
phenol, respectively.
Commercial synthesis of 2-nitrophenol and 4-nitrophenol is accomplished
through the hydrolysis of the appropriate chloronitrobenzene isomers with
aoueous sodium hydroxide at elevated temperatures (Howard, et al. 1976).
Production of 3-nitrophenol is achieved through the diazotization and hy-
drolysis of m-nitroani1ine (Matsuguma, 1967). The mononitrophenol isomers
are used in the United States primarily as intermediates for the production
of dyes, pigments, Pharmaceuticals, rubber chemicals, lumber preservatives,
photograohic chemicals, and pesticidal and fungicidal agents (U.S. Inter-
national Trade Commission, 1976). As a result of this use pattern, the ma-
jor source for environmental release of mononitrophenols is likely to be
from production plants and chemical firms where the compounds are used as
intermediates. The mononitrophenols may also be inadvertently produced via
microbial or photodegradation of pesticides which contain mononitrophenol
moieties. Approximately 10 to 15 million pounds of 2-nitrophenol are pro-
duced annually (Howard, et al. 1976) for uses including synthesis of o-ami-
nophenol, o-nitroanisole, and other dye stuffs (Matsuguma, 1967; Howard, et
al. 1976). Although production figures for 3-nitrophenol are not available,
Hoecker, et al. (1977) estimate that production is less than one million
pounds annually. 3-Nitrophenol is used in the manufacture of dye intermedi-
A-l
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ates SUCH as anisidine and Ti-aminophenol (Kouris ana Northcctt, 1963; >v(at-
suguma, 1967). 4-Nitrophenol is probably the most important of the mononi -
trophenols in terms of quantities used and potential environmental contamin-
ation. Demand for 4-nitrophenol was 35,000,000 pounds in 1976 and produc-
tion is projected to increase to 41,000,000 pounds by 1980 (Chemical Market-
ing Reporter, 1976). Most of the 4-nitrophenol produced (87 percent) is
used in the manufacture of ethyl and methyl parathions. Other uses (13 per-
cent) include the manufacture of dye-stuffs and n-acetyl-p-aminophenol
(APAP) and leather treatments. A possible source of human exposure to
4-nitrophenol is as a result of microbial or photodegradation of the para-
thions. _In_ vivo production of 4-nitrophenol following absorption of para-
thion or other pesticides by humans is another possible source of human ex-
posure.
Physical and chemical properties of the mononitrophenols are summarized
in Table 1.
Qini trpphenols
Six isomeric forms of dinitrophenol are possible, distinguised by the
position of the nitro-groups on the phenolic ring. Of the six possible
dinitrophenol isomers, 2,4-dinitrophenol is by far the most important. The
most recent production figure for 2,4-dinitrophenol is 863,000 lb. reported
by the U.S. International Trade Commission (1968). Approximate consumption
per year is estimated at 1,000,000 Ibs. (Howard, et al. 1976). 2,4-Oinitro-
phenol is used primarily as a chemical intermediate for the production of
sulfur dyes, azo dyes, photochemicals, pest control agents, wood preserva-
tives, and explosives (Matsuguma, 1967; Perkins, 1919; Springer, et al.
1977a,b).
A-2
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TABLE 1
Properties of Mononitrophenols*
connula
Molecular Weight
Melting Doint (*C)
Boiling Point (°C)
Density
Water Solubility
fg/i)
Vapor Pressure
Ka
2-Nitrophenol
C6H5N03
139.11
44 -45
214-216
1.485
0.32 at 38*C
1.08 at 100'C
1 mm Hg at 49.3°C
7.5x10-8
3 -Mi trophenol
C6H5N03
139.11
97
279
1.485
1.35 at 25°C
13.3 at 90*C
5.3x10-9
4-Ni tropheno
CsH5N03
139.11
113-114
279
1.479
0.804 at 15"
1.6 at 25'C
7x10-8
1
C
*Sources: Windholz, 1976; Weast, 1975; Matsuguma, 1967.
A-3
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ion *igures and usage data for tne remaining f;ve dinit
are not available. It is reasonable to assume that production and
usage of these compounds are extremely limited in the United States.
Commercial synthesis of 2,4-dinitrnnhenol is accomplished by the hydrol-
ysis of 2,4-
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TABLE 2
Properties of Dinitrophenol Isomers*
Isomer
2,3-Dinitrophenol
2,4 -Cini trophenol
2,5-Dinitrophenol
2,6-Oinitrophenol
3, 4 -Oini trophenol
3,5-Dinitrophenol
m.p.
( C)
144
114-115
(sublimes)
104
63.5
134
122-123
K
(at 25'C)
1.3 x 10-5
1.0 x 10-4
7 x 10-5
2.7 x 10-4
4.3 x 10~5
2.1 x 10-4
Water
Solubility
(9/1)
2.2
0.79
0.68
0.42
2.3
1.6
Density
1.681
1.683
1.672
1.702
*Source: Harvey, 1959; Wirdholz, 1976; Ueast, 1975.
A-5
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"ABLE 3
Properties of Trinitrophenols*
Molecular Weight
229.11
2,3.5-Trim'trophenol
Molecular Weight
Melting Point
229.11
119-120°C
2,3,6-Trinitrophenol
Molecular Weight
Melting Point
Water Solubility
Room Temperature
Hot Water
229.11
119'C
Slightly Soluble
Very Soluble
2,4,5-Trini trpphenol
Molecular Weight
Melting Point
Water Solubility
Room Temperature
Hot Water
229.11
96'C
Slightly Soluble
Soluble
2,4,5-Trinitrophenol
Molecular Weight
Melting Point
Boiling Point
Vapor Pressure
Density
Water Solubility
Room Temperature
100'C
229.11
122-123*C
Sublimates: Explodes at
300'C
1 mm Hg at 195°C
1.763 g/cm3
1.28 g/1
6.7 g/1
*Source: Windholz, 1976; Weast, 1975; Matsuguma, 1967.
A-6
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According to Vatsuguma (1967) picric acid has found usage as: a dye in-
termediate, explosive, analytical reagent, germicide, fungicide, staining
agent and tissue fixative, tanning agent, photochemical, pharmaceutical, and
a orocess material for the oxidation and eching of iron, steel and copper
surfaces. The extent to which picric acid finds usage in any of these ap-
plications at the present time is unknown.
Dinitrocresols
Dinitro-ortho cresol is a yellow crystalline solid derived from o-cre-
sol. There are six possible isomers but the 4,6-dinitro-o-cresol isomer is
the only one of any commercial importance. In fact, a comprehensive search
of the literature failed to reveal information on any of the other five
dim'trocresol isomers.
4,6-Oinitro-o-cresol (hereafter referred to as DNOC) is produced either
by sulfonation of o-cresol followed by treatment with nitric acid or by
treatment of o-cresol in glacial acetic acid with nitric acid at low temper-
ature. Some important chemical and physical properties of DNOC are shown in
Table 4.
The U.S. Environmental Protection Agency (U.S. EPA) has no record of
DNOC being currently manufactured in the United States for use as an agri-
cultural chemical. Imports of DNOC have also decreased in recent years;
from 217,899 Ibs. in 1972 to 146,621 Ibs. in 1973 and then to 30,442 Ibs. in
1976 (National Institute for Occupational Safety and Health (NIOSH), 1978).
Since DNOC is not manufactured in the U.S., pesticide formulators and spray-
ers are the major groups with potential occupational exposure to DNOC.
DNOC is used primarily as a blossom-thinning agent on fruit trees and as
a fungicide, insecticide, and miticide on fruit trees during the dormant
A-7
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TABLE 4
Properties of 4,6-Dinitro-o-cresol*
Molecular Weight 198.13
Appearance Yellow Solid
Melting Point 85.8'C
Vapor Pressure 0.000052 mm Hg at 20*C
Water Solubility 100 mg/1 at 20*C
pKa 4.46
*Source: Windholz, 1976; Weast, 1975; Matsuguma, 1967.
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season. NIOSH (1973) estimates that' 3,000 workers in the U.S. are poten-
tially exposed to ONOC. In view of the small amount of DNOC used in the
U.S., exposure of the general public is expected to be minimal.
In general, few data are available regarding the breakdown of nitro-
phenols by natural communities of microorganisms. A number of researchers
have isolated microorganisms capable of using nitrophenols as a sole source
of carbon in pure culture (Simpson and Evans, 1953; Raymond and Alexander,
1971; Chambers, et al. 1963; Guillaume, et al. 1963). However, the signifi-
cance of such studies as related to the stability of nitrophenols in the en-
vironment is not known.
Several investigators have shown that individual species of aerobic and
anaerobic bacteria, including Azotabacter chroococcum and Clostriduim butyr-
ium, and the fungus Fusarium, are capable of reducing 2,4-dinitrophenol in
culture (Radler, 1955; Lehmber, 1956; Madhosingh, 1961). However, the pre-
cise pathway for metabolic degradation is not known. Jensen and Lautrup-
Larson (1967) found that Arthrobacter simplex, Pseudomonas, and Arthrobacter
were able to metabolize 2,4-dinitrophenol and 2,4,6-trinitrophenol, forming
nitrite.
The actual degradation pathway of dinitro-o-cresol has been investigated
by Tewfik and Evans (1966) in pure cultures of microorganisms. It was re-
ported that in Pseudomonas sp. degradation proceeded by way of formation of
an aminocresol. In Arthrobacter simplex, a hydroxylated catechol is formed
prior to ring cleavage.
The significance of such studies as related to the stability of nitro-
phenols in the environment is not known. Certain investigators have postu-
lated that ambient nitrophenol concentrations may be too low to induce the
appropriate microbial enzymes necessary to facilitate population growth and
metabolism of the compounds (U.S. EPA, 1976).
A-9
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-nat i 3n regadding the TObilitv an
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REFERENCES
Chambers, C.W., et al. 1963. Degradation of aromatic compounds by phenol-
adapted bacteria. Jour. Water Pollut. Control Fed. 35: 1517.
Chemical Marketing Reporter. 1976. Chemical profile: p-nitro-phenol.
Gomaa, H.W. and S.D. Faust. 1972. Chemical hydrolysis and oxidation of
parathion and paraoxon in aquatic environments. Adv. Chem. Ser. Vol. III.
Iss. Fate Org. Pestic. in the Aquatic Environ.
Guillaume, J, et al. 1963. Oxidation of p-nitrophenol by certain Mycobac-
teria. Comp. Rend. 256: 1634.
Harvey, O.G. 1959. On the metabolism of some aromatic nitro compounds by
different species of animal. Part III. The toxicity of the dinitrophenols,
with a note on the effects of high environmental temperatures. Jour. Pharm.
Phamiacol. 11: 462.
Hoecker, J.E., et al. 1977. Information profiles on potential occupational
hazards. Nat. Inst. Occup. Safety Health, Cincinnati, Ohio.
Howard, H., et al. 1976. Investigation of selected potential environmental
contamination: Nitroaromatics. Off. Tox. Subst. U.S. Environ. Prot.
Agency, Washington, O.C.
A-ll
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Jensen, 'J.L. and G. Lautrup-larson. 1967. Microorganisms that decorroose
nitro-aromatic compounds, with special reference to dinitro-o-creso 1. Acta
Agric. Scand. 17: 115.
KouHs, C.S. and J. Northcott. 1963. Aniline and its derivatives. _In_:
Kirk-Othmer Encyclopedia of Chemical Technology. 2nd ed. 2: 411.
lehmber, C. 1956. Untersuchungen uber die Winbung von Ascorbunsaure, Stof-
fwechselgifren and Anderen Faktoren auf den Staffwechsel von Clostridium
butyrium. Beif. Arch. Mikrobiol. 24: 323.
Madhosinggh, C. 1961. The metabolic detoxification of 2,4,-dinitrophenol
by Fusarium oxysponim. Can. Jour. Microbiol. 7: 553.
Matsuguma, H.J. 1967. Nitrophenols. lr±: Kirk-Othmer Encyclopedia of Chem-
ical Technology. 2nd. ed. 13: 888.
Mosinska, K. and A. Kotarski. 1972. Determination of 2-isopropyl-4,6-dini-
trophenol and 2,4-ONP in herbicides and in technical 2-isopropyl-4,6-ONP by
TCL. Chemia Analltyezma. 17: 327.
Perkins, R.G. 1919. A study of the munitions intoxications in France. Pub.
Health Rep. 34: 2335.
Padler, F. 1955. Untersuchunger uber den verlaug der stoffwech Selvorgan-
gebei Azotobacter chroococcum. Beig. Arch. Microbiol. 22: 335.
A-12
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Raymond, C. ,v. and vi. Alexancer. 1971. Vicrobial ~etabc'"S.T anc :o~e:a-
bolism of nitrophenols. Pestic. Siochem. Physio!. 1: 123.
Saltzman, S. and S. variv. 1975. Infrared study of the sorbtion of pneno.
and p-nitrophenol by montmori1lonite. Soil Sci. Soc. Am. ?roc. 39: 4/4.
Simpson, J.S. and W.C. Evans. 1953. The metabolism of nitropheols by cer-
tain bacteria. Siochem. Jour. 55: 24.
Springer, E.L., et al. 1977a. Chemical treatment of chips for outdoor
storage. Evaluation of sodium n-methyldithiocarbomate and sodium 2,4-dini-
trophenol treatment. Tapi. 60: 88.
Springer, E.L., et al. 1977b. Evaluation of chemical treatments to prevent
deterioration of wood chips during storage. Tapi. 60: 93.
Tewfik, M.S. and W.C. Evans. 1966. The metabolism of 3,5^initro-o-crescl
(DNOC) by soil microorganisms. Biochem. Jour. 99: 31.
U.S. EPA. 1976. Investigation of selected potential environmental contami-
nants: nitroaromatics. Final Sep. Off. Tox. Subst. Washington, D.C.
U.S. International Trade Commission. 1967-73. Synthetic organic chemicals:
U.S. production and sales. Washington, D.C.
U.S. International Trade Commission. 1976. Imports of benzenoid chemica's
and products, 1974. Publ. No. 762. Washington, O.C.
A-13
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Weast, 3.C., ed. 1975. Handbook of Chemistry and Physics. 57th ed. C3C
Press.
, i*., ed. 1976. The Merck Index. 9th ed. Merck and Co., Rahway,
Mew Jersey.
v, S., et al. 1966. Infrared study of the absorotion of benzoic add
and nitrobenzene in montomori llonite. Isr. Jour. Chem. 4: 201.
A-1
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Aquatic Life Toxicology*
INTRODUCTION
Although fish and invertebrate acute toxicity data and plant toxicity
data are available for the groups of organic compounds which contain various
numbers of nitro groups substituted into the aromatic ring of a phenol or
cresol, collectively referred to as nitrophenols, there are only limited
data available for any individual nitrophenol. There are no data available
dealing with chronic effects of any nitrophenol on freshwater aauatic organ-
isms, and no suitable substitute chronic value can be determined from avail-
able toxicity information. The limited data available preclude deriving a
criterion for any of the individual nitrophenol compounds. The derivation
of a single criterion which would protect freshwater aauatic organisms from
all nitrophenols is also impractical because of the wide difference in tox-
icity of individual nitrophenols.
The saltwater data base available for the various nitrophenols is also
limited. For 4-nitrophenol there are acute test results for one fish and
one invertebrate species, and one fish chronic test. In addition, there are
two invertebrate lethal threshold values. The data base for 2,4-dinitro-
phenol consists of acute tests, one algal test, and miscellaneous effects on
one invertebrate species. Information on 2,4,6-trinitrophenol is even more
limited, consisting of acute results for one fish, one algal, and one in-
vertebrate species.
*The reader is referred to the Guidelines for Deriving Water Quality Cri-
teria for the Protection of Aquatic Life and Its Uses in order to better un-
derstand the following discussion and recommendation. The following tables
contain the appropriate data that were found in the literature, and at the
bottom of each table are calculations for deriving various measures of tox-
icity as described in the Guidelines.
3-1
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~~TECT$
Acute Toxicity
The data base for freshwater invertebrate species (Table 1) contains
seven data points for four nitrophenol compounds with two invertebrate spe-
cies. An unspecified dinitromethylphenol (reported by Sanders and Cope,
1968 as dinitrocresol) is the most toxic compound with an ICcg value of
320 ug/l for a stonefly, Pteronarcys californica. This compound is followed
in order of decreasing toxicity by 2,4-dinitro-6-methylpheno1, 2,4-dinitro-
phenol, 4_nitrophenol, and 2,4,6-trinitrophenol. The 2,4-dinitro-6-methy1-
phenol LCgQ for Daphnia magna is 3,120 ug/l (U.S. EPA, 1978). It appears
that stoneflies may be more sensitive to dinitromethylphenol than are daph-
nids, although this is impossible to verify since the compound tested with
stoneflies is inadeauately identified and may be a slightly different com-
pound than that tested with daphnids. For 2,4-dinitrophenol, the two LC,-n
values for daphnids are Quite close and are 4,710 ug/1 (Kopperman, et al.
1974) and 4,090 ug/l (U.S. EPA, 1978). The toxicity of 4-nitrophenol to
daphnids shows greater variation between investigators with reported LC50
values of 8,396 ug/1 (Kopperman, et al. 1974) and 21,900 ug/l (U.S. EPA,
1978). The least toxic nitrophenol to daphnids is 2,4,6-trinitrophenol with
a reported LC5Q value of 84,700 ug/l (U.S. EPA, 1978).
The freshwater fish acute toxicity data base (Table 1) consists of
seven 1C values for four nitrophenols and two fish species. Although
differences in test methods make comparisons difficult, it appears tluegills
are more sensitive than fathead minnows with all three nitrophenols for
which data are available for both species. Comparisions of LCgQ values
for fishes indicate that 2,4-dinitro-6-methylphenol is the most toxic nitro-
phenol with LC5Q va1ues of 230 ug/1 (ILS< EPA^ 1978) and 2,030 ug/l
8-2
-------
(Phipps, et al. Manuscript) for the bluegill and fathead minnow, respective-
ly; 2,4-dinitro-6-methylphenol is followed in order of decreasing toxicity
by 2,4-dinitrophenol, 4-nitrophenol, and 2,4,6-trinitrophenol. The largest
variation in species sensitivity occurred with 2,4-dinitrophenol where
LC5Q values are 620 ug/1 for bluegills (U.S. EPA, 1978) and 16,700 ug/l
for fathead minnows (Phipps, et al. Manuscript). The LCcn values reported
for 4-nitrophenol for bluegills and fathead minnows are 8,280 ug/1 (U.S.
EPA, 1978) and 60,500 ug/1 (Phipps, et al. Manuscript), respectively. The
high 2,4,6-trinitrophenol LC5Q of 167,000 ug/1 for bluegills (U.S. EPA,
1978) indicates the toxicity of nitrophenols does not increase directly with
increasing nitro-group substitution.
The order of toxicity of the four nitrophenol compounds tested with
both freshwater fish and invertebrate species is the same, and it appears
from the limited data available that there are no large sensitivity differ-
ences among these species with any of these nitrophenols. Invertebrate tox-
icity values for 4-nitrophenol and 2,4-dinitrophenol fall between those cal-
culated for bluegills and fathead minnows. Daphnia magna appeared slightly
more sensitive to 2,4,6-trinitrophenol than were bluegills, and slightly
less sensitive to 2,4-dinitro-6-methylphenol than were fathead minnows.
As seen in Table 1 for the saltwater acute data, 4-nitrophenol resulted
in a 96-hour 1C of 7,170 ug/1 with mysid shrimp, whereas the sheepshead
minnow showed greater resistance with a 96-hour LC5Q value of 27,100
ug/1. Both the mysid shrimp and the herring embryos exhibited greater sen-
sitivity to 2,4-dinitrophenol (96-hour LC5Q values of 4,850 and 5,500
ug/1, respectively) than did the sheepshead minnow at 29,400 ug/1. Of the
three nitrophenols tested, 2,4,6-trinitrophenol appeared to be the least
toxic, producing 96-hour LCcg values of 19,700 ug/1 for the mysid shrimp
B-3
-------
and 134,000 ug/1 for the sheesshead Tiinnow. This nitroohenol was also t^e
least toxic of the tested compounds to freshwater fish and invertebrate spe-
cies. Although the invertebrate species was consistently more sensitive- to
all these compounds than was the sheepshead minnow, the herring embryos also
were very sensitive to the toxic effects of 2,4-dinitrophenol.
Chronic Toxicity
There are no data available on the chronic effects of any of the var-
ious nitrophenol compounds on freshwater aouatic life.
The chronic effects of 4-nitrophenol and 2,4-dinitrophenol on hatching
and survival in an early life stage test with the sheepshead minnow have
been determined (U.S. EPA, 1978). Chronic values of 12,650 and 7,900 ug/1
were obtained for 4-nitrophenol and 2,4-dinitrophenol, respectively (Table
2). Although 4-nitrophenol may be somewhat less toxic in the chronic test,
the compounds were of similar acute toxicity to the sheepshead minnow (Table
1).
Acute-chronic ratios for these nitrophenols may be derived for the
sheepshead minnow. For 4-nitrophenol the acute-chronic ratio is 2.1, and
for 2,4-dinitrophenol the ratio is 3.7.
There are no nitrophenol chronic effects data for any saltwater inver-
tebrate species.
Plant Effects
Freshwater plant toxicity values (Table 3) are lower, in certain in-
stances, than acute LC-- values for fish and invertebrate species. Expo-
sure to 4-nitrophenol produced toxic effects in ChloreT •- vulgaris at 6,950
ug/l (Dedonder and Van Sumere, 1971) and in Selenastrum capricornutum at
4,190 ug/1 (U.S. EPA, 1978). These plant effect levels for 4-nitrophenol
are both below the lowest fish or invertebrate LC5Q values for this com-
pound. A 50 percent reduction in chlorophyll a_ also occurred in Selenastrum
B-4
-------
cap^icoroutjn in 95 hours at a 2,4,6-trini trooheno' concentrat ; on ,'ai,7CC
ug/1) t^at is below the lowest LC-0 value for fish or invertebrate spe-
cies. As observed with fish and invertebrate species, 2,4,5-trinitropheno '
is less toxic than d_nitrophenol and 2,4-dini trophenol to Selenastrum capri-
(U.S. EPA, 1973).
Dedonder and Van Sumere (1971) determined that 9,200 ug/1 of 2,4-dini-
trophenol caused a 70 percent growth inhibition in Chlorella vulgaris in 80
hours, although this concentration caused only a 25 percent growth inhibi-
tion in 160 hours.
Results of tests which examined the relative, toxicity of the three iso-
meric foms of mononi trophenol s to an alga (Huang and Gloyna, 1967) indi-
cated that chlorophyll synthesis in Chlorella pyrenoidosa was inhibited to a
point below initial control levels at concentrations of 25,000 ug/1 by 4-
nitrophenol, 35,000 ug/1 by 2-nitrophenol , and 50,000 ug/l by 3-nitro-
phenol. Studies with three species of algae indicate that 4-nitropheno 1 is
slightly more toxic to plants than is 2,4-dinitrophenol (Table 3). The one
exception to this toxicity trend was observed by Simon and Blackman (1953),
who found that 50 percent growth reduction in duckweed, Lemna minor, occur-
red at 2,4-dinitrophenol and 4-nitrophenol concentrations of 1,472 ug/1 and
9,452 ug/1, respectively. However, these observed effects on duckweed oc-
curred under conditions of low pH (5.2 and 5.4) and effects at a more neu-
tral pH were not measured.
All of the available freshwater plant effects data for nitrophenols are
based on unmeasured concentrations and therefore there is no Freshwater Fin-
al Plant Value.
Only one saltwater algal species was examined for its response to three
nitrophenols (U.S. EPA, 1978). The effects of these compounds on Skeletone-
ma_ costatum are summarized in Table 3. The 96-hour LCrQ concentrations of
B-5
-------
4-nitrophenol (about 7,000 ug/1) and 2,4-dinitroonenol (about 95,000 yg/1)
were very similar for effects on both chlorophyll a_ production and reduction
in cell numbers. However, 2,4,6-trinitrophenol seemed to have a greater ef-
fect on chlorophyll a_, with a 96-hour EC5Q of 62,7000 ug/1, than on cell
numbers where the effective concentration was 141,000 ug/1. Skeletonema
costatum was most sensitive to 4-nitrophenol.
Residues
No measured, steady-state bioconcentration factors are available for
saltwater or freshwater organisms and any nitrophenol.
Miscellaneous
No miscellaneous data were found that would be a suitable substitute
for a Final Chronic Value for any nitrophenol compound (Table 4), although
some data are the lowest acute values or the only acute values available for
several nitrophenol compounds. Bringmann and Kuhn (19_78) calculated the 8-
day toxicity thresholds (the pollutant concentration causing the onset of
cell multiplication inhibition) for nine nitrophenol compounds with two
algal species, Microcystis aeruginosa and Scenedesmus ouadricauda. Data
from the most sensitive species tested in this study are the lowest plant
values available for 2-nitrophenol (4,300 ug/1), 3-nitrophenol (7,600 ug/1),
2,4,6-trinitrophenol (40,000 ug/1), and 2,4-dinitro-6-niethylphenol (150
ug/1) and are the only plant data available for 2-nitro-£-cresol (3,800
ug/1), 4-nitro-m-cresol (7,000 ug/1), and 6-nitro-m-cresol (7,000 ug/1). In
another study, Bringmann and Kuhn (1977) determined the 24-hour LC5Q
values for the same nine nitrophenols with the cladoceran, Oaphnia magna.
This study provides the only invertebrate or fish acute data available for
2-nitro-£-cresol (130,000 ug/1), 4-nitro-m-cresol (33,000 ug/1), and 6-ni-
tro-m-cresol (43,000 ug/1). and also provides a toxicity comparison of the
3-5
-------
three isomeric forms of mononitrophenols with daphnids. They found 4-nitro-
phenol to be the most toxic compound to daphnids, followed in order of de-
creasing toxicity by 3-nitrophenol and 2-nitrophenol. Gersdorff (1939) also
found the same relative order of toxicity for the three mononitrophenols
with goldfish (8,000 ug/1 for 4-nitrophenol, 24,000 ug/1 for 3-nitrophenol,
and 33,300 ug/l for 2-nitrophenol). While these are not LC5Q values, they
represent the lowest acute effects available for these three mononitrophenol
compounds. Although the relative order of toxicity is the same for the
three mononitrophenols with both fish and invertebrate species, the toxicity
order appeared to be slightly different in the alga, Chlorella pyrenoidosa
(Huang and Gloyna, 1967, Table 3).
Juvenile Atlantic salmon were much more sensitive to 2,4-dinitrophenol,
with a lethal threshold value of 700 ug/1 (Zitko, et al. 1976). (Lethal
threshold is the geometric mean of the highest concentration with no deaths
and the next higher concentration at which all animals died). The authors
tested a variety of compounds with both freshwater and saltwater species.
They did not specify whether the Atlantic salmon was tested in salt or
freshwater, but the size of the tested organism leads us to believe that the
test was probably conducted in freshwater.
The only saltwater datum for 2-nitrophenol is listed in Table 4. This
result is very similar to that for 4-nitrophenol and the shrimp, Crangon
septemspinosa. The soft shell clam responded to the toxic effects of 4-ni-
trophenol at about the same concentration (McLeese, et al. 1979). 2,4-Oini-
trophenol also adversely affected sperm and embryo of two sea urchin species
at concentrations as low as 46,000 ug/1.
8-7
-------
Summary
For the four nitrophenols for which freshwater acute data are avail-
able, the order of most to least toxic is 2,4-dinitro-6-methylpheno1, 2,4-
dinitrophenol, 4-nitrophenol, and 2,4,6-trinitrophenol. Acute LC,-0 values
ranged from a low of 230 ug/1 for bluegills exposed to 2,4-dinitro-6-
nethylphenol to a high of 167,000 ug/1 for bluegills exposed to 2,4,6-trini-
trophenol. There are no freshwater chronic data available for any nitro-
phenol. Plant values and miscellaneous data for the various nitrophenols
showed that effects occurred at exposure concentrations ranging from a low
of 150 »g/l for 2,4-dinitro-6-methy1phenol, based on an 8-day toxicity
threshold with algae, to a high of 210,000 ug/1 for 2-nitrophenol, based on
a 24-hour LCg. with daphnids.
For the three nitrophenols having a saltwater acute toxicity data base,
the mysid shrimp was consistently more sensitive than the sheepshead min-
now. In comparing the acute toxicity of these three compounds, the least
toxic for both organisms was 2,4,6-trinitrophenol. Acute and early life
stage tests conducted on the sheepshead minnow yielded acute-chronic ratios
of 2.1 for 4-nitrophenol and 3.7 for 2,4-dinitrophenol. There are no chron-
ic data for 2,4,6-trinitrophenol.
Plant values and miscellaneous data for the various nitrophenols showed
a concentration range of effects from a low of 7,370 ug/1 for 4-nitrophenol
on chlorophyll a^ production in an alga to a high of 141,000 ug/1 for an ef-
fect of 2,4,6-trinitrophenol on cell numbers of the same species.
CRITERIA
The available data for nitrophenols indicate that acute toxicity to
freshwater aauatic life occurs at concentrations as low as 230 yg/1 and
would occur at lower concentrations among species that are more sensitive
3-8
-------
than fiose tested. Mo data are available concerning the cnronic toxicity of
nitropnenols to sensitive freshwater aquatic life out tcxicity to one spe-
cies of algae occurs at concentrations as low as 150 ug/1.
The available data for nitrophenols indicate that acute toxicity to
saltwater aouatic life occurs at concentrations as low as 4,350 ug/1 and
would occur at lower concentrations among species that are more sensitive
than those tested. No data are available concerning tne chronic toxicity of
nitroohenois to sensitive saltwater aquatic life.
3-9
-------
Table t. Acute values for nltrophenols
Species
Cladoceran,
Daphnla magna
Cladoceran,
Daphnla Miagna
Cladoceran,
Daphnla magna
Cladoceran,
Daphnla magna
C 1 adoceran ,
Daphnla magna
Cladoceran,
Daphnla magna
Stonefly (naiad),
Pteronarcys call for n lea
Fathead minnow
( juvenl le).
Plmepnales promelas
Fdttiead minnow
(juvenl le).
Plroephales promelas
Fathead minnow
(juvenl le).
Plmephales promelas
Bluegill,
Lepomls macrochlrus
Bluegj 1 1,
Lepomis macrochlrus
Bluegill,
Ltipomls macrochlrus
Method*
S, U
S, U
S, U
S, U
S, U
S, U
S, U
FT, M
FT, M
FT, M
S, U
S, U
S. U
Chemical
FRtSHWAftR
4-nl trophenol
4-nl 1r ophenol
2,4-dlnltro-
phenol
2,4-dlnltro-
pnenol
2,4,6-trlnltro-
phenol
2,4-dlnltro-6-
methy (phenol
Olnltromethyl-
phenol**
4-nl trophenol
2,4-dlnltro-
phanol
2,4-dinitro-6-
methy (phenol **
4-nl trophenol
2,4-dlni tro-
phonol
2,4,6-trlnl tro-
phonol
LC50/EC50
(M9/D
SPECItS
8,396
21,900
4,710
4,090
84.700
3,120
320
60,500
16,700
2,030
8,280
620
167,000
Species Mean
Acute Value
(ug/l)
_
13,560
-
4,389
84 , 700
3,120
320
60.500
16,700
2,030
8,280
620
167.000
Reference
Koppurman, et al.
1974
U.S. EPA, 1978
Kupperman, ot al.
1974
U.S. EPA, 1978
U.S. EPA, 19/8
U.S. EPA, 1978
Sanders & Cope, 1968
Phlpps, et al.
Manuscript
Phlpps, et al.
Manuscript
Phlpps, et al.
Manuscr Ipt
U.S. EPA, 1978
U.S. EPA. 1978
U.S. EPA, 1978
B-10
-------
Table 1. (Continued)
Species
Liluegl 1 1,
tupomts macrochlrus
Mysid shrlnp,
Mysldopsls bah ia
Mysid shrimp,
Mysldopsis bah la
Mysid shrimp.
Mysldopbis Uih i a
Herring (embryo),
Clupea harengus
Sheepbhoad minnow,
C/pr inodon varieyatus
ShHupbliuad in if mow,
Cypr inodon varieyatus
Slieupbhfcjd minnow,
Cypr inodon varieyatus
Method"
S, U
S, U
S, U
S, U
S, U
S, U
S, U
S, U
Chemical
2,4-dinitr 0-6-
mulhy t ptiunol
4-ni 1r ophonol
2,4-diul tro-
phbllol
2,4,6-1i Inl tro-
phonol
2,4-dlnitro-
phuliol
4-di tr'ophenol
2,4-dinltro-
phenol
2,4,6-tr Inl Iro-
pllaflol
LC50/LC50
(Mi/'J
230
UK SHEClES
7,170
4.050
19.700
5,500
27,100
29,400
134.000
Species Mean
Acute Value
(ug/D
230
7,170
4,050
19,700
5,'j()0
27,100
29,400
134,000
Reference
U.S. tt'A. 1970
U.S. EPA, 19/0
U.S. EHA, 1970
U.S. EHA, 19/0
Kosorilliol i Slc-l^or
1970
U.S. EHA, 1976
U.S. Ef'A, 19/0
U.S. EHA, 19/0
* S = italic, FT = flow-through, (I = unmeasured, M - measured
""Authors fuported results as 4,6-dlni tro-o-cresol or as only d lui 1ro<_rov>ol (Sanders and Cope, 1960).
3-a.l
-------
Table 2. Chronic values tor nltrophenols (U.S. tPA, 1970)
Species
Sheepshead minnow,
Cyprlnodon varleqatus
Sheepshead minnow,
Cyprlnodon varlegatus
* ELS = early life staye
Spec 1 os
Sheepshead minnow,
Cyprlnodon varlegatus
Sheepshead minnow,
Cypr Inodon varlegatus
Method"
SAL TW AUK
ELS
hLS
Chemical
SPEC Its
4-nl trophenol
2,4-dlnltro-
phenol
Limits
(My/I)
10.000-
16.000
5,200-
12,000
Chronic Value
(M9/I)
12.650
7,900
Acute-Chronic Ratio
Chemical
4-nl Trophenol
2,4-dlnltro-
phenol
Acute
Value
(ug/i)
27,100
29,400
Chronic
Value
tut}/ I )
12,650
7,900
Ratio
2.1
3.7
3-L2
-------
Table 3. Plant values for nltrophenols
Species
AI yd.
Chloral la pyrenoldosa
Alga,
Cn I or e 11 a pyrenoldosa
Alga.
Chloral la pyrenoldosa
Alga.
ChIore I la pyrenoldosa
Alga,
Chior el la pyrenoldosa
Alga,
Chi or t, I la vulgar is
Alga.
Ch I or el la vulgar Is
Alga,
ChIoreIIa vuIgar is
Alija,
Selenastrum caprI cornuturn
Alga.
Sfelenastrum caprI cor nutum
Chemical
H.|
2-nltr ophenol
3-iiMrophbnol
4-nl trophenol
2,4-dinitro-
phenol
2,4-dlnitro-6-
methy I phenol *
4-ni trophenol
2,4-dlni tro-
phenol
2,4-dlnitro-
phenol
4-nl trophenol
2,4-dlnltro-
phenol
Effect
SMWAjifL^tc.!!
I nh Ibi t ion of
chlorophy 1 1
synthesis after
3 days
Inhibit Ion of
chlorophy 1 1
synthesis after
3 days
Inhibit Ion of
ch lot ophy I I
synthesis after
3 days
Inhibition of
ch lot ophy I I
synthesis after
3 days
Intrlbi t Ion of
chlorophy I I
synthesis after
3 days
50* growth Inhi-
bition lit bO hrs
70* growth Inhi-
bition In «0 hrs
25* growth Inhi-
bition In I60 hrs
96-hr EC50,
chlorophy 1 1 a
96-hr EC50,
ch lor ophy 1 1 a
Result
(M9/I)
35,000
50,000
25,000
50,000
50,000
6,950
9,200
9,200
4,190
9,200
Reference
Huang & G loyna.
Huang & G loyna.
Huang 4 G loyna.
Huang & G loyna.
Huang & G loyna.
Uedonder & Van
Sumero, 1971
Oedonder & Van
Sumere, 1971
Oedonder & Van
Suniere, 19/1
U.S. EPA, I97b
U.S. EHA, 197tt
1967
1967
1967
1967
1967
3-13
-------
Table 3. (Continued)
Species
Alga,
Sulunastrum capr Icornutuin
Duckweed,
lomna minor
Duckweed,
Lbmna minor
Duckweed,
Lemna mlnoi
Alga,
Skeletonema costatum
Alga,
Skeletonema costatum
Alga,
Skeletonema costatum
Alga,
Skeletonema costatum
Alga,
Skelfetonema costatum
Alga,
Skeletonemd costatum
Chemical
2.4,6-tr Inl Iro-
ptvenol
Effect
%-hour EC 50
ch lorophy 1 1 a
2-nl trophenol 50J growth
ruducl Ion
4-nl trophunol 501 growth
reduct ion
2,4-dinitro- 501 growth
phenol reduction
SALTWATER SPECIES
4-nl trophenol
4-nl trophenol
2,4-dlnl h-o-
phdnol
2,4-dlnl tro-
phenol
2,4,6-trlnltro-
pheiiol
2,4,6-trlnltro-
phenol
96-hr EC50,
ch 1 orophy 1 1 a
96-hr EC50,
eel 1 number
96- hr EC50,
ch lorophy 1 1 a
96-hr EC50,
ce 1 1 number
96- hr EC50,
ch lorophy 1 1 a
9<}-hr EC50,
eel 1 number
Result
(ug/l) Reference
41,700 U.S. EPA, 19/8
62,550 Simon & Blackmail,
1953
9,452 Simon & bldckman,
1953
1,472 Simon & blackmail.
1953
7,370 U.S. EPA, 1978
7,5/0 U.S. EPA, 1978
93,200 U.S. EPA, 1978
98,700 U.S. EPA, 19/8
62,700 U.S. EPA, 1978
141,000 U.S. EPA, 1978
* Authors reported results as 4,6-dlnltro-o-cresol.
B-14
-------
Table 4. Other data for nltrophenols
Species
Chemical
Duration
Effect
Result
(ug/l>
Reference
FKtSHWATEK SPtCltS
Cli lomydomonas sp.
Alya,
Microcyst Is
Alga,
Scenedesmus
Alga,
Microcyst Is
Alga,
Scenedesmus
Alga,
Microcyst Is
Alga,
Scenedesmus
Alga,
Microcyst Is
Alga,
Scenedesmus
Alga,
Microcyst is
Alga,
Scenedesmus
Alga,
Microcyst is
Alga,
Scenedesmus
aeruglnosa
quadr Icauda
aeruglnosa
quadr Icauda
aeruglnosa
quadr Icauda
aeruglnosa
quadr icauda
aeruglnosa
(juadr icauda
aeruginosa
quadr Icauda
D Inl trophenol
2-nl trophoni!
2-nl trophenol
3- nl trophenol
3-nl trophenol
4-nl trophenol
4-ni trophenol
2,4-dlnitro-
phenol
2,4-dlnltro-
phenol
2,4,6-tr I ni tro-
phenol
2, 4, 6-trlnl tro-
phenol
2-nl tro-p-
cresol
2-nltro-p-
cresol
30
8
8
8
8
8
8
8
8
8
8
8
8
sec
days
days
days
days
days
days
days
days
days
days
days
days
50* Inhibition
of f lagel lar
motl 1 1 1/
Toxlclty
threshold*
Toxlclty
tires hold*
Toxiclty
threshold*
Toxlclty
threshold*
Toxlclty
threshold*
Toxlclty
threshold*
Toxlcily
threshold*
Toxlcity
threshold*
Toxlcity
threshold*
Toxlclty
threshold"
Toxiclty
threshold*
Toxlclty
1 tires hold*
18,400
27
4
17
7
56
7
33
16
40
61
32
3
,000
,300
,000
,600
,000
,400
,000
,000
,000
,000
,000
,800
Marcus & Mayor, 1963
Br Inymdim
Brlngmann
1978
Brlngmann
1978
Br Ingmann
1978
Brlngmann
1978
Brlnc^nann
1978
Brlngmann
1978
Br Inymann
1978
Brlngmann
1978
Br Ingmann
1978
Br Inymann
1978
Brlnqmanri
19 10~
& Kulm,
& Kuhn ,
& Kuhn,
& Kuhn,
& Kul«i.
& Kuhn,
& Kuhn,
& Kulm,
& Kuhn,
& Kuhn,
& Kuhn,
& Kuhn,
B-15
-------
Table 4. (Continued)
Species
Alga,
Mlcrocystls aeruyioosa
Al.ja,
Scenedesmus quadrlcauda
Alga,
Hlcrccystls aerutjlnosa
Alga,
Scenadesmus quadrlcauda
Alga,
Mlcrocystls aerujjlncsa
Alga,
Scenedesmus quadrlcauda
A/ooo ba,
Amoeba proteus
Amoeba,
Amoeba proteus
Cladoceran,
Daphnla mayna
Cladoceran,
Daphnla magna
Cladoceran,
Daphnla macjna
Cladoceran,
Oaphnla magna
Cladoceran,
Daphnla magna
Cladoceran,
Udphnia nuitina
Chemical
4-ni tro-m-
cr osol
4-nl tro-m-
crosol
6-nltro-m-
cresol
6-nltro-m-
crt»sol
2,4-dinltro-6-
rnethyl phenol **
2,4-dinltro-6-
methyl phenol**
01 ni trophenol
Dial trophenol
2-nl trouhenol
3-nltrophenol
4-nl trophenol
2,4-dinltro-
phenol
2,4,6-trinltro-
phenof
2-ni tro-g-
cresol
Duration
6 d'tys
ti days
6 days
8 days
8 days
8 days
24 hrs
48 hrs
24 hrs
24 hrs
24 hrs
24 hrs
24 hrs
24 hrs
Effect
Toxlcl ty
threshold*
foxlcl ty
thrushold*
Toxic Ity
threshold*
Toxic Ity
thrushold*
Toxic Ity
threshold*
Toxlcl ty
threshold*
A6f reduction in
amoeba containing
go 1 g 1 bod i as
18* mortality
IC50
LC50
LC50
IC50
LC50
L.50
Result
(M9/D
13,000
7,000
34,000
7.000
150
13,000
92,000
92,000
210,000
39,000
35,000
19,000
145,000
130,000
Reference
UrinijirtdiiM &
1976
brlnijinaiin &
19/8
Br Inijihdfiu &
1978"
Brlnymann &
1978
Brlnynidnn &
1976
brlntjmann &
1978
Ft lckiny«r.
K 1 Icktnger,
brlngmann &
1977
Brinymann &
1977
iirlngmann &
1977
Brlnymann A
1977
Orln
-------
Table 4. (Continued)
Spec 1 es
Cladoceran,
Ddphnla mayna
Cladoceran,
Daphnla magna
Cladoceran,
Daphnla magna
Southern bullfrog
(tadpole),
Kana gry 1 lo
Atlantic salmon (juvenile).
Sal mo salar
Goldfish,
Carasslus auratus
Goldfish,
Carasslus auratus
Goldfish,
Carasslus auratus
Bluegl 1 1 ( juvenl le),
Lepomls macrochlrus
blueglll (juvenile),
LepcMils macrochlrus
Shrimp,
C ran yon septemsplnosa
Shrimp,
Crangon septemsplnosa
Clam (soft-she! 1),
Mya arenarla
Chemical
4-n i tro-ni-
cresol
6-nl tro-m-
cresol
2,4-dlnl1ro-6-
methy (phenol *"
2,4-dlnltro-
phenol
2,4-dlnltro-
phenol
2-nl trophenol
3-nltrophenol
4- n I trophenol
2-nl trophenol
2-nl trophenol
2-nl Irophenol
4-nl trophenol
4-nl trophenol
Duration
24 hrs
24 hrs
24 hrs
7 hrs
96 hrs
8 hrs
8 hrs
8 hrs
24 hrs
48 hrs
SA11WAUR
96 hrs
96 hrs
96 hrs
Effect
LC50
LC50
LCt>0
Increased
resplrat Ion
Lethal threshold
value
38} mortality
53> mortality
42* mortality
LC50
LC50
SPECIES
Lethal threshold
value
Lethal threshold
value
Lethal threshold
value
Result
(U9/D
33,000
43,000
6,600
5,520
700
33, 300
24,000
8,000
66,900
46,300-
51,600
32,900
26,400
29,400
Reference
br Ifiijrtiann &
1977
Brlnymanri &
I9//
Urintjmann &
19/7
Kullll,
Kuhn ,
Kuhn,
Lewis 4 Frleden, 1959
Zltko, et al. 1976
Gersdorff, 1939
GersdorH, 1939
Gersdorff, 1939
Lammorlncj & burtwnk,
1960
Ldnmierlny & BurUanK,
I960
McLeese, et al. 1979
McLeese, tit
McLeese, et
al. 1979
al. 1979
B-17
-------
Table 4. (Continued)
Species
Sud urchin (sporni),
Sfroiujy locentrotus
£urj)urdlus
bud urchin (ombryo),
Pseudocdiitrotus dupressus
Chemlcdl
2,4-dinltro-
plionol
2,4-Ulnltro-
phono 1
Ourdtlon tttect
|i Irs Inhibit cosplra-
t UMI, mul flit/
2 lirs AUiornial cluavdtjo
Result
w.ooo
4b,000
Refer enco
,»M*t.,«,
* Toxlclty threshold = the pollutant conctmti at ion ciiusin^ the onsot of col \ mul t Ip 11 ccit ion Inhibition.
"Authors reported results as 4,6-dini tro-o-cresol.
B-I8
-------
REFERENCES
Bernstein, G.S. 1955. Effect of 2,4-dinitrophenol on sea urchin sperm.
Proc. Soc. Exp. Biol. Med. 90: 28.
Bringmann, G. and R. Kuhn. 1977. Befunde der Schadwirkung wassergefahrden-
der Stoffe gegen Oaphnia magna. Z.F. Waser-und Abwasser-Forschung. 5: 161.
Br-fngman, G. and R. Kuhn. 1978. Testing of substances for their toxicity
threshold: Model organisms Microcystis (Diplocytis) aeruginosa and Scenedes-
mus quadricauda. Mitt. Inter. Verein. Limnol. 21: 275.
Qedonde-r, A. and C.F. Van Sumere. 1971. The effects of phenolics and re-
lated compounds on the growth and respiration of Chiore 11 a vulgaris. Z.
Pflanzen. Physiol. 65: 70.
Flickfnger, C.J. 1972. Influence of inhibitions of energy metabolism on
the formation of Golgi bodies in Amebae. Exp. Cell Res. 73: 154.
Gersdorff, W.A. 1939. Effect of the introduction of the nitro group into
the phenol molecule on toxicity to goldfish. Jour. Cell. Comp. Physiol.
14: 61.
Huang, J. and E. Gloyna. 1967. Effects of toxic organics on photosynthetic
reoxygenation. Environ. Health Eng. Res. Lab. PB 216-749.
8-19
-------
•'7"''~3, v.<. I960. ~^e er~fect cf DN? anc \aN-> on '"erti • : zee eaas of tne
^ ~* "
sea j*-cMn with sDecia* reference to the induction cf the abncrma' cleav-
age. Embryologia. 5: 71.
Kcope'-re", -.!_., et a1. 197^. Aoueous chlorination and ozonation studies.
I. Structure -toxicity correlations of phenolic compounds to Daphnia magna.
Oem. 3ioT. Interact. 9: 245.
-ing, M.W. and N.C. Surbank. 1960. The toxicity of phenol, o-chloro-
o^enol and o-nitrophenol to bluegill sunfish. Eng. Bull. Purdue Univ., Eng.
Ext. Serv. 106: 5*1.
Lewis, E.J.C. and E. Frieden. 1959. Biochemistry of amphibian metamorpho-
sis: Effect of triiodothyronine, thyroxin, and dinitrophenol on the respira-
tion of the tadoole. Endocrinology. 65: 273.
Marcus, M. and A.M. Mayer. 1963. Flagellar movement in Chlamydomonas snow-
iae and its inhibition by ATP and dinitrophenol. Iri; Studies on Microalgae
and PHotosynthetic Bacteria. Jap. Soc. Plant Physio!., Univ. Tokyo Press,
Tckyo, Jaoan.
eese, O.W., et al. 1979. Structure-lethality relationships for phenols,
an-j lines, and other aromatic compounds in shrimp and clams. Chemosphere.
q:
ph'oos, G.L., et al. The acute toxicity of phenol and substituted phenols
to the fathead minnow. (Manuscript)
B-20
-------
Rosenthal, H. and R. Stelzer. 1970. Wirkungen von 2,4- und 2,5-dinitro-
phenol auf die Embryonalentwicklung des Herings Clupea harengus. Mar.
B1ol. 5: 325.
Sanders, H.O. and O.B. Cope. 1968. The relative toxicities of several
pesticides to naiads of three species of stoneflies. Limnol. Oceanogr.
13: 112.
Simon, E.W. and G.E. Blackman. 1953. Studies in the principles of phyto-
toxicity. IV. The effects of the degree of nitration on the toxicity of
phenol and other substituted benzenes. Jour. Exp. Bot. 4: 235.
U.S. EPA. 1978. In-depth studies on health and environmental impacts of
selected water pollutants. U.S. Environ. Prot. Agency. Contract No. 68-01-
4646.
Zitko, V., et al. 1976. Toxicity of alkyldinitrophenols to some aauatic
organisms. Bull. Environ. Contam. Toxicol. 16: 508.
3-21
-------
MONONITROPHENOLS
san Toxicology and uuman ^ealth Effects
INTRODUCTION
has three isomeric forms, distinguished by the oosition
of the m'tro- group on the phenolic rinq. Three isomeric fcrms are oossi-
ble, namely 2-nitroohenol, 3-nitrophenol, and 4-nitrophenol . The comoounds
are also commonly referred to as o-m'troohenol, m-nitroohenol, and o-m'tro-
ohenol, respectively.
Commercial synthesis of 2-nitroohenol and 4-nitroohenol is accomplished
tHrouoh the hydrolysis of the appropriate chloronitrobenzene isomers with
aqueous sodium hydroxide at elevated temperatures (Howard, et al. 1976).
Production of 3-nitropheno 1 is achieved through the diazotization and hy-
drolysis of ii-ni troani 1 ine (Matsuguma, 1967). The mononitroohenol isomers
are used in the United States primarily as intermediates for the production
of dyes, pigments, Pharmaceuticals, rubber chemicals, lumber preservatives,
photoaraohic chemicals, and pesticidal and fungicidal agents {U.S. Int.
Trade 7omi. 1976). As a result of this use pattern, the major source for
environmental release of rnononitroonenols is likely to be from production
plants and chemical firms where the comoounds are used as intermediates.
The mononitrophenols may also be inadvertently produced via microbial or
photodearadation of pesticides which contain mononitrophenol moieties.
Approximately 10 to 15 million pounds of 2-nitrophenol are produced annually
(Howard, et al. 1976) for uses including synthesis of o-aminophenol, o-
nitroanisole, and other dye stuffs (Matsuquma, 1967; Howard, et al. 1976).
Although production figures for 3-m'trophenol are not available, Hoecker, et
al. (1977) estimate that production is less than one million pounds annual-
ly. 3-Nitroohenol is used in the manufacture of dye intermediates such as
C-l
-------
anis^dine and Ti-a^inoohenol >Kouris and Nortncott, 1953; ^atsusuma, '.9571.
4-Nitrophenol is Probably the most important of the monoitroohencls in terms
of quantities used and ootential environmental contanination. Demand for
-------
TABLE 1
Properties of Mononitrophenols*
Formula
Molecular Weight
Melting =>oint (°C)
Soiling Point
Density
Water Solubility
(g/D
Vapor Pressure
Ka
2-Nitrophenol
C6H5N03
139.11
44-45
214-216
1.485
0.32 at 38*C
1.08 at 100'C
1 mm Hg at 49.3'C
7.5x10-8
3-Nitroohenol
C6H5N03
139.11
97
279
1.485
1.35 at 25'C
13.3 at 90"C
5.3x10-9
4-Nitrophenol
C6H5N03
139.11
113-114
279
1.479
0.804 at 15'C
1.6 at 25°C
7x10-3
*Sources: Windholz, 1976; Weast, 1975; Matsuguma, 1967.
c-3
-------
CH
2-nitrophenol
OH
MO,
3-nitrophenol
4-nitrophenol
FIGURE 1
MononitrophenoU
C-4
-------
operation around 1930. 2-Nitrophenol was detected at unidentified levels in
2 river water samples and in 4 samples of chemical plant effluent; 3-nitro-
phenol was found in one chemical plant effluent sample (U.S. EPA, 1976).
Systematic monitoring for mononitrophenols in the environment has not been
done. It is reasonable to assume that measureable (although perhaps trans-
ient) levels of the mononitrophenols may be present in localized areas where
organophosphate pesticides are in use.
Little data are available regarding the breakdown of mononitrophenols by
natural communities of microorganisms. Alexander and Lustigman (1966) stud-
ied the degradation of mononitrophenols by a mixed population of soil micro-
organisms. The inoculum was derived from a suspension of Niagara silt loam
soil. Their results indicated that 2-nitrophenol was more resistant to de-
oradation than either 3-nitrophenol or 4-nitrophenol. Utilizing the absorb-
ancy of small soil inoculums to estimate the loss- of mononitrophenol,
3-nitrophenol was found to degrade completely within a 4-day period.
4-Nitrophenol degraded fully within a 16-day period, while 2-nitrophenol re-
sisted degradation over a 64-day period.
Brebion, et al. (1967) examined the ability of microoganisms to attack
4-nitrophenol. The bacteria were derived from soil, water or mud and grown
on a porous mineral bed and were cultivated in a mineral nutrient solution
to which nitrophenols were added as the sole source of carbon. The experi-
mental findings revealed no significant removal of the compound under these
conditions.
In contrast to these reports, a number of investigators have found that
the mononitrophenols are readily and rapidly degraded by acclimated popula-
tions of microorqanisms. Tabak, et al. (1964) studied the ability of ac-
climated cultures derived from garden soil, compost, and river mud to de-
C-5
-------
grade the morionitroonenols. Phenol-adapted bacteria derived from these
sources were found to readily degrade all three rcionitroghenol isomers.
Ninety-five percent degradation (measured spectrophotometrically) occurred
within three to six days. Pitter (1976) reported greater than 95 percent
degradation of the three mononitrophenol isomers in an acclimated sludge
system. The nitrophenols served as the sole source of organic carbon and
degradation was complete within 120 hours.
A recent study (Haller, 1978) reports on the ability of unacclimated
microorganisms to degrade the mononitrophenols. Either sludge obtained from
the primary settling tank of the city of Ithaca, N.Y. wastewater treatment
plant or a Windsor loamy fine sand soil were used as the source of the ino-
culum. 2-Nitro-, 3-nitro-, and 4-nitrophenol (16 mg/1) were completely de-
graded in three to five days by the sludge system. Soil inocula degraded 16
mg of 3-nitrophenol/1 in three to five days while a similar concentration of
2-nitrophenol and 4-nitrophenol required 7 to 14 days for complete degrada-
tion.
Although definitive conclusions cannot be derived from this limited num-
ber of studies, it appears that the mononitrophenols are readily and rapidly
degraded by microbial populations present in the environment.
Ingestion from Food
No data were found demonstrating the presence of mononitrophenols' in
food. One possible source of mononitrophenol exposure for humans is through
the food chain as a result of the ingestion of food crops contaminated with
pesticides containing the nitrophenol moiety. The production of 4-nitro-
phenol by microbial metabolism of parathion is well documented (Munnecke and
Hsieh, 1974, 1976; Siddaramappa, et al. 1973; Sethunathan and Yoshida, 1973;
Katan and lichtenstein, 1977; Sethunathan, 1973). Microbial metabolism of
C-6
-------
*luoridifen (o-nitroonenyl, a,a,a-trifljoro-2-oitro-p-tolyl ether-) results
in the intermediate formation of 4-nitroohenol (Tewfik and Hamdi, 1975).
The major degradation product of fluoridifen following uptake by peanut
seedling roots was 4_nitroohenol (Eastin, 1971). 4-,Nitropheno 1 was also
detected in soyoean roots following absorption of fluoridifen (Rogers,
1971). °hotodecomDO"sition of the herbicide nitrogen (2,4-dichloroohenyl-o-
nitrophenyl ether) in anueous susoensions under sunlight or simulated sun-
light is characterized by rapid cleavage of the ether linkage to form 2,4-
dichloroohenol and 4-nitrophenol (Nakagawa and Crosby, 1974). El-Refai and
Hopkins (1966) have investigated the metabolic fate of parathion following
foliar applicatin or root absorption by bean plants, Phaseolus vulgaris.
Detectable amounts of 4-nitroohenol were found in chloroform rinses of para-
thion-treated leaves after four days.
In another experiment, analysis of nutrient solutions containing para-
thion in which plants were grown for root absorption studies revealed
4-nitroohenol, oaraoxon, and traces of degradation products. Since these
compounds were also detected in control solution which did not contain
olants, the authors concluded that possible photochemical oxidation process-
es had occurred in the aaueous medium. The authors believed that the
4-nitrophenol detected following foliar application of parathion was due to
ohotochemical degradation. 4-Nitrophenol was not detected in bean plants
followinq injection of parathion directly into the stems of bean plants (El-
Refai and Hook ins, 1966).
4-Nitrophenol has also been detected as a photoalteration product of
parathion following application to cotton plants (Joiner and Baetcke, 1973).
Archer (1974) has examined the dissipation of parathion and its metabol-
ites from field spinach. Field plots were sprayed with either 0.5 or one
C-7
-------
oound of active oarathion per acre. Application r-econneidations for
thion are: not less than 14 days before harvest at the -ate of 0.5 pounds
active ingredient oe" acre. Spinach samples were analyzed daily for para-
thion residues and a number of known metabolites including 4-nitrooheno i.
Levels of
-------
TABLE 2
Levels of 4-Nitrophenol Following Application of Parathion to
Field Spinach at Two Different Application Rates*
4-Nitrophenol Residue (wg/kg)a»b
Sample Day
1
2
3
4
5
6
7
8
9
10
11
12
13
14
0.5 Ib. Parathion/Acre
172
88
73
76
73
72
35
40
34
34
31
38
28
33
1.0 Ib. Parathion/Acre
453
305
240
188
136
216
117
18
19
18
18
22
16
19
*Source: Archer, 1974.
^Calculated on a fresh weight basis. Percent moisture from 86.4 to 89.2.
bllnsprayed spinach control samples taken prior to any spray treatments con-
tained 95 ug/kg.
C-9
-------
that the average urine void is 1.4 I/day per 70 kg person, initial exposure
levels can be estimated from residual levels found in urine. For example,
the exposure level leading to the 1.0 yg/1 residue can be calculated as
fo"lows:
Exposure = (10.0 ug nitrpphenol/1) (1.41 of urine/day) = Q.Q2 ug/kg/day
70 kg man
A similar calculation using the maximum urine residue level observed by
Kutz, et al. (1978) (113 ug/1) gives an exposure of 2.26 pg/kg/day.
Knowles, et al. (1975) have demonstrated the production of a wide number
of mononitroohenols including 2-nitrophenol in a model system simulating
gastric digestion of smoked bacon. These studies, utilizing nitrosated
liquid smoke, were conducted under conditions favorable to nitrosation, and
since the temperature, pH, and duration emoloyed approximated those encount-
ered during gastric digestion, their results indicated that nitrosation of
phenols in smoked bacon may occur in the stomach with resultant production
of 2-nitrophenol.
Mononitrophenols may also be formed in vivo via metabolic degradation of
pesticides such as parathion by humans. Excretion of 4-n-;trophenol, a meta-
bolite of the organophosphorous pesticides, parathion, methylparathion,
0-ethyl 0-(p-nitrophenyl) phenylphosphonothioic acid (EPN), and dicapthon is
a good indicator of human exposure to these pesticides (Wolfe, et al. 1970;
Broadway and Shafik, 1973; Elliott, et al. 1960; Roan, et al. 1969). 4-Ni-
trophenol has also been detected as a urinary metabolite of nitrobenzene in
humans (Myslak, et al. 1971).
A bioconcentration factor (BCF) relates the concentration of a chemical
in aauatic animals to the concentration in the water in which they live.
The steady-state 3CFs for a 1ioid-soluble compound in the tissues of various
C-10
-------
snuatic animals seem to be proportional to the percent Hpid in the tis-
sue. Thus the oer capita inqestion of a 1ioid-soluble chemical can be esti-
mated from the oer capita consumption of fish and shellfish, the weighted
average percent lipids of consumed fish and shellfish, and a steady-state
3CF for the chemical.
Data from a recent survey on fish and shellfish consumption in the
United States were analyzed by SR! International (U.S. EPA, 1980). These
data were used to estimate that the per capita consumption of freshwater and
estuarine fish and shellfish in the United States is 6.5 g/day (Stephan,
1930). In addition, these data were used with data on the fat content of
the edible portion of the same species to estimate that the weighted average
percent lipids for consumed freshwater and estuarine fish and shellfish is
3.0 percent.
No measured steady-state bioconcentration factor tBCF) is available for
any nitrophenol, but the eiuation "Log BCF = (0.85 Log P) - 0.70" can be
used (Veith, et al. 1979) to estimate the steady-state BCF for anuatic or-
ganisms that contain about 7.6 percent lipids (Veith, 1980) from the oc-
tanol/water partition coefficient (P). The log P values were obtained from
Hansch and Leo (1979) or were calculated by the method described therein.
The adjustment factor of 3.0/7.6 = 0.395 is used to adjust the estimated BCF
from the 7.6 percent lipids on which the eouation is based to the 3.0 per-
cent lipids that is the weighted average for consumed fish and shellfish in
order to obtain the weighted average bioconcentration factor for the edible
portion of all freshwater and estuarine anuatic organisms consumed by Ameri-
cans.
Chemical
2-nitrophenol
4-nitrophenol
C-ll
loa P
1.73
1.91
Estimated
Steady-State
BCF
5.89
8.38
Weighted
Averaae
BCF
2.33
3.31
-------
Inhalation
Quantitative data were not found regarding the presence of rnononitro-
phenols in air. Lao, et al. (1973) discussed the application of a gas chro-
matograph quadrapole Tiass soectrometer-data processor combination for rou-
tine analysis of air pollutants. During a sample run of urban ambient
particulate matter (location not designated) these investigators identified
the presence of 4-nitrophenol as well as a large number of other air pollu-
tants. Quantitative data were not provided, however. Ambient air levels of
4-nitrophenol in a Boeing plant where the compound was used for the preser-
vation of the cork surfaces of the Minuteman Missile were equal to or less
than 0.05 mg/m of air (Butler and Bodner, 1973).
4-Nitrophenol may be produced in the atmosphere through the photochemic-
al reaction between benzene and nitrogen monoxide. Nojima, et al. (1975)
irradiated a combination of benzene vapor and nitrogen monoxide gas for five
hours with a xenon lamp and characterized the resulting photochemical prod-
ucts. The production of nitrobenzene, 2-nitrophenol, 4-nitrophenol, 2,4-
dinitrophenol and 2,6-dinHrophenol was described by the authors. Identity
of the compounds was confirmed using thin layer chromatography, gas chromat-
ography, gas chromatography-mass spectrometry, and infrared spectrometry.
The authors suggested that these nitro-compounds may be the cause of the
characteristic symptoms of seriously stricken victims of photochemical smog
in Japan including: headache, breathing difficulties, vomiting, rise in body
temperature, and numbness in the estremeties.
In a second paper (Nojima, et al. 1976), the photochemical reaction of
toluene with nitrogen monoxide was investigated. It was felt that the prod-
ucts of photochemical reaction of toluene with nitrogen monoxide might be
C-12
-------
more important >n the production of photochemical smog since the concentra-
tion of toluene in urban air is higher than that of benzene. Compounds pro-
duced as a result of this reaction included o-cresol, m-nitrotoluene, 4-ni-
troohenol, 2-methyl-5-nitrophenol, 3-methyl-4-nitrophenol , 2-methyl-4-ni-
troohenol, and 2-methy1-4-nitrooheno1. These compounds were identified by
gas chromatography-mass spectrometry. In another experiment, the investiga-
tors examined the organic compounds present in rain. An analysis of rain-
water yielded 4-nitrophenol, 2-methyl-6-nitrophenol and 2-methyl-4-nitro-
phenol. The authors suggested that the nitrophenols produced by the photo-
chemical reactions, described above, dissolve in rain. It seems likely that
in areas where severe photochemical smog exists, humans may be exposed to
substantial levels of mononitrophenols. However, it is impossible to esti-
mate the levels at which humans are exposed to these compounds via inhala-
tion, based on available data.
Dermal
Roberts, et al. (1977) used human autopsy epidermal membranes in an _m
vitro system to determine the permeability of human skin to various com-
pounds. Both 3-nitrophenol and 4-nitrophenol were shown to permeate the
skin and to produce damage at threshold concentrations of 0.8 and 0.9 per-
cent (w/v), repsectively. According to Patty (1963)i 2-nitrophenol may be
absorbed through the intact skin. No information on possible human dermal
exposure to the mononitrophenols was found.
PHARMACOKINETICS
Absorption and Distribution
Data specific to the absorption and tissue distribution of the monoitro-
phenols were not available. It is reasonable to assume, based on the rapid
urinary elimination of the mononitrophenols, that the compounds may be re-
stricted primarily to the blood and urine following absorption by humans.
C-13
-------
Metabol ^sm
Metabolism of the rnononitrophenols prcoably occurs via one of three
•nechanisms in humans. The major route of mononitrophenol metabolism is un-
Jouotealy conjugation and the resultant formation of either glucuronide or
sulfate conjugates. Other possible routes of metabolism include reduction
of amino-compounds or oxidation to dihydric-nitrophenols.
Sulfate and glucuronide conjugative processes are two of the major de-
toxification mechanisms in many species, including mammals (Quebbemann and
Anders, 1973). In recent years, 4-nitrophenoi has been used as a preferred
substrate for biochemical analysis of the giucuronidation reaction in a wide
number of species (Aitio, 1973; Sanchez and Tephly, 1974; Ranklin, 1974;
Heenan and Smith, 1974; Yang and Wilkinson, 1971). This use reflects the
simple techniques available for quantitating the disappearance of 4-nitro-
phenol and the synthesis of the glucuronide conjugate. The relevance of
many of these in vitro studies towards an assessment of the metabolic fate
of tne mononitropnenols in humans is questionable; thus only those _j_n vivo
studies with direct relevance to the metabolic fate of mononitrophenols in
humans or experimental animals are discussed here.
It has been known for some time that levels of the mixed function oxi-
dases and the enzymes responsible for conjugation of many compounds are gen-
erally highest in the mammalian liver. Litterst, et al. (1975) assayed
liver, lung, and kidney tissue from the rat, mouse, rabbit, hamster, and
guinea pig for standard microsomal and soluble fraction enzymes involved in
drug biotransformation. These studies included an analysis of glucuronide
conjugation of 4-nitrophenol by these tissues-. For all species, liver was
the most active organ. Kidney and lung activities were usually 15 to 40
C-14
-------
percent of that found in liver, with kidney slightly Tiore active than lung.
UDP-glucuronyl-transferase activity toward the acceotor, 4-nitrophenol, was
higher in hamsters and rabbits than in other species.
Conjugation activity need not be constant even within the same species.
Pulkkinen (1966b) noted that sulfate conjugation of 4-nitrophenol is de-
creased during pregnancy in rabbits. The author suggested that large
amounts of estrogens may cause more protein binding, thus inhibiting the re-
action. In another study (Pulkkinen, 1966a) it was noted that conjugation
capacity increases with age in the rat, guinea pig, and man. The human
fetus does not have a very high capacity to form sulfate or glucuronide con-
jugates of mononitrophenols or other compounds. In addition, Moldeus, et
al. (1976) noted that the relative rate of glucuronide versus sulfate conju-
gation of 4-nitrophenol may depend on the levels of substrate present. In
in vitro tests utilizing isolated rat liver cells, ttre investigators noted
that at 4-nitrophenol concentrations of 25 uM, the rate of glucuronide con-
jugation was low and over 75 percent of the conjugation products were found
to be sulfates. The glucuronidation increased more rapidly than did the
sulfate conjugation with increasing substrate conjugation. At 250 uM, con-
jugation of 4-nitrophenol with sulfate was inhibited almost completely and
more than 95 percent of the conjugates formed were found to be glucuronides.
Robinson, et al. (1951) studied the metabolic detoxification of the
mononitrophenol isomers in rabbits. They showed that, with doses of 0.2 to
0.3 g/kg, conjugation _in_ vitro with glucuronic and sulfuric acids was almost
complete. Only small amounts (less than one percent) of the unchanged
nitrophenol were excreted. With all three of the mononitrophenol isomers,
the major conjugation product was nitrophenyl-glucuronide, which accounted
for about 70 percent of the dose. The corresponding sulfate conjugates were
C-15
-------
also excreted. Reduction of the nitroohenols occurre: to a small extent,
the reduction of the 4- isomer being slightly greater than that of the 2-
and 3- isomers. The mononitrophenols were also shown to undergo oxidation
to a very small extent (less than one percent). 2-NitroDhenol yields traces
of nitroouinol; 3-nitroohenol yields nitroouinol and 4-nitrocatechol; and
4-nitrophenol yields 4-nitrocatechol.
A summary of the metabolism of the mononitrophenols is shown in Table
3. Data directly addressing the metabolic fate of the mononitrophenols in
humans are not available. However, it is expected that following exposure
to the mononitrophenols humans will rapidly excrete both glucuronide and
sulfate conjugates in the urine.
Excretion
Data directly addressing the excretion of the mononitrophenols following
exposure of humans were not found in the literature. However, excretion
patterns for 4-nitrophenol following human exposure to parathion may shed
some liaht on their elimination kinetics. Arterberry, et al. (1961) studied
the pharmacodynamics of 4-nitrophenol excretion following exposure to para-
thion. They noted that the excretion of 4-nitrophenol in the urine was
nuite rapid "as might be expected in the case of a water-soluble metabolite
of a substance which is luickly broken down by th-e animal organism."
4-Nitrophenol usually had disappeared from the urine within about 48 hours
after cessation of exposure. In a similar study of orchard spraymen in-
volved in the application of parathion, Wolfe, et al. (1970) noted that
urinary levels of 4-nitrophenol rose promptly in response to parathion and
returned to the nondetectable level after several days. Myslak, et al.
(1971) reported on the excretion of 4-nitrophenol from a 19-year-old female
subject following a suicidal oral dose of nitrobenzene. Large quantities of
C-16
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TABLE 3
Urinary Metabolites of Mononitrophenols in Rabbits*
Percentage of Dose Excreted as
Nitrophenol
2-Nitrophenol
3-Nitrophenol
4-Nitrophenol
Nitro
Compounds
(N)
82
74
87
Amino
Compounds
(A) (N + A)l
3 85
10 84
14 101
Glucuronides
(G)
71
78
65
Ethereal
Sulfates
(E)
11
19
16
(G + E)
82
98
81
*Source: Robinson, et al. 1951.
1(N + A) should be roughly equal to (G + E) since the amounts of free phenols excreted were
very small. Both glucuronides and ethereal sulphates include nitro and amino conjugates.
C-17
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4-nitrooherol and 4-aminoohenol were detected in the urine. Elimination of
d-nitroonenol in the urine was exoressed by the aquation V /y
i.' 0
—0 '"'OP*1
e~ "" " where V and 7 denote the excretion rate at the interval
time 0 and t measured in hours. THe half-life for excretion corresoonded to
about 34 hours.
Shafik, et al. (1973) studied the urinary excretion of 4-nitroohenol
following administration of the pesticide EPN. Following oral administra-
tion of the pesticide for three days, animals were maintained and urine sam-
oles collected at 24-hour intervals. Three days were required 'or complete
excretion of 4-nitropnenol under these conditions. The foregoing studies
indicate that 4-nitroohenol is rapidly excreted following its production _i_n_
vivo *rom other organic compounds.
Only one study was found that examined excretion of 4-nitrophenol fol-
lowing direct administration of the compound. Lawford, et al. (1954) stud-
ied the elimination of various nitrophenolic compounds from the blood of ex-
oeriTiental animals. Elimination of 4-nitrophenol by the monkey following
oral and intraoeritoneal doses of 20 mg/kg body weight was complete within
five hours. Elimination by mice, rats, rabbits, and guinea pigs was also
rapid. Vost doses were eliminated completely from the blood within two
hours of administration. Experimental animals eliminated 4-nitrophenol from
the blood in the following descending order of efficiency: mouse, rabbit,
guinea pig, rat, and monkey.
In summary, the available data indicate that the mononitrophenols are
excreted rapidly via the urinary route and that total elimination time is
likely not to exceed one week. The mononitrophenols are highly water solu-
ble and accumulation or bioconcentration in various tissues is not expected
to occur to a larae extent. However, much more data are needed to precisely
define the transport distribution and elimination of these compounds in
humans.
C-13
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EFFEC~S
Threshold concentrations for odor, taste, and color for 2-nitro-,
3-m'tro-, and 4-nitrophenols in reservoir water have been reported in an ab-
stract of a oaoer from the Russian literature (Makhinya, 1964). Reported
threshold concentrations for 2-nitroohenol were 3.83 mg/1 for odor, 3.6 mg/1
for taste, and 0.6 mg/1 for color. Concentrations for 4-nitroohenol were
53.3, 43.4, and 0.24 mg/1 for odor, taste, and color, respectively. The
values for 3-nitrophenol were given as 339, 164.5, and 26.3 mg/1. Accepta-
bility thresholds from the standpoint of human consumption were not reported
by these investigators.
Acute, Subacute, and Chronic Toxicity
Known effects of 4_nitrophenol demonstrated in animal experiments are
methemoglobinemia, shortness of breath, and initial stimulation followed by
proaressive deoression (von Oattinqen, 1949).
Acute toxicity information for the mononitrophenol isomers has been corn-
oiled and presented as Table 4. 4-Nitrophenol is the most toxic of the
mononitrophenols followed by 3-nitroohenol and 2-nitroohenol in relative
toxicity. Toxicologic symptoms of mononitrophenol poisoning have not been
well described in the literautre. Sax (1968) noted that 2-nitrophenol expo-
sure produced kidney and liver injury in experimental animals. Methemoglob-
in formation as a result of mononitrophenol exposure has also been reported
(Patty, 1963). Grant (1959), however, was unable to detect methemoglobin
formation after oral administration of 3-nitro- and 4-nitroohenols to rats.
Small inconstant amounts of methemoglobin were formed after 3-nitrophenol
administration. Smith, et al. (1967) were able to show that the reduction
products of mononitrophenols, 2- and 4_aminophenol, would produce methemo-
globin in female mice. Methemoglobin formation, therefore, may depend on
C-19
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TABLE A
Acute Toxic ity of Mononitrophenol Isomers
Species
Dose
(mg/kg)
Route of
Administration Effects
References
Frog
Mouse
Rabbit
Cat
Doq
Rat
Mouse
Guinea Pig
Doq
Rat
Mouse
Frog
Rabbit
Cat
Dog
Rat
Mouse
Rat
300
600
1700
600
100
2830
1300
900
83
930
1410
50
600
197
10
620
470
350
2-Nitrophenol
s.c
i.m.
s.c.
s.c.
i .v.
Oral
Oral
s.c.
3-Nitrophenol
i.v.
Oral
Oral
4-Nitrophenol
s.c.
s.c.
s.c.
i.v.
Oral
Oral
Oral
Lethal Dose
Lethal Dose
Lethal Dose
Lethal Dose
Lethal Dose
LD50
L050
Lethal Dose
Minimum Lethal Dose
L050
LD50
Minimum Lethal Dose
Minimum Lethal Dose
Minimum Lethal Dose
Lethal Dose
LD50
LH50
Spector, 1956
Spector, 1956
Spector, 1956
Spector, 1956
Spector, 1956
Vernot, et al. 1977
Vernot, et al. 1977
Spector, 1956
Spector, 1956
Vernot, et al. 1977
Vernot, et al. 1977
Spector, 1956
Spector, 1956
Spector, 1956
Spector, 1956
Vernot, et al. 1977
Vernot, et al. 1977
Fairchild, 1977
:-20
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the capacity of the organism to reduce the morionitroohenoIs. As mentioned
in the metabolism section of this document, reduction of the nitroohenols
does not normally occur to any large extent.
Oqino and Yasukura (1957) reoorted the development of cataracts in vita-
min C-deficient guinea Digs following administration of 4-m'troohenol.
Cataracts developed in two of three guinea Digs on days 7 and 11 following
daily intraperitoneal adminstration of 8.3 to 12.5 mg 4-nitroohenol/kg body
weight. Subchronic administration of 4-nitrophenol over a 20-day test
period produced cataracts while 2- and 3-nitroohenols did not. The authors
concluded that the oara-oositioning of the hydroxyl- and nitro- grouos is
necessary for cataract induction.
Several deficiencies in this study preclude definitive conclusions on
the cataractogenic properties of 4-nitrophenol. The investigators failed to
report results on control animals, either totally untneated or treated with
the nitrophenols and a vitamin C supplement. Thus, it is oossible, based on
the results reported, to conclude that vitamin C deficiency itself caused
cataracts in some of the animals tested. The small size of the experimental
grouos (three animals/test compound) also make definitive conclusions diffi-
cult. The reported conclusions must be taken with considerable caution
based on the above considerations.
In contrast, Dietrich and Beutner (1946) found 2-nitro- and 4-nitro-
phenol s to be devoid of cataract-forming activity in seven-day-old chicks.
Animals were fed on a commercial brand of chick food containing 0.25 percent
nitrophenol. Although cataracts developed rapidly (within 24 to 48 hours)
when the animals were fed 2,4-dinitrophenol, no cataracts developed within a
three-week period when animals were fed the mononitrophenol isomers. The
capacity for cataract formation in humans following mononitrophenol exposure
is unclear.
:-2i
-------
Both 2-nifo- and 4-nitroohenol have been shown to inhibit oorcine
malate denydrogenase in vit^o (Wedding, et al. 1967). The compounds acted
as comoetitive inhibitors for NAD in the forward direction of the enzynatic
reaction. The clinical significance of these findings is unknown.
The ventilatory effects of the mononitrophenols have been examined in
anesthetized rats (Grant, 1959). Test comcounds were administered by stom-
ach tube: 2-nitroohenol, 60 to 120 mg; 3-nitrophenol, 20 to 45 mg; 4-nitro-
phenol, 7 to 12 mg. Significant increases in respiratory volume ranging
from 15 to 30 percent were reported in these experiments.
Neither carbon dioxide output nor oxygen uptake were affected by •sub-
lethal doses of 2-nitrophenol in rats (Cameron, 1953). In contrast, oxygen
uptake was decreased in 3-nitrophenol-treated rats while carbon dioxide out-
put was increased following 4-nitroPhenol administration. Rectal tempera-
ture was depressed in rats receivina any of three isomers. These results
suggest that mononitrophenol isomers are not potent uncouplers of oxidative
ohosohorylation, in contrast to the chemically similar compound 2,4-dini-
trophenol.
Although the mechanism of toxic action of the mononitrophenols is not
well understood, the following studies suggest that an action directly on
cell membranes may occur. 3-Nitroohenol binds readily to red blood cell
(R8C) membranes. Expansion of RBC ghosts occurs following nitrophenoi
treatments, as measured by the resistance of such ghosts to hemolysis (Macn-
leidt, et al. 1972). 2-Nitrophenol and 4-nitroohenol inhibit chloride
transport in red blood cells (a metabolism-independent process) suggestion a
direct action on the cell membrane (Motais, et al. 1979). Further informa-
tion on the acute or chronic toxicity of the mononitrophenols to humans w
not found.
C-22
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The National Institute for Occupational Safety and Health (NIOSH),
recently undertook a health hazard evaluation determination at the request
of an erioloyee of the Boeina Company who had routinely handled 4-nitrophenol
(Sutler and Sodner, 1973). A 15 percent solution of 4-nitrophenol and
methylphenol was painted on the exposed cork surfaces of the Minuteman Mis-
sile before arrival at the assembly plant. If the surface was damaged in
transit it was necessary to apply small amounts of the 4-nitrophenol solu-
tion to the repaired areas of cork. The worker in auestion was engaged in
such touch-up operations. Workers routinely wore an organic vapor cartridge
respirator, a face shield, cotton gloves, rubber gloves, and were completely
covered with protective clothing. The employee complained of fatigue, joint
pain, abdominal cramos and diarrhea, and attributed these symptoms to his
exposure to both the treating solution and the dried cork impregnated with
4-nitrophenol durina his work as a mechanic. Medical~examination failed to
detect 4-nitrophenol in the urine but revealed a complete absence of the im-
munoalobins IqA and IqQ in the employee. Based on medical judgement and the
existing literature, the study concluded that the employee's condition stem-
med from the lack of IgA and IgD and that this deficiency was not caused by
exposure to 4-nitrophenol.
Gabor, et al. (1960) reported a uniaue effect of 2-nitrophenol on blood
platelet levels. When 31 rats were administered 2-nitrophenol by intraperi-
toneal injection at 1 mg/kg body weight, the platelet count increased sig-
nificantly. Even at doses of 0.1 mg/kg a similar effect was produced. Ad-
ministration of 3-nitro or 4-nitrophenol did not produce a rise in platelet
levels. Additional data are not available to explain this unioue pheno-
menon, nor is the clinical significance of these findings known.
C-23
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A reoort from the Russian literature (Makhinya, 1969) reports that
2-nitro-, 3-m'tro-, and 4-nitroohenols possess distinct cumulative proper-
ties. Chronic administration of any of the mononitroohenols to mammals
caused alterations of neurohumoral regulation and patholoaical changes in-
cluding colitis, enteritis, hepatitis, gastritis, hyperplasia of the spleen,
and neuritis. Limiting doses for the disruption of conditioned reflex ac-
tivity were establised as 0.003 mg/kg (equivalent to 0.006 mg/1 of water)
for 2-nitroohenol and 3-nitroohenol and 0.00125 mg/kg (eouivalent to 0.0025
•ng/1 of water) for 4-nitrophenol. Unfortunately a report of this study was
available in abstract form only. Details of the experiment including animal
species, mode of administration, duration of the treatment, and a complete
description of the observed biological effects, were not reported. The re-
sults must be considered Questionable until evaluation of the experimental
protocol is possible.
Synergism and/or Antagonism
Only one report was found dealing with possible synergistic effects of
the mononitroohenols. Cairns, et al. (1976) studied the effects of a sub-
lethal exposure to zinc and subseouent toxicity of 4-nitrophenol to snails,
Goniobasis livescens. Snails were exposed for 96 hours to two sublethal
concentrations of zinc (1.54 mg/1 and 3.08 mg/1 corresponding to 0.2 and 04
of the 48-hour LC50 dose, respectively) followed by an acutely lethal dose
of 4-nitrophenol (1,000 mq/1). A significantly reduced survival time fol-
lowing exposure to 4-nitrophenol was reported. In a second experiment,
snails were exposed to sublethal levels of 4-nitrophenol (13.2 mg/1) and
subjected to a lethal temperature shock 96 hours later. A significant de-
crease in the median survival time of the snails during the temperature
shock was noted. The applicability of these data to humans or mammals is
unknown. Data regarding synergistic or antagonistic effects of the the
•nononitrophenols in mammals were not found.
C-24
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Teratocenici -•/
Pertinent information could not be located in the available literature
regarding teratogenic properties of the mononitrophenols.
Mutaoem'c'ity
Szybolski (1953) tested the three mononitrophenol isomers for their
ability to induce streptomycin-independence in streptomycin-independent E_.
co 1 i. All three isomers gave negative results.
Buselmaier, et al. (1976) tested 4-nitrophenol for mutagenic activity in
mice with the host mediated assay and the dominant lethal method, using Sal-
monella typhimurium G46 His", Serratia marcescens a21 leu~, and Serratia
marcescens a31 His", as indicator organisms. Spot tests j_n vitro were al-
so performed. Mutagenic activity was not demonstrated.
4-Nitropnenol also failed to induce mutations in Salmonel1 a both with
and without microsomal activation (McCann, et al. 19751^
Fahrig (1974) demonstrated a weak mutagenic activity when 4-nitroonenol
was tested for mitotic gene conversion in Saccharomyces cerevisiae. This
test system allows the detection of a genetic alteration whose molecular
mechanism is presumably based on the formation of single-strand breaks of
DNA.
Adler, et al. (1976) used the difference in growth inhibition of wild
type Proteus mirabilis and the corresponding repair-deficient strain as an
indication of DNA damage. 4-Nitrophenol showed some evidence of DNA damage
in this system.
Effects on mitosis and chromosome fragmentation have been reported in
plants. Sharma and Ghosh (1965) examined the mitotic effects of the mononi-
troohenol isomers in root tips of A11i urn cepa. Inhibition of mitosis in
root tips was reported for all three mononitrophenol isomers but only
4-nitrophenol induced detectable chromosome fragmentations. Amer and Ali
C-25
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^Ia69j studied the ejects of 2- and i-ni trooheno 1 s on the "ate-'a1 '•oot
mitoses of V i c • a f aba seedlirgs. The -nitotic Index was reduced at con-
centrations of these compounds ranging from 0.025 oercent to O.I percent.
Induction of anaohase bridges by both isomers was noted but (in agreement
with the work of Levin and Tjio (19*3) with A11ium ceoa) chromosome fragmen-
tation was not detected. The relationship of these changes in plants to al-
terations in mammalian cells has not been established. Based on the avai"-
able data, the mononitrophenols do not appear to pose a mutagenic hazard to
humans.
Carcinogenicity
Data on the possible carcinogencity of the mononitrophenols are scant in
the literature. Soutwell and Bosch (1959) have studied the ability of a
number of phenolic compounds to promote tumor formation on mouse skin fol-
lowing a single initiating dose of dimethylbenzanthracene. Although phenol
itself has demonstrated a promoting capacity in this system, both 2- a^c
4-nitroohenols failed to promote tumor development in mice. No othe»- data
on possible carcinogenic potential of the mononitrooherols were found.
4-Nitrophenol has been selected by the National Cancer Instritue (NCI)
for testing under the Carcinogenesis Bioassay Program.
C-26
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OINITSOPHENOLS
Mammalian Toxicology ana Human Health Effects
INTRODUCTION
Six isomeric forms of dinitrophenol are possible, distinguished by the
position of the nitro- groups on the phenolic ring. Of the six possible
dinitrophenol isomers, 2,4-ainitrophenol is by far the most important. The
most recent production figure for 2,4-dinitrophenol is 363,000 Ibs. reported
by the U.S. International Trade Commission (1968). Approximate consumption
per year is estimated at 1,000,000 Ibs. (Howard, et al. 1976). 2,4-Dinitro-
phenol is used primarily as a chemical intermediate for the production of
sulfur dyes, azo dyes, photochemicals, pest control agents, wood preserva-
tives, and explosives (Matsuguma, 1967; Perkins, 1919; Springer, et al .
1977a,b).
Production figures and usage data for the remairnng five dinitrophenol
isomers are not available. It is reasonable to assume that production ana
usage of these compounds are extremely limited in the United States.
Commerical synthesis of 2,4-dinitrophenol is accomplised by the hydroly-
sis of 2,4-dinitro-l-chlorobenzene with sodium hydroxide at 95 to 100°C
(Matsuguma, 1967). As a result of the use pattern of 2,4-dinitrophenol
(2,4-DNP) the major source for environmental release of 2,4-DNP is likely to
be from production plants and chemical firms where the compound is used as
an intermediate. It is possible that 2,4-DNP may also be produced via
microbial or photodegradation of compounds which contain the nitropnenol
moiety, such as parathion (Gomaa and Faust, 1972). 2,4-DNP has also been
identified as a impurity in technical preparations of the herbicide DN?
(2-isooropyl-4,6-dinitrophenol) by Mosinska and Kotarski (1972).
The physical and chemical properties of the dinitrophenol isomers are
summarized in Table 5 and Figure 2.
C-27
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TABLE 5
Properties of Dinitrophenol Isomers*
Isomer
2,3-Oinitrophenol
2,4-Dinitrophenol
2,5-Oinitrophenol
2,6-Oinitrophenol
3,4-Dinitrophenol
3,5-Oinitrophenol
m.p.
CO
144
114-115
(sublimes)
104
63.5
134
122-123
K
(at 25*
1.3 x
1.0 x
7 x
2.7 x
4.3 x
2.1 x
C)
10-5
10-4
10-6
10-4
10-5
10-4
Water
Solubility
(9/1)
2.2
0.79
0.68
0.42
2.3
1.6
Density
1.681
1.683
1.672
1.702
*Source: Harvey, 1959; Windholz, 1976; Weast, 1975.
C-28
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MO,
OH
NO,
2,3-dinitrophenol
2,4'-dinitrophenol
2,5-dinitrophenol
OH
OH
2,6-dinitrophenol
3,4-dinitrophenol
3,5-dinitrophenol
FIGURE 2
Oinitrophenols
C-29
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EXPOSURE
Ingestion from Water
No data were available regarding human exposure via ingestion of dini-
troohenols from water.
The enhancement of biological waste water treatment by 2,4-ONP has been
examined (Shah, 1975; Shah, et al. 1975). Addition of 0.92 mg/1 2,4-ONP to
waste water systems results in an increase of 85 percent in waste degrading
rate and a decrease of 70 percent in cell growth. Shah, et al. (1975) note
that the optimum concentration for 2,4-DNP in this system (0.92 mg/'l) is un-
dersirably high from the standpoint of current Federal effluent regulations
but that the compound is completely eliminated by adsorption on activated
carbon which Generally follows biological treatment of waste waters. It is
not known whether this treatment method is currently used in the United
States. Theoretically, such usage might result in 2,4-ONP contamination of
surface waters.
Games and Hites (1977) detected dinitrophenol (isomer not identified) in
the effluent waters of a dye manufacturing plant. Dinitrophenol at 300 to
400 ug/1 was detected in raw waste water, prior to biological treatment.
The final plant effluent contained dinitrophenol at 42 to 270 ug/1. Mud and
river water samples downstream from the effluent point were analyzed by gas
chromatoqraphy/mass spectrometry. Dinitrophenol was not detected in these
samples.
The persistence of dinitroohenol isomers in ambient waters has not been
well studied. A number of investigators have studied the bacterial degrada-
tion of the dinitrophenols utilizing acclimated populations of microorgan-
isms. Phenol-adapted bacteria obtained from garden soil, compost, and river
C-30
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mud degraded 2,--dinitropreno1 in seven to ten days (Tabak, et al. 196^;.
2,5-DinitroDhenol was degraded very slowly in this system. 2,4-, 2,5-, and
2,6-Dinitrophenols were tested for biological degradability by an activated
sludge culture obtained from a sewage treatment plant (Fitter, 1976). 2,5-
Dinitro- and 2,5-dinitrophenols were not degraded in this system although 35
percent removal of 2,4-dinitrophenol was achieved within 20 days. Further
degradation of 2,4-dinitrophenol did not occur in this system, however.
Bacteria isolated from parathion-treated flooded soil (Sudhakar-Barik, et
al. 1976) degraded 2,4-dinitrophenol after an exceptionally long lag
period. Nitrite was produced only in trace amounts after 25 days. Even
after 50 days, only eight percent nitrogen was accounted for as nitrite.
The available data indicate that dinitrophenols are susceptible to par-
tial degradation by certain microorganisms. Of the dinitrophenol isomers,
2,4-ONP appears to be most easily degraded. It may be speculated that dini-
trophenols will be subject to microbial attack in environmental situations
where acclimated mirobiological populations exist (e.g., sewage treatment
plantsK The persistence of dinitrophenols in the environment where accli-
mated microbial populations do not exist is speculative.
Ingestion from Food
Pertinent data could not be located in the available literature regarding
exposure to dinitrophenols via ingestion of food.
No measured steady-state bioconcentration factor (BCF) is available for
any nitrophenols, but the equation "Log BCF » (0.85 Log P) - 0.70" can be
used (Veith, et al. 1979) to estimate the steady-state BCF for aquatic or-
ganisms that contain about 7.6 percent lipids (Veith, 1980) from the oc-
tanol/water partition coefficient (P). The log P values were obtained from
Hansch and Leo (1979) or were calculated by the method described therein.
C-31
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The adjustment factor of 3.0/7.5 = 0.295 is jsea to adjust tne est;~ateJ 3CF
from the 7.6 percent lipids on which the equation is basea to the 3.0 oer-
cent lipids that is the weighted average for consumea fish and shellfisn in
order to obtain the weighted average bioconcentration factor for the edible
portion of all freshwater and estuarine aquatic organisms consumed oy Ameri-
cans.
Inhalation
Dinitrophenol isomers may be produced in the atmosphere through a photo-
chemical reaction between benzene and nitrogen monoxide. Nojima, et al.
(1975) irradiated a combination of benzene vapor and nitrogen monoxide for
five hours with a xenon lamp and characterized the following resulting
photochemical products: nitrobenzene, 2-nitrophenol, 4-nitrophenol, 2,4-
dinitrophenol, and 2,6-dinitrophenol. The authors suggested that these
nitro- compounds may be the cause of the characteristic symptoms of serious-
ly stricken victims of photochemical smog in Japan, which include headache,
breathing difficulties, vomiting, rise in body temperature and numoness in
the extremities. In the absence of monitoring data it is impossible to
estimate the extent of human exposure to dinitrophenols as a result of their
photochemical production in the atmosphere.
Dermal
2,4-ONP is rapidly absorbed through the intact skin (Gleason, et al.
1969). Although no direct information on the other dinitrophenol isomers is
available, it is reasonable to suppose that dermal absorption will readily
occur with these compounds as well. Since 2,4-ONP is used primarily as a
chemical intermediate, dermal exposure is expected to occur most often in an
inductrial setting. 2,4-ONP is also used occasionally as a spray against
aphids and mites, as a fungicide for certain molds and mildews, as a weea
C-32
-------
killer, and as an ingredient in some wood preservative formulations (Glea-
son, et al. 1969). Dermal exposure to humans may occur among individuals
handling 2,4-DNP in these aoolications. Data on the importance of the der-
•nal exposure route of dinitrophenols in humans are not available.
PHARMACOKINETICS
Absorption
Absorption of dinitroohenol (isomer unspecified) through the skin and
following inhalation occurs, readily (von Oettingen, 1949).
Gehring and Buerge (1969b) reported that 2,4-ONP is absorbed very rapid-
ly by ducklings and rabbits followina intraperitoneal adminstration. In
fact, imnature rabbits absorbed the administered DNP so rapidly that an ab-
sorption constant could not be calculated from the data. ONP concentration
is serum peaked within five minutes of administration.
Other Quantitative information on the rate of absorption of the dinitro-
ohenol isomers was not found.
Distribution
Blood levels of the dinitrophenols rise rapidly following absorption
(Gehring and Buerge, 1969b; Harvey, 1959) suggesting that the dinitrophenol
isomers are transported by the blood regardless of the mode of absorption.
2,4-ONP binds to serum proteins following intraoeritoneal administration to
rabbits and ducklings. Early after the administration of 2,4-ONP, the con-
centration of free DNP in serum is much greater than the bound form, and at
later times the reverse is true (Gehring and Buerge, 1969b).
Based on the available data, the dinitrophenol isomers do not appear to
be stored to any significant extent in the tissues of human or experimental
animals followino absorption. Gisclard and Woodward (1946) unsuccessfully
attempted to extract 2,4-dinitrophenol or its metabolites from the tissues
of two human victims of fatal intoxication.
C-33
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It seems likely, based on the short half-lives of these compounds in
mammals, that the large majority of any dose will be rapidly excreted via
the urine. On the other hand, von Oettingen (1949) reported both dinitro-
phenol (unspecified isomer) and aminonitrophenol in the liver, kidney,
brain, blood, and spinal fluid of dogs after fatal doses of dinitrophenol.
Recent work on the tissue distribution of the dinitrophenols following ab-
sorption in mammals was not found.
Metabolism
In a study of the munitions industry in France (Perkins, 1919) it was
reported that the urine of men fatally poisoned by 2,4-DNP contained:
2-amino-4-nitrophenol, 4-amino-2-nitrophenol, diamino-phenol, and a number
of nitrogen compounds resulting from a combination of two molecules of ami-
nonitrophenol or of diaminophenol. It has frequently been reported that
2-amino-4-nitrophenol invariably exists in the urine of persons suffering
from serious intoxication by 2,4-DNP. Williams (1959) stated that 2,4-DNP
is excreted in mammals in the following forms: partially unchanged; partial-
ly conjugated with glucuronic acid; reduced to 2-amino-4-nitrophenol,
2-nitro-4-aminophenol and probably 2,4-diaminophenol. Rats orally dosed
with 2,4-DNP at 1.5 to 12 rag/kg excreted both free dinitrophenol (78 per-
cent) and 2-amino-4-nitrophenol (17 percent) (Senszuk, et al. 1971).
Although the jji vitro metabolism of 2,4-dinitrophenol has not been ex-
tensively studied in mammalian systems, Parker (1952) examined the enzymatic
reduction of 2,4-DNP by rat liver homogenates and found 4-amino-2-nitro-
phenol to be the major metabolite. The metabolite 2-amino-4-nitrophenol
comprised less than 10 percent of the total metabolites formed; 2,4-diami no-
phenol was found in trace amounts. Presumably the latter metabolite was
formed from the reduction of the remaining nitro- group of one of the two
aoove compounds.
C-34
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In contrast, Eiseman, et a!. (1974) reported that 2-amino-i-m'troonenoi
was the major metabolite (75 percent of total ami no). In the latter study
4-amino-2-nitrophenol was formed in considerably smaller amounts (23 per-
cent) when ?,4-QNP was enzymatically reduced in vitro by rat liver homoaen-
ates. These investigators also detected only traces of diaminoohenol indi-
catinq that it nay be a secondary reduction product as suagested by Barker
(1952). A precise definition of the metabolic fate of the dinitroohenoIs in
humans awaits further investigation.
Excret ion
Data on the elimination kinetics of the dinitrophenols or their metabol-
ic products in humans were not found. Edsall (1934) stated: "Judging from
the metaoolic response, DNP appears to be eliminated entirely in three or
four days; in the presence of liver or kidney damage it is possible that the
drug will be retained over a longer oeriod." Information on the elimination
kinetics of the dinitrophenols from experimental animals is also scant in
the literature.
Genring and 3uerge (1969b) have developed enuations which describe the
elimination of 2,4-DN? from the serum of ducklings, mature rabbits, and im-
mature rabbits following intraperitoneal administration of the compound.
Serum levels of 2,4-ONP in the mature rabbit declined to less than one per-
cent of their original high values within seven hours. Twenty-four hours
were renuired before the serum levels in the immature rabbit declined to two
percent of their orignial values. Ducklings eliminated 2,4-DNP from the
serum over a similar time frame (96 percent elimination in 24 hours).
Lawford. et al. (1954) also studied the elimination of various nitro-
phenolic compounds (including 2,4-dinitrophenol). Elimination from the
blood of mice, rabbits, guinea pigs, rats, and monkeys was complete within
C-35
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30 hours. Harvey (1959) calculated the elimination rates of all six dini-
trophenol isomers from the blood of mice and rats following a single large
dose given intraoeritoneally. Data are presented in Table 6. The data de-
velooed by these investigators must be taken with caution since the actual
elimination of the dinitrophenols or their metabolits in urine was not di-
rectly measured. In view of the lack of data suggesting concentration of
the dinitrophenols in mammalian tissues and the high water solubility of
these compounds, their elimination via the urine may be a rapid process in
humans.
EFFECTS
Acute, Subacute, and Chronic Toxicity
All of the dinitrophenol isomers are potent metabolic poisons. Most of
the literature available deals with 2,4-dinitrophenol since this compound
has been used extensively for more than 70 years. A number of excellent re-
views on the uses, chemistry, mode of action, and mammalian toxicity of 2,4-
dinitrophenol are available (Edsall, 1934; Metcalf, 1955; Homer, 1942;
Simon, 1953; Slater, 1962; Parascandola, 1974; Howard, et al. 1976) and no
attempt will be made to duplicate the information found in these documents.
2,4-Oinitrophenol is considered a classic uncoupler of oxidative phos-
phorylation and is widely used by biochemists to determine whether a given
biochemical process is energy dependent. Hence, an enormous body of litera-
ture has been generated dealing with the biochemical effects of 2,4-dinitro-
phenol on cellular and biochemical processes both _in_ vivo and j£ vitro.
Only those studies with direct relevance to the acute or chronic effects of
the dinitrophenols on humans are reviewed in this document.
The toxic action of the dinitrophenols is generally attributed to their
ability to uncouple oxidative phosphorylation. These compounds prevent the
C-36
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TABLE 5
Elimination Rates of Dinitroohenol Isomers from the Slood of Mice
and Rats Following a Single Large Intraperitoneal Dose*
Isomer
2,3-Dinitrophenol
2,4-Dinitrophenol
2,5-Dinitrophenol
2,6-Dinitrophenol
3,4-Dinitrophenol
3,5-Dinitrophenol
2,3-Dinitrophenol
2,4-Dini troohenol
2,5-Oinitrophenol
2,5-Dinitrophenol
3,4-Dinitrophenol
3,5-Dinitrophenol
Dose
(mg/kg)
MICE
90
20
ISO
30
60
30
RATS
90
20
90
25
90
30
Half-time for
El imination
( m i n . )
2.7
54.0
3.3
233.0
3.5
2.7
12.5
225.0
13.0
210.0
11.5
2.1
*Source: Harvey, 1959.
C-37
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utilization of the energy orovided by cellular respiration and glyco'vsis b/
inhibiting the formation of high energy phosphate bonds. All energy depen-
dent biochemical orocesses are therefore affected by the action of the com-
oounds (Metcalf, 1955). The large number of clinical effects attributed to
dinitrophenol toxicity result essentially from the shortcircuiting of
metabolism in cells which absorb sufficient dinitrophenol.
All six dinitroohenol isomers are ootent uncouplers of oxidative phos-
phorylation. The relative potencies of the six dinitrophenols in uncoupling
phosphorylation in rat liver mitochondria were found to be (in declining
order): 3,5-> 2,4-> 2,6- = 3,4-> 2,3- = 2,5-dinitrophenol (Burke and White-
house, 1967). 3,5-Dinitrophenol is approximately five times more potent
than 2,5-dinitophenol as measured in this system. The relative _in_ vivo tox-
icities of the dinitrophenol isomers have been determined by a number of in-
vestigators (von Oettingen, 1949; Harvey, 1959; Cameron, 1958; Grant, 1959;
Levine, 1977) and the order of relative potency of the isomers determined in
these investigations frequently differs form the order developed by Burke
and Whitehouse (1967). Several explanations for these discrepancies are
possible: (1) differential tissue absorption of the isomers or (2) different
metabolic detoxification mechansims for the isomers or (3) the presence of
cellular or biochemical effects unrelated to the uncoupling of oxidative
phosphorylation. Resolution of this Question awaits further investigation.
At concentrations higher than those necessary to uncouple oxidative
phosphorylation, a number of inhibitory effects of the dinitrophenol isomers
on certain enzymatic reactions may occur.
Both 2,4-dinitro- and 3,5-dinitroohenol inhibit porcine heart malate
dehydrogenase in vitro (Wedding, et al. 1967). Inhibition of the reaction
C-38
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occurred -at nitrophenoi concentrations 10 to 100 times tnose causing jn
ling, and resulted from a competitive inhibition with NAD in the forward
direction of the malate dehydrogenase reaction. In a similar study Stock-
dale and Selwyn (1971) reported in the j_n_ vitro inhibition of both lactate
dehydrogenase and hexokinase by 2,4-dinitro-, 2,5-dintro-, 2,6-dinitro-
phenols.
The dinitrophenols may also act directly on the cell membrane, thus
causing toxic effects oh ceils which do not depend on oxidative phosphoryla-
tion for their energy requirements. 2,4-Dinitro-, 2,5-dinitro-, and 2,5-
dinitroohenols inhibit oassive oermeabi1ity to chloride (a metabo lically in-
dependent process) in red blood cells (Motais, et al. 1978).
Acute toxicity information for the dinitroohenols has been compiled and
oresented in Table 7.
Numerous occasions of human poisoning by 2,4-DNP -have been reported in
the literature. The earliest cases of fatal 2,4-DN? intoxication relate to
its usage as a component of explosives during World War I. Thirty-six cases
of *atal occupational dinitrooh^nol poisoning occurred among employees of
the munitions industry in France between 1916 and 1918 (Perkins, 1919). A
literature review by von Oettinqen (1949) revealed 27 reported cases of
fatal occupational dinitrophenol poisoning in the United States for the
years 1914 to 1916.
Gisclard and Woodward (1946) reported two fatal cases of dinitrophenol
poisoning during manufacture of picric acid where 2,4-DNP was produced as an
intermediate. Swamy (1953) describes a case of suicidal poisonina by
2,4-DNP.
Early in the 1930s, 2,4-dinitrophenol was widely recommended as a treat-
ment for obesity. Oinitrophenol was received with overwhelming popularity
C-39
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TABLE 7
Acute Toxicity of Oinitrophenol Isomers
Soecies
Oose
(mg/lcg)
Route of
Administration
Effects
References
2,4-Dinitrophenol
Rat
Rat
Rat
Rat
Rat
Mouse
Mouse
Guinea Pig
Rabbit
Rabbit
Rabbit
Ooq
Dog
Doa
Dog
Dog
°iqeon
Pi aeon
Human
Human
Human
25
35
30
23.5
31
36
26
700
30
200
100
30
20-30
22
20
30
7
15-20
40 mg/m^
1-3 a
d.3 '
s.c.
i.o.
Oral
i.p.
i.p.
i .p.
i.p.
Dermal
s.c.
Oral
i.p.
UNK
Oral
s.c.
i .m.
i.v
i.m.
i.v.
Inhalation
Oral
Oral
LD50
l°50
L05Q
L050
LDlOO
LD50
i-050
Lethal Oose
LD50
LOso
Lethal Oose
MLD
L050
LD50
L050
LD50
Lethal Dose
Lethal Dose
Lethal Cone.
Lethal Dose
Lethal Dose
von Oettingen, 1949
Harvey, 1959
Spector, 1956
Lawford, et al. 1954
Gatz and Jones, 1970
Harvey, 1959
Lawford, et al. 1954
Spencer, et al . 1948
von Oettingen, 1949
Spector, 1956
Spector, 1956
Harvey, 1959
Spector, 1956
Spector, 1956
Spector, 1956
Spector, 1956
Spector, 1956
Spector, 1956
MacBryde and Taussig,
1935
Sax, 1968
Geiger, 1933
2,3-Oinitrophenol
Rat
Mouse
Dog
190
200
1000
i.p.
i.o.
UNK
L050
LD50
MLD
Harvey, 1959
Harvey, 1959
Harvey, 1959
C-40
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TABLE 7 (continued)
Acute Toxicity of Dinitrophenol Isomers
Species
Dose
(mg/kg)
Route of
Administration
Effects
References
Rat
Mouse
Dog
Rat
Mouse
Dog
150
273
100
38
45
50
2,5-Dinitrophenol
i.D.
i.p.
UNK
LD50
MLD
2,6-Dinitrophenol
i.p.
i.p.
UNK •
LD50
LD50
MLD
3,4-Oim'trophenol
Harvey, 1959
Harvey, 1959
Harvey, 1959
Harvey, 1959
Harvey, 1959
Harvey, 1959
Rat
Mouse
Dog
Rat
Mouse
Dog
s.c.
i.p.
i .m.
i.v.
UNK
MLO
98
112
500
45
50
500
subcutaneous
intraperitoneal
intramuscular
intravenous
unknown
minimum lethal dose
i
i
U
i
i
U
.p.
• P.
NK
3?
.p.
• P.
NK
LDSO
MLO
5-Dinitroohenol
LDSO
MLO
Harvey,
Harvey,
Harvey,
Harvey,
Harvey,
Harvey,
1959
1959
1959
1959
1959
1959
C-41
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(Homer, 1942) as a slimming agent in spite of warnings of harmful sicie ef-
fects caused by disruption of the metabolic rate. It was estimated that
during the first 15 months follwoing its introduction, 100,000 persons took
the drug for weight reduction (Homer, 1942). More than 1,200,000 capsules
of 0.1 g each were dispensed from a single clinic in San Francisco. More
than 20 drug houses offered to supply both dinitrophenol and mixtures con-
taining the drug. Many of these remedies could be procured without pre-
scription and with no further directions than to take "one capsule three
times daily after meals." In view of this widespread and uncontrolled usage
of the compound, it is not surprising that both toxic side effects and
fatalities resulted. Horner (1942) reported a total of nine deaths result-
ing from the use of dinitrophenol as a slimming agent.
. Parascandola (1974) reviewed the history and public concern which de-
veloped over dinitrophenol in the United States. An article appearing in
Newsweek (1933) entitled "Diet and Die with Excess Alpha Dinitrophenol" was
typical of public concern generated by misuse of dinitrophenol. In the wake
of reports that cataract development in humans attributable to dinitrophenol
was occurring, the drug was finally withdrawn from use in 1937.
The toxic manifestations of dinitrophenol exposure as reviewed by Horner
(1942), included subacute symptoms such as gastrointestinal distrubances
(nausea, vomiting, colic, diarrhea, anorexia), profuse sweating, weakness,
dizziness, headache, and loss of weight. Acute poisoning has resulted in
the sudden onset of pallor, burning thirst, agitation, dyspnea, profuse
sweating, and hyperpyrexia. Intense and rapid onset of rigor mortis after
death has also been described. A physician who ingested a fatal overdose of
dinitrophenol (estimated at 2.5 to 5 g) was literally "cooked to death"
(Geiger, 1933). Rectal temperature at death exceeded 110'F.
C-42
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Perkins (1919) made the interesting observation that postmortem examina-
tion of dinitrophenol victims demonstrated no characteristic lesions. Acute
edema of the lungs was mentioned but was believed to be secondary to the
toxic effects on the vasomotor system. Microscopic lesions of the liver and
kidney cells were inconstant and typical changes were lacking elsewhere.
Spencer, et al. (1948) studied the chronic toxicity of 2,4-dinitrophenol
in rats. Male rats were fed diets containing 0.01, 0.02, 0.05, 0.10, or
0.20 g of 2,4-dinitrophenol per 100 g of food. Rats were maintained on
diets containing 2,4-dinitrophenol for six months and both hematological
pathological investigations on surviving animals were performed. Hematolog-
ical examination included erythrocyte counts, hemoglobin concentrations,
leukocyte counts, differential counts, and bone marrow counts at autopsy.
Both gross and microscopic examination of liver, kidney, spleen, lung,
heart, adrenal, pancreas, and stomach tissues were also performed. Rats
maintained on diets containing 0.02 percent 2,4-DNP (corresponding to 5.4 to
20 mq/kg body weight/day) grew at a normal rate and the investigators failed
to detect discernible ill effects of pathological changes at autopsy. Sim-
ilarly, pathological changes were not found upon microscopic examination of
tissues from rats receiving diets containing 0.05 percent 2,4-DNP (corres-
ponding to 13.5 to 50 mg/kg/day) although growth of these rats fell five to
ten percent below that of the controls throughout the six-month experimental
period. At autopsy the only changes observed in these animals were a very
slight deoletion of body fat and a very slight increase in the average
weight of the kidneys. At higher doses of 2,4-dinitrophenol in their diets
(54 to 200 mg/kg body weight/day) rats occasionally died and survivors lost
weight rapidly. Examination of surviving animals revealed marked emacia-
tion, an empty gastrointestinal tract, a slightly enlarged and dark spleen,
C-43
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and undersized testes. Microscopic examination showed slight congestion and
cloudy swelling of the liver, very slight parenchymatous degeneration of the
epithelium of the renal tubules, slight congestion and hemosiderosis of the
spleen and testicular atrophy. No significant pathological changes were ob-
served in the lung, heart, adrenals, pancreas, or stomach of these animals.
Based on the work of Spencer, et al. (1948), a no-observable-effect-level
for 2,4-DNP in rats lies between 5.4 and 20 mg/kg body weight/day.
Information on the subacute or chronic effects of the other dinitro-
phenol isomers in experimental animals was not found. Langerspectz and
Tarkkonen (1961) failed to detect histological changes in the adrenals or
the liver during 2,4-dinitrophenol treatment of Swiss albino male mice.
2,4-Oinitrophenol was administered via the subcutaneous injection of 10 mg
of 2,4-ONP/kg twice daily for 30 days.
Arnold, et al. (1976) examined the effects on the kidney of a single
large dose of 2,4-DNP. Although a dose close to the ID5Q was chosen, (20
mg/kg) only small areas of cortical tubular necrosis were observed in a few
of the '•ats treated.
Tainter and Cutting (1933) administered 2,4-DNP to dogs at intervals of
three or more days over a period of two to three months. Abnormal liver and
kidney pathology were not detected but an effect on spleen tissue was
noted. Over large areas of the material containing "numerous large faintly
staining cells with vesicular polyhedral nuclei."
The widespread use of 2,'4-dinitrophenol as a weight reducing agent in
humans during the 1930s provides some information regarding the chronic ef-
fects of this compound in man. Recommended theraputic doses of 2,4-DNP for
weight control on humans ranged from 2 to 5 mg/kg body weight/day (Dunlop,
1934; Horner, 1942; Tainter, et al. 1933). Tainter, et al. (1933) adminis-
tered 2,4-DNP to 113 obese patients for as long as four months without
C-44
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demonstrating evidence of cumulative or toxic effects. The most important
side effect noted by the investigator was a skin rash observed in about 7
percent of the patients treated. The rash was manifested usually after a
one-day period of mild itching and consisted of a maculopapular or urticar-
ial type of rash. The itching was intense and in some cases there was con-
siderable swelling. Symptoms subsided in two to five days following with-
drawal from the drug. The next most important side effect noted by the
authors was a loss of taste for salt and sweets observed in 5.3 percent of
the patients. This effect also subsided following withdrawal from 2,4-DNP.
The investigators failed to detect any effect of 2,4-ONP on liver or kidney
function, pulse, blood pressure, or oxygen capacity of the blood. No cases
of anemia, agranulocytosis, or malignant neutropenia appeared. Three cases
of mild gastrointestinal upset were reported, however.
The development of cataracts following dinitrophenol therapy was first
described by Horner, et al. (1936). In a later publication, Homer (1942)
reviewed the acute and chronic toxicity of use of 2,4-DNP (including catar-
act formation) resulting from therapeutic use of the compound. Gastrointes-
tinal symptoms consisting of nausea, vomiting, and loss of appetite were
common as a result of 2,4-DNP administration. Cutaneous lesions were the
most frequent side effect with an incidence of 8 to 23 percent. Although
the majority of lesions were mild, others were severe. Bone marrow effects
of dinitrophenol have also been reported. Eight cases of agranulocytosis
were reported, with three fatalities. Thirty cases of neuritis including
aberrations of taste and multiple regional involvement, particularly affect-
ing the feet and legs were recorded. Symptoms appeared after an average of
ten weeks, followed ordinary therapeutic doses and persisted for weeks or
C-45
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months. Electrocardiograohic evidence of functional heart damage was o*-
fered by several investigators and fragmentation of the heart muscle was re-
oorted at autopsy in one fatal case. It was generally agreed that 2,4-ONP
was Barely injurious to the liver and kidneys when administered in thera-
oeutic doses.
Over 100 cases of cateract formation following dinitrophenol therapy
were reviewed by Homer (1942). Horner described the following characteris-
tic features of 2,4-DNP induced cataracts: (1) they occurred in young women
who were physically normal save for varying degrees of obesity and were in
an age group in which senile cataracts do not occur; (2) they were bilateral
and appeared either during of after periods of dinitrophenol treatment; (3)
an interval of months or years might elapse between the time the last dose
was taken and the onset of blurred vision; (4) lenticular changes were
strikingly similar and could be demonstrated with the biomicroscope at a
time when vision for distance and reading was still normal; (5) after arad-
ual onset, the lenticular changes progressed with startling rapidity until
the vision was obscured; (6) treatment was without effect in staying their
progress; and (7) surgical removal of the lens was uniformly successful in
restoring vision.
The length of time that 2,4-DNP was taken and the amount of the drug
consumed varied widely amonq cataract victims. In 29 cases, the duration of
treatment varied from 3 months to 24 months with an average of 11 months.
Neither the length of treatment nor the total dose seemed to have any bear-
ing on the occurrence of cataracts. Individual susceptibility appeared to
be a more important factor. Horner (1942) estimated that the incidence of
cataracts in patients who had taken dinitrophenol exceed one percent.
C-46
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Formation of cataracts by acute exposure to ONP was first demonstrated
in animals almost ten years after the problem was known to exist in humans
(Gehring and Buerge, 1969a; Ogino and Yasukura, 1957; Feldman, et al. 1959,
1960; Settman, 1946). Experimental cataracts, first produced in ducks and
chickens, differ from DNP-induced human cataracts in that they can be formed
in acute exposures and may appear in less than one hour. Furthermore, these
lesions will disappear spontaneously in animals with 25 hours (Howard, et
al. 1976). Hence, the usefulness of data on the formation of cataracts in
experimental animals following ONP administration in assessing human hazard
to dinitrophenol is questionable.
The available data do not allow the calculation of a minimum effect
level for 2,4-DNP-induced cataract formation in man. Cataractogenic activ-
ity in humans has been observed in a small proportion of patients receiving
as little as 2 mg/kg body weight/day. An assessment of the no-effect-level
for cataract formation awaits further investigation. Such an assessment is
further complicated by the fact that cataract formation in humans, following
ONP administration, differs significantly from the situation seen in experi-
mental animal studies.
Synergism and/or Antagonism
A report of teratogenic synergism following the combined administration
of 2,4-dinitrophenol and insulin to chicks was made by Landauer and Clark
(1964). The injection of 2,4-dinitrophenol at 100 yg/egg was nontoxic and
nonteratogenic after 96 hours of incubation. However, the combined adminis-
tration of insulin (a known teratogen) with 100 ug of 2,4-dinitrophenol
raised the incidence of embryo mortality from 16 to 19 percent and shortened
the upper beak by 1.4 to 18.5 percent.
C-47
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Both thyroid hormones and 2,4-dinitroohenol decrease the efficiency of
mitochondrial oxidative phosphorylation in_ vivo and _m vitro. The _j_n_ yiyp
administration of both 1-thyroxine and 2,4-ONP results in larger changes in
metabolic rate and body temperature than are accounted for by the sum of the
separate effects of each agent (Hoch, 1965).
Other direct information on possible synergism between the
dinitrophenols and other chemical compounds is not available.
Teratogenicity
Wulff, et al. (1935) examined the effects of 2,4-dinitrophenol on the
fertility, gestation, and fetal life of rats. They administered 20 mg of
2,4-DNP/kg body weight to female rats eight days prior to the introduction
of males. Oinitrophenol was administered intragastrically twice daily until
the respective litters were weaned. The average number born in each litter
was not affected by the use of dinitrophenol, and the treatment did not ap-
preciably affect the body weight gains of mothers during pregnancy. Neonat-
al malformations were not detected. Among 2,4-dinitrophenol treated rats,
however, 25 percent of the total number of young were stillborn while only
6.8 percent of the young were stillborn in the control group. In addition,
the mortality during the nursing period of viable young born to mothers ad-
ministering 2,4-ONP was 30.9 percent as compared with 13.4 percent for young
of control mothers. Two possible explanations for this latter phenomenon
were offered: treated mothers neglected their young while in a febrile
state, and only the more vigorous of the offspring managed to reach the
mother for nursing; or, a toxic agent was passed to the young through the
milk. Data to distinguish between the two possibilities are not available.
Intraperitoneal (7.7 or 13.6 mg/kg) or oral (25.5 or 38.3 mg/kg) admin-
istration of 2,4-ONP to mice during early organogenesis does not produce
C-48
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morohological defects in the young, but embryo toxicity occurs at the higher
dose levels (Gibson, 1973). The higher doses also oroduced overt toxic
signs (hyoerexcitability and Hyoerthermia) in the dams, but were not lethal.
Bowman (1957) has studied the effect of 2,4-DNP on the develooino chick
embryo j_n vitro. At 2,4-DNP concentrations of 18 mg/1 or 370 mg/1 a syn-
drome of abnormal ities resulted consisting of degeneration and sometimes
complete absence of neural tissue accompanied by a reduction in the number
of somites. The 2,4-ONP concentrations used in this study are extremely
hiqh and the relevance of the experimental findings to the j_n vivo situation
in mammals is unknown.
Malformations such as hemiophthalmus and cross beak were induced in
chick embryos following administration of 0.5 uM/egg (92 ug/egg) into the
yolk sack at 48 hours of incubation (Miyamoto, et al. 1975). Based on'exam-
ination of purified myelin in the malformed embryos the investigators sug-
gested that 2,4-DNP administration resulted in deficient embryonic myelina-
tion.
Based on the available data it appears unlikely that the dinitrophenols
pose a teratogenic hazard to humans. Further investigations on this nues-
ti'on are warranted.
Mutaaenicitv
Friedman and Staub (1976) have developed an approach to mutaoenic test-
incj which utilizes the measurement of induction of unscheduled ONA synthesis
in testes. These investigators found a good correlation between a reduction
in the residual level of cell cycle-associated DNA synthesis and the pres-
ence of known mutagenic compounds. Testicular ONA synthesis in mice was un-
affected by administration of 2,4-DNP suggesting a lack of mutagenic activ-
ity.
C-49
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Bacterial mutaqenesis of 2,4-DNP has been tested by Demerec, et a".
(1951), based on the production of back mutations from streptomycin deoen-
dence to independence in E_. col i. Mutations were increased severalfold over
control values.
A recent study has been conducted on the effect of various phenolic com-
pounds including 2,4-DNP on chromosomes of bone marrow cells from mice
(Mitra and Manna, 1971). Mice were injected intraperitoneally with satu-
rated aoueous solutions of 2,4-DNp and the bone marrow tissue was collected
24 hours after trea:~ent. The results suggest that 2,4-DNP may produce
chromatid type breaks in bone marrow cells. However, there was no linear
relationship between the frequency of chromosome aberrations and the dose of
2,4-DNP.
It is possible to make a rough estimate of the 2,4-DNP doses adminis-
tered to the mice by these investigators. The water solubility of 2,4-ONP
at 75.8*F is 3.01 mg/ml (Windholz, 1976). If this value approximates the
saturated solution used by Mitra and Manna (1971) and a three-to-four-month-
old mouse weighs approximately 40 g, the following calculations result in
three 2,4-DNP dose levels expressed as mg/kg body weight.
(0.25 ml) (3.01 mq/ml) = 13.3 mg/kg
0.04 kg
(0.5 ml) (3.01 mg/ml) s 37.5 mg/|
-------
Data addressing the possible mutagenicity of the other dinitrophenol
isomers were not found.
Carcinogenicity
In a study designed to measure tumor promoting activity, Boutwell and
Bosch, (1959) examined the ability of 2,4-ONP to promote tumor formation
following a single initiating dose of dimethylbenzanthracene. Although
phenol itself has a promoting activity in this system, 2,4-DNP failed to
promote skin tumors in mice under similar conditions. In a similar experi-
ment, Stenback and Garcia (1975) examined the ability of 2,4-DNP to promote
skin tumor formation in mice. No promoting activity was demonstrated.
Spencer, et al. (1948) failed to detect tumor formation during chronic
administration of 2,4-DNP to mice (over a six month period).
The available data suggest the 2,4-DNP does not possess carcingenic pro-
perties. Information on the other isomeric dinitrophenols is not available.
C-51
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TRINITROPHENOLS
Mammalian Toxicology and Human Health Effects
INTRODUCTION
Six isomeric forms of trinitrophenol exist distinguished by the position
of the nitro groups relative to the hydroxy group on the six carbon benzene
ring. The five isomers are: 2,3,4-, 2,3,5-, 2,3,6-, 2,4,5-, 2,4,6-, and
3,4,5-trinitrophenols. Production volumes for the trinitrophenols are not
available. Usage of the trinitrophenol isomers is apparently limited to
2,4,6-trinitrophenol, other wise known as picric acid. In fact, a compre-
hensive search of the literature failed to detect a single citation dealing
with any of the trinitrophenol isomers except picric acid. Consequently,
the only information on these isomers presented in this document are the
chemical and physical properties found in Table 8 and Figure 3.
According to Matasuguma (1967) picric acid has found use as a dye inter-
mediate, explosive, analytical reagent, germicide, fungicide, staining
agent, and tissue fixative, tanning agent, photochemical, pharmaceutical,
and a process material for the oxidation and etching of iron, steal, and
copper surfaces. The extent to which picric acid finds usage in any of
these applications at the present time is unknown.
EXPOSURE
Ingestion from Uater
Monitoring data on the presence or absence of 2,4,6-trinitrophenol
(2,4,6-TNP) in water were not found, however, a single report of 2,4,6-TN?
contamination of ground water was found (Cole, 1974). In 1955, 2,4,6-TNP
(0.7 mg/1) was detected in a well approximately one mile from the former
site of an expolsives manufacturing plant in England. The plant was engaged
in the manufacture of explosives from 1914 to 1918. The brief report by
C-52
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TABLE 8
Properties of Trinitrophenols*
2,3,4-Tn'nitrophenol
Molecular Weight
229.11
2,3,5-TrinitroDhenol
Molecular Weight
Melting Point
229.11
119-120*C
2,3,6-Trim'trophenol
Molecular Weight
Melting Point
Water Solubility
Room Temperature
Hot Water
229.11
119"C
Slightly Soluble
Very Soluble
2,4,5-Trim'trophenol
Molecular Weight
Melting Point
Water Solubility
Room Temperature
Hot Water
229.11
96'C
Slightly Soluble
Soluble
2,4,6-Trim'trophenol
Molecular Weight
Melting Point
Boiling Point
Vapor Pressure
Density
Water Solubility
Room Temperature
100'C
229.11
122-123'C
Sublimites: Explodes at
300 *C
1 mm Hg at 195*C
1.763 g/cm3
1.28 g/1
6.7 g/1
*Source: Windholz, 1976; Weast, 1975; Matsuguma, 1967.
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NO,
NO,
OH
OH
2,3,4-trinitrophenol 2,3,5-trinitrophenol 2,3,6-trinitrophenol
OH
OH
wo
2,4,5-trinitrophenol 2,4,6-trinitrophenol 3,4,5-trinitrophenol
FIGURE 3
Trinitrophenols
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Cole (1974) failed to describe either the types of explosive manufactured by
the plant or the disposition of the waste water during the period the
explosives were manufactured.
Harris, et al. (1946) described an outbreak of hematuria which resulted
from ingestion of 2,4,6-TNP in the drinking water of U.S. Navy personnel
aboard ships anchored at Wakayama, Japan. Approximately three weeks prior
to the outbreak, more than 100 tons of confiscated Japanese ammunition, (in-
cluding 2,4,6-TNP) had been dumped in the immediate vicinity of the anchor-
age. 2,4,6-TNP was apparently pumped into the ships' drinking water stills
and carried over with the vapor phase into the freshwater supply, inducing
hematuria among those who drank the water. The investigators failed to
detect 2,4,6-TNP in the sea water; however, analysis of the distilled drink-
ing water yielded 2,4,6-TNP levels to 2 to 20 mg/1.
Although it is not possible to precisely estimate either the TNP water
levels or duration of exposure necessary to induce hematuria, Harris, et al.
(1946) detected levels of 10 mg/1 and 20 mg/1 in drinking water aboard two
ships at the time of the hematuria outbreak.
Hoffsommer and Resen (1973) have shown that the highly explosive tetryl
(N-methyl-N,2,4,6-tetranitroanil ine) dissolved in sea water at pH 8.1 and at
temperature 25°C is largely converted to 2,4,6-TNP in a few months. Al-
though tetryl is no longer manufactured in the U.S. (Howard, et al. 1976),
these experiments Indicate that 2,4,6-TNP may be produced in water as a re-
sult of degradation of other organic compounds. The nature of other com-
pounds which may give rise to 2,4,6-TNP following degradation is speculative.
The persistence of 2,4,6-TNP following release to the environment is now
well understood. Fitter (1976) failed to detect degradation of 2,4,6-TNP
using an acclimated activated sludge system with 2,4,6-TNP as a sole source
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of carbon for the microbes in the inoculum. Tabak, et al. (1964) on the
other hand were able to demonstrate 95 percent degradation of 2,4,6-TNP (250
ma/1) in three to six days by acclimated cultures of microorganisms de-
rived from garden soils, compost, and river mud. The extent to which micro-
bial populations capable of degrading 2,4,6-TNP exist in the environment is
unknown.
No other data on possible ingestion of 2,4,6-TNP from water by humans
were found.
Inqestion from ?
Pertinent data could not be located in the available literature
concerning exposure to 2,4,6-TNP via ingestion of food.
No measured steady-state bioconcentration factor (BCF) is available for
any nitrophenols, but the equation "Log BCF = (0.85 Log P) - 0.70" can be
used (Veith, et al. 1979) to estimate the steady-state BCF for aauatic or-
ganisms that contain about 7.6 percent lipids (Veith, 1980) from the oc-
tanol/water partition coefficient (P). The log P values were obtained from
Hansch and Leo (1979) or were calculated by the method described therein.
The adjustment factor of 3.0/7.6 = 0.395 is used to adjust the estimated BCF
from the 7.6 percent lipids on which the enuation is based to the 3.0 per-
cent lipids that is the weighted average for consumed fish and shellfish in
order to obtain the weighted average bioconcentration factor for the edible
portion of all freshwater and estuarine aauatic organisms consumed by Ameri-
cans.
Inhalation
Pertinent information could not be located in the available literature
on the presence or absence of trinitrophenols in air.
Dermal
Information on the dermal absorption of 2,4,6-TNP is scant in the liter-
ature. During the 1920s and 1930s, 2,4,6-TNP was used both alone and in
C-56
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combination with butesin (di-n-butyl-p-aminobenzoate trinitroohenol) as an
antiseotic surgical dressing for the treatment of burns. Ehrenfried (1911)
remarked on the dangers of poisoning by absorption of 2,4,6-TNP in dermal
ointments, but added that, if the ointments were properly used, there was no
danger of toxic symptoms developing in humans.
A serious case of central nervous system dysfunction following the topi-
cal aoolication of 2,4,6-TNP was reported by Oennie, et al. (1929). The
patient recovered rapidly following cessation of the 2,4,6-TNP treatment.
No other information on dermal absorption of the trinitrophenols by humans
or experimental animals was found.
PHARMACOKINETICS
Absorption
Quantitative information on the absorption of 2,4,6-TNP by humans or ex-
oerimental animals is not available.
Neurological complications following the topical administration of
2,4,6-TNP (Oennie, et al. 1929) indicate that the compound may be absorbed
through the skin. Since the compound was applied to a burned area of the
patient, the relevance of this data to the absorption of 2,4,6-TNP through
intact skin in unknown.
The occurrence of human cases of microscopic hematuria resulting from
ingestion of 2,4,6-TNP in drinking water (Harris, et al. 1946) and the known
oral toxicity of 2,4,6-TNP in experimental animals indicate that absorption
by the gastrointestinal tract readily occurs.
Oi stribution
Examination of dogs after a lethal dose of 2,4,6-TNP (Dennie, et al.
1929) revealed yellow staining of the subcutaneous fat, lungs, intestines,
C-57
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and the olood vessels, indicating tnat 2,4,6-TNP is distributed to nany tis-
sues in the body. These investigatores also demonstrated the presence of
2,4,6-TNP in the blood and suggested that the compound may be bound to serum
proteins. It seems likely that distribution of 2,4,5-TNP would occur via
the olood. No other data on the tissue distribution of 2,4,6-TNP following
absorption were found.
Metaoolism
In a review of the early literature, Burrows and Dacre (1975) indicated
that elimination of 2,4,6-TNP from humans occurs in both the free form and
as picramic acid. In perfusion experiments with liver, kidney and spleen,
the liver exhibited the strongest capacity for reduction of 2,4,6-TNP.
Other studies dealing with the metabolism of 2,4,6-TNP in humans or in
experimental animals were not found.
Decomposition of 2,4,6-TNP by an atypical strain of Corynebacterium sim-
plex with the production of nitrites has been reported by Gunderson ana Jen-
sen (1956). This alternative metabolic pathway for 2,4,6-TNP has not been
reported in mammals.
Excretion
The presence of 2,4,6-TNP in blood and urine within 1.5 hours after ad-
ministration of a lethal dose in dogs was reported by Dennie, et al.
(1929). The presence of 2,4,6-TNP in the urine of humans following oral ex-
posure was reported by Harris, et al. (1946). These studies indicate tnat
2,4,6-TNP is partially excreted in the urine following exposure. Other data
on the excretion of 2,4,6-TNP were not found.
EFFECTS
Acute, Subacute, and Chronic Toxicity
According to Windholz (1976) ingestion or percutaneous absorption of
2,4,6-TNP may cause nausea, vomiting, diarrhea, abdominal pain, oliguria,
C-58
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anurea, yellow staining of skin, pruritus, skin eruptions, stupor, convul-
sions, and death.
Although Dennie, et al. (1929) stated: "The application of a solution of
trinitrophenol to burned or abraded skin is dangerous even for nonsensitive
persons since many deaths have been reported from its application," no re-
ports of human fatalities resulting from 2,4,6-TNP exposure were found in
the literature. Gleason, et al. (1968) reported the lowest recorded lethal
dose for 2,4,6-TNP in humans as 5 mg/kg body weight, however, details of the
poisoning episode were not provided. It is reasonable to assume, based on
the known toxicity of 2,4,6-TNP in experimental animals, that exposure to
sufficient amounts of the compound would be lethal in humans. The limited
acute toxicity information for experimental animals has been compiled and is
presented in Table 9.
Following acutely lethal doses of 2,4,6-TNP, dogs die from respiratory
paralysis (Dennie, et al. 1929). Autopsy results demonstrate the presence
of yellow staining of the subcutaneous fat, the lungs, the intestines, and
the blood vessels. Swelling of the liver and glomerulitis of the kidneys
were also seen.
The major effect of nonlethal doses of trinitrophenol (TNP) appears to
be an allergic or irritative dermatitis (Anon. 1937; Ehrenfried, 1911). Ac-
cording to Dennie, et al. (1929) about four percent of people treated with
TNP are sensitive and develop a local dermatitis. Reactions may also appear
in unexposed areas. An intense itching and burning, pruritis, skin erup-
tions, and irritability are common. Skin eruptions are characterized by ir-
regular-shaped macules, papules, vesicles, blebs, excoriations, and edema,
as well as dried yellow crusts which are sources of reabsorption. In the
maculopapular stage, a purplish-yellow color is characteristic.
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TABLE 9
Acute Toxicity of Trinitrophenol Isomers3
Species
Dog
Dog
Dog
Rabbit
Frog
Frog
Cat
Human
Dose
(mg/kg)
100-125
60
60
120
200
200-300
500b
5
Route of
Administration
2,4,6-Trinitroohenols
s.c.
s.c.
UNK
Oral
s.c.
s.c.
Oral
Oral
Effects
Lethal Dose
MLD
MLD
Lethal Dose
Lethal Dose
MLD
Lethal Dose
Lethal Dose
References
Dennie, et al .
Spector, 1956
von Oettingen,
von Oettingen,
Windholz, 1976
Spector, 1956
von Oettingen,
Gleason, et al .
1929
1949
1949
1949
1968
3Acute toxicity data for trinitrophenol isomers other than 2,4,6-TNP were not
found.
bTotal dose in milligrams.
MLO = Minimum Lethal Dose
UNK = Unknown
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More severe reactions can lead to diffuse, often severe erythema and
desiuamation of affected areas (Sulzburger and Wise, 1933; Am. Conf. Gov.
!nd, Hyg., (ACGIH) 1971). The reaction may last from several weeks to al-
most a year (Sulzburqer and Wise, 1933).
Effects on the skin are apparent at concentrations well below those
necessary for oral systemic poisoning. Of 71 individuals exposed at concen-
trations of 0.0088 to 0.1947 mg/m , dermatitis developed only among those
exposed to the lower concentrations. Desensitization or adaptation reac-
tions may occur (ACGIH, 1971).
Guinea pigs tested for allergic reactions gave similar results (Land-
steiner and OiSomma, 1940; Maguire and Chase, 1972; Maguire, 1973; Chase and
Maguire, 1972). Using a split-adjuvant method of sensitization, reactions
have been noted at concentrations of less than one percent, with weaker sol-
utions often giving stronger reactions. A boosting effect was also noted on
subsequent tests with sensitized animals (Maguire and Chase, 1972).
Sub-lethal doses of less than or enual to 50 mg/kg body weight in dogs
have resulted in transitory changes in the kidney which include glomerulitis
and involvement of TNP ranging from cloudy swelling to gelatinous degenera-
tion. The liver also showed cloudy swelling with no staining while the
lungs were stained brownish-yellow in some animals (Dennie, et al. 1929).
Other reactions in humans include central nervous system effects result-
ing in temporary impairment of speech, memory, walking, and reflexes
(Dennie, et al. 1929) and microscopic hematuria caused by ingestion of TNP
(2 to 20 mg/1) in water distilled from sea water (Harris, et al. 1946).
Althouah it is not possible to estimate precisely either the TNP water
levels or duration of exposure necessary to induce hematuria, Harris, et al.
(1946) detected levels of 10 mg/1 and 20 mo/1 in drinking water aboard two
ships at the time of the hematuria outbreak.
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Crenation of intact erythrocytes by 1 mM 2,4,6-TNP has been noted i-j
Sheetz and Singer (1976) with lysing occurring at higher concentrations.
2,4,6-TNP has also been observed to affect glycolysis in human red blood
cells u^ vitro by affecting ATP production and consumption, although the ex-
act mechanism has not been determined (Vestergaard-Bogind and Lunn, 1977).
Pugh and Stone (1968) noted that from 6 to 11 mg/kg 2,4,6-TNP administered
intravenously to anesthetized dogs results in a moderate increase in bile
flow and a rise in body temperature. The clinical significance of these ef-
fects is unknown.
Synergism and/or Antagonism
Information on synergistic or antagonistic effects involving 2,4,6-TNP
is scant in the literature. An interesting study by Huidobro (1971) demon-
strated that administration of 50 mg of 2,4,6-TNP kg of body weight 30
minutes before the administration of a number of analgesic drugs resulted in
a significant increase in the area of analgesia induced by the opioids and
the minor analgesics employed. 2,4,6-TNP did not, itself, evoke analgesia.
The compounds tested included: morphine, meperidine, methadone, pentazocine,
aminopyrine, sodium salicilate, and etonitazene. The investigators suggest-
ed that the elimination or metabolism of the analgesics may be modified by
an effect of 2,4,6-TNP on enzymatic systems. However, further investigation
is needed to definitively answer this question.
Teratogenicity
Pertinent information could not be located in the available literature
on possible teratogenic effects of 2,4,6-TNP.
Mtitagenicity
Streptomycin-independent mutants were induced in streptomycin-requiring
E_. col i B/Sd-4 after preincubation in the presence of 0.18 percent 2,4,6-T'i?
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for three hours before olating on streptomycin free agar (Demerec, et al.
1951) Yoshikawa, et al. (1976) reported that 2,4,6-TNP was capable of in-
ducing mutations in Salmonella, when tested in a system which included mic-
rosomal activation. In contrast, Auerbach and Robson (1947) failed to
demonstrate sex-linked lethals in Drosophila after bathing the eggs in an
aoueous solution of 2,4,6-TNP. Other data on possible mutagenic properties
of 2,4,6-TNP were not found.
Carcinoaenicity
Pertinent data could not be located in the available literature on pos-
sible carcinogenic effects of 2,4,6-TNP.
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DINITROCRESOLS
Mammalian Toxiciloqy and Human Health Effects
INTRODUCTION
Dinitro-ortHo-cresol is a yellow crystalline solid derived from o-cre-
sol. There are six possible isomers but the 4,6-dinitro-o-cresol isomer is
the only one of any commercial importance. In fact, a comprehensive search
of the literature failed to reveal information on any of the other five
dinitrocresol isomers.
4,6-Oinitro-o-cresol (hereafter referred to as ONOC) is produced either
by sulfonation of o-cresol followed by treatment with nitric acid or by
treatment of o-cresol in glacial acetic acid with nitric acid at low temper-
ature. Some important chemical and physical properties of DNOC are shown in
Table 10 and Figure 4.
An excellent review of the toxicological effects of ONOC on human and
labortory animals has recently been published by the National Institute for
Occupational Safety and Health (NIOSH, 1978). In view of the comprehensive
coveraae of both English and foreign language literature, no attempt will be
made to duplicate this impressive effort within this criterion document.
Key papers and frenuent reference to the NIOSH review will be used where the
available literature does not contain information directly relevant to cri-
teria formulation.
ONOC usage in the U.S. has declined in recent years because the compound
is highly toxic to plants in the growth stage and nonselectively kills both
desirable and undesirable vegetation. Additionally, the compound is highly
toxic to humans and is considered one of the more dangerous agricultural
pesticides.
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TABLE 10
Prooerties of 4,6-Dinitro-o-cresol
Molecular Weight 198.13
Appearance Yellow Solid
Melting Point 85.8*C
Vapor Pressure 0.000052 mm Hg at 20*C
Water Solubility 100 mg/1 at 20*C
pKa 4.46
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4,6-Dinitro-o-cresol (ONOC)
FIGURE 4
Oinitrocresols
C-66
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The Environmental Protection Agency has no record of DNOC being current-
ly manufactured in the United States for use as an agricultural chemical.
Imports of DNOC have also decreased in recent years; from 217,899 Ibs. in
1972 to 146,621 Ibs. in 1973 and then to 30,442 Ibs. in 1976 (NIOSH, 1978).
Since DNOC is not manufactured in the U.S., pesticide formulators and spray-
ers are the major groups with potential occuoational exposure to DNOC.
DNOC is used primarily as a blossom-thinning agent on fruit trees and as
a fungicide, insecticide, and miticide on fruit trees during the dormant
season. NIOSH (1978) estimates that 3,000 workers in the U.S. are potent-
ially exposed to DNOC. In view of the small amount of DNOC used in the
U.S., exposure of the general public is expected to be minimal.
EXPOSURE
Ingestion from Water
Monitoring data on the presence of DNOC in ambient water are not avail-
able. An unspecified amount of DNOC was detected in the waste waters of
Fison's Pest Control Limited in Harston, Cambridge, England (Jenkins and
Hawkes, 1961). Webb, et al. (1973) detected 18 mg DNOC/1 in the waste water
of a specialty chemical plant. The extent to which human exposure to DNOC
results from the ingestion of contaminated water is unknown.
Inqestion from Food
No data are available on the presence or absence of DNOC residues in
food for human consumption. Since the primary usage of the compound in-
volves treatment of fruit trees during the dormant season, it appears un-
likely that contamination of human food stuffs would occur to any large ex-
tent.
No measured steady-state bioconcentration factor (BCF) is availahle for
any nitrophenols, but the equation "Log BCF = (0.85 Log P) - 0.70" can be
C-67
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used (Veith, et al. 1979) to estimate the steady-state BCF for aouatic or-
ganisms that contain about 7.6 percent lipids (Veith, 1980) from the oc-
tanol/water partition coefficient (P). The log P values were obtained from
Hansch and Leo (1979) or were calculated by the method described therein.
The adjustment factor of 3.0/7.6 = 0.395 is used to adjust the estimated BCF
from the 7.5 percent lipids on which the eouation is based to the 3.0 per-
cent lipids that is the weighted average for consumed fish and shellfish in
order to obtain the weighted average bioconcentration factor for the edible
portion of all freshwater and estuarine anuatic organisms consumed by Ameri-
cans.
Inhalation
An evaluation of the literature (NIOSH, 1978) indicates that occupation-
al injury and disease associated with exposure to DNOC results primarily
from inhalation of, and skin contact with, the aerosol form. A large number
of human intoxications, including fatalities, have been reported resulting
from such DNOC exposure. Persons at risk include those manufacturing, form-
ulatina, or applying the compound as an aerosol. Inhalation exposure to the
general public is expected to be minimal although data addressing this point
are not available.
Dermal
As mentioned in the preceeding section, occupational intoxication by ex-
posure to DNOC has occurred as a result of inhalation and dermal exposure
where the compound is manufactured, formulated or applied. Dermal exposure
of the general public is considered unlikely, however, direct data bearing
on this point were not found.
PHARMACOKINETICS
Absorption
DNOC is readily and rapidly absorbed through the skin, the gastrointest-
inal tract and respiratory tract in humans (NIOSH, 1978). Although most
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cases of occupational intoxication resulting from D'iOC exposure contain both
a respiratory and a dermal component, human intoxication has been reportsd
as a result of dermal contact with DNOC alone.
In a report from the Russian literature (3uchinskii, 1974) a four-year-
old boy was fatally intoxicated after a rash had been treated with 50 g of
an ointment to which 25 percent DNOC was added by mistake. Stott (1956) re-
ported two cases of DNOC poisoning resulting from skin absorption. The two
men were involved in the cleaning and maintenance of aircraft booms used to
spray solutions of DNOC. Since neither man worked near the actual opera-
tion, and both denied blowing into the spray jet to clean them, Scott (1956)
concluded that the major route of exposure was skin contact.
Work by Harvey, et al. (1951) indicates that DNOC is rapidly absorbed by
the human gastrointestinal tract. These investigators described the effects
of DNOC taken orally by five male volunteers. It was noted the DNOC levels
in the blood increased steadily after administration and were maximal from
two to four hours after ingestion. Van Noort, et al. (1960) investigated
the effectiveness of personal protective equipment used by 24 sprayers in
Holland. Serum DNOC levels and the quanitity of DNOC used were determined
in a three-week spraying period. Their findings indicated that both inhala-
tion and dermal contact with DNOC can lead to an appreciable absorption into
the blood stream.
Experimental animal studies, reviewed by NIOSH (1978), also have con-
firmed the toxicity of DNOC in humans exposed by the oral, inhalation, and
dermal routes.
Distribution
Whether absorption of DNOC occurs through the skin, gastrointestinal
tract, or respiratory tract, the compound is transported in and distributed
by the blood (NIOSH, 1978). Harvey, et al. (1951) described the effect of
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DNOC taken orally by five male volunteers. Caosules containing 75 T,q Of
oure DNOC were administered daily for five consecutive days amounting to a
total dose of from 0.95 to 1.27 mg/kg/day. The concentration of ONOC in the
blood increased in the first three to four days and reached concentrations
of 15 to 20 mq/kg. After concentrations of 15 to 20 mg/kg had been ob-
tained, additional doses aopeared to cause temporary high blood concentra-
tions which were associated with toxic symptoms.
Blood analysis of humans displaying symptoms of ONOC toxicity has invar-
iably revealed concentrations exceeding 10 mg/kg (NIOSH, 1978).
In studies conducted to determine the kinetics of absorption and distri-
bution, DNOC has not been shown to accumulate in the blood of .-arious animal
species (King and Harvey, 1953a; Parker, et al. 1951). In rats and rabbits
that were given two or more daily injections of DNOC subcutaneously, serum
levels on succeeding days were no higher than they were 24 hours after the
first dose (Parker, et al. 1951). Serum levels in dogs rose for the first
three days but then decreased despite the administration of two additional
doses.
ONOC is more rapidly eliminated from the blood of animals than from the
blood of humans (King and Harvey, 1953b; Parker, et al. 1951; Harvey, et al.
1951). Within a 24-hour period following a single subcutaneous injection of
ONOC, elimination from the serum of rabbits was nearly complete. Four days
were necessary for serum clearance in rats and cats, while six days were re-
nuired for elimination from the serum of dogs (Parker, et al. 1951). DNOC
accumulated only slightly in the blood when given to rats by stomach tube or
i.p. injection and did not accumulate in the blood of rabbits after adminis-
tration by stomach tube (King and Harvey, 1953a).
C-70
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The accumulation of DNOC in the blood of humans following DNOC exposure
has been we:l documented (^arvey, et al. 1951; Bidstruo, et al. 1952). The
accumulative effect may reflect the binding of DNOC with albumin in the
blood and a subsenuent slow rate of excretion in humans (Harvey, et al.
1951).
ONOC is slowly eliminated from humans. The investigations by Harvey, et
al. (1951) indicated detectable amounts of DNOC in the blood (1 mg/kg) as
long as 40 days following the last of five consecutive daily oral doses in
human volunteers. Another study (Van Noort, et al„ 1960; reviewed by NIOSH,
1978) showed that it took two to eight weeks for ONOC to be cleared from the
serum.
Parker, et al. (1951) studied the tissue distribution of ONOC following
subcutaneous injection in the rat. They noted that a single dose of 10
mg/kg DNOC produced very high levels in the serum (100 mg/1 at 30 min) but
no accumulation in other tissues was detected. The lungs and heart con-
tained high levels of DNOC but the investigators postulated that these
levels were the highest due to the h.igh blood content of these organs. The
investigators calculated that within 30 minutes of the injection, 83 percent
of the DNOC that could be accounted for was present in the blood. Six hours
after the injection, 0.37 mg of the 1.5 mg dose of ONOC could be accounted
for, of which 72 percent was in the blood.
ONOC content of a number of tissues was determined in rats receiving a
single subcutaneous injection of the compound (Parker, et al. 1951). The
results, presented in Table 11, clearly indicate the ONOC failed to accumu-
late in the tissues.
In another experiment Parker, et al. (1951) failed to detect significant
DNOC accumulation in liver or kidney tissue of rats after 40 successive
daily injections of 20 mg/kg DNOC.
C-71
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TABLE 11*
ONOC Content of Blood and Tissues** of Rats Killed at Intervals After
Subcutaneous Injection of One Dose of 1.5 mg DNOC*
Time
After Injection
30 min.
1 hr.
2 hrs.
3 hrs.
4 hrs.
5 hrs.
6 hrs.
Serum
(rag/1)
100
89
97
93
79
76
45
Brain
1.5
3.5
2.0
4.0
3.5
2.0
3.0
Spleen
4.0
4.0
4.5
8.0
3.0
4.0
1.5
Kidney
7.5
7.5
11.0
11.0
4.5
4.5
7.5
Liver
14.0
12.0
10.5
11.5
13.5
8.5
8.5
Muscle
0.5
2.0
0.0
3.5
0.5
2.0
1.5
Heart
98.0
13.5
19.0
14.0
13.0
14.0
10.5
Lung
18.0
20.0
20.5
15.5
14.0
14.5
30.0
*Source: Parker, et al. 1951.
**DNOC content of tissue mg/kg net weight.
C-72
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In a single study reviewed by NIOSH (1978) Sovljanski, et al. (1971)
discussed tissue distribution of DNOC in humans. Autopsy results of two
victims, who had commmitted suicide by ingestion of DNOC, yielded detectable
DNOC in the stomach, intestines, liver, kidneys, heart, and brain, with the
stomach containing the greatest amount. Neither blood DNOC levels nor quan-
titative data on tissue levels were reported.
Steer (1951), on the other hand, demonstrated that the tissues of a
fatal case of DNOC poisoning contained no more than 5 mg/kg of DNOC and many
contained 1 mg/kg or less.
According to King and Harvey (1953b) the accumulation of DNOC in man can
be explained in two ways; either the detoxification and excretion are very
slow or there is some storage of DNOC in body tissues. Based on their cal-
culation of excretion kinetics in man, the investigators suggested that de-
toxification and excretion of DNOC are inefficient and slow in humans.
None of the available data suggest significant accumulation of DNOC in
specific tissues of humans or experimental animals (NIOSH, 1978).
Metabolism
The metabolism of DNOC in humans has not been studied. However, several
investigators have conducted experiments to determine the rate of DNOC after
its administration to animals.
Truhaut and De Lavaur (1967) reported on the metabolism of DNOC in rab-
bits. Following the administration of DNOC by gastric intubation, both DNOC
and 6-amino-4-nitro-o-cresol were detected in liver, kidney, brain, and
urine of animals. 4-Amino-6-nitro-o-cresol was not detected in the ani-
mals. It was concluded by the investigators that the ratio of 6-amino-4-
nitro-o-cresol to DNOC in the tissue and urine was a function of the dose of
DNOC administered to the animal. When a low dose of DNOC was administered,
C-73
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ve*-y litt'e 5-amino-d-nitro-o-cresol *as detected in eitner the urine or
tissues. The authors considered the metabolism of ONOC to 6-amino-4-nitro-
o-cresol a detoxification mechanism that plays an important role only when a
toxic dose of DNOC is administered. They further suggested that the ratio
of 5-amino-4-nitro-o-cresol to DNOC might be a useful indicator in evalua-
tion of the severity of exposure to DNOC.
The metabolic fate of DNQC in rabbits was also investigated by Smith, et
al, (1953). Following administration of 20 to 30 mg/kg ONOC to rabbits by
stomach tube, urinary metabolites were identified by paper chromatography
and spectrophotometry. Less than 20 percent of the dose was recovered in
the urine in two days. Between 5 and 5.5 percent was detected as free DNOC,
and 0.7 percent as DNOC conjugates. The conjugates were not characterized
by the investigators. Most of the urinary metabolites (about 12 percent of
the dose) were derivatives of 6-amino-4-nitro-o-cresol. About 1.5 percent
of the dose was excreted as 6-acetamido-4-nitro-o-cresol, and 9 to 10.5 per-
cent as the hydroxyl group conjugate. Traces of 6-amino-4-nitro-o-cresol,
4-amino-6-nitro-o-cresol, and 3-?amino-5-nitrosal icyl ic acid were also de-
tected.
Since the detoxification and excretion of DNOC in man are very slow com-
pared to rats or rabbits (King and Harvey, 1953b), the applicability of the
experimental animal detoxification mechanism to the human situation is un-
known. The elucidation of DNOC detoxification mechanism in humans awaits
further investigation.
Excretion
Available data indicate that DNOC is rapidly excreted following adminis-
tration to experimental animals. Parker, et al. (1951) found that DNOC in-
jected subcutaneously disappeared from the blood at various rates in differ-
C-74
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ent species. Single 10 mg/kg doses of DNOC were administered subcjtaneously
to an unspecified number of dogs, cats, rabbits, and rats. DNOC given in
one injection was completely eliminated from the serum of rabbits within 24
hours, while blood DNOC levels were between 30 and 40 mg/1 in the rats,
cats, and dogs at this time. It took four days for DNOC blood levels to
fall to zero in rats and cats, and six days in dogs. The half-time for
elimination of DNOC from the blood after a single injection of 10 mg/kg 3NOC
was approximately three hours in the rabbit, 15 hours in the rat, 20 hours
in the cat, and 36 hours in the dog.
Lawford, et al. (1954) reported that animals eliminated DNOC from the
blood in the following descending order of efficiency: mouse, rabbit, guinea
pig, rat, and monkey.
DNOC is eliminated in the blood of animals faster than it is from the
blood of humans (King and Harvey, 1953b; Parker, et al. 1951). King and
Harvey (1953b) calculated the half-time for elimination of DNOC from the
blood of rats, rabbits, and humans. The values were 28.5 hours, 6.6 hours,
and 153.6 hours, respectively.
Pollard and Filbee (1951) reported on the urinary excretion of DNOC from
a seriously poisoned man in Great Britain. The man was admitted to the hos-
pital and full biochemical investigations were performed immediately after
admission. The man recovered almost totally from the poisoning episode
within five days. However, DNOC levels of 4 mg/1 were still detected in the
blood one month following the exposure. Blood DNOC level was reported to
fall in an exponential fashion.
Van Noort, et al. (1960) measured the serum DNOC levels in ten spraymen
on a weekly basis for two months after the spraying period ended. They
found the ONOC was eliminated from the serum slowly and that the rate varied
C-75
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from individual to individual. TA/O to e^gnt weeks elapsed before DNOC
cleared comoletely from the serum of these workers. The amount of tir^.e
needed for QNOC to be totally eliminated was directly related to the quan-
tity of DNOC in the serum on the last day of exposure.
In experiments whe^e DNOC was orally administered to five human volun-
teers, Harvey, et al. (1951) demonstrated that DNOC, absorbed by ingestion
at 24-hour intervals accumulates in the human body and is excreted slowly.
Forty days after the last dose of DNOC was orally administered by mouth, 1
to 1.5 mg/1 ONOC was still present in the blood.
The experimental evidence suggests, therefore, that a substantial dif-
ference in the excretion patterns of humans vs. experimental animals ex-
ists. Since storage of DNOC in the tissues of humans has not been reported,
it is concluded that slow and inefficient detoxification or excretion prob-
ably occurs in humans.
Occupational studies (NIOSH, 1978) have long utilized serum levels of
DNOC in order to assess exposure of humans to dangerous amounts of the com-
pounds. A review of the literature (NIOSH, 1978) indicates that workers
with DNOC concentrations of 40 mg/kg of whole blood (approximately 80 mg/1
of serum) or greater will most likely develop toxic effects. In addition,
in the concentration range between 20 and 40 mg/kg of whole blood (probably
because of variation in individual susceptibility) some workers are affected
and others show no adverse effects. Most workers with blood DNOC levels be-
low 20 mg/kg are not affected, although because of individual susceptibil-
ity, some exhibited mild effects. The blood level of 20 mg/kg has been used
as a maximum permissible level for industrial or agricultural workers uti-
lizing the compound during employment.
C-76
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Bidstruo, et al. (1952) recommeded that a person should be removed fron
further contact with ONOC for at least six weeks if the blood level eight
hours after the last exposure was 20 mg/kg or higher.
Other data on the elimination of DNOC from humans were not found.
EFFECTS
Acute. Subacute, and Chronic Toxicity
The acute toxic dose of DNOC with different routes of administration,
has been determined for a number of different experimental animal species.
These data have been compiled and presented in Table 12.
Although the available human toxicity data do not permit the calculation
of the acute lethal dose for ONOC in humans, it has been estimated (Fair-
child, 1977) that 5 mg/kg may prove lethal to humans.
A large number of occupational and nonoccupational poisonings of humans
by DNOC have been reviewed by NIOSH (1978). The available literature con-
cerning humans indicates that DNOC may be absorbed in acutely toxic amounts
though the respiratory and gastrointestinal tracts and through the skin, and
that it accumulates in the blood. Individuals exposed to DNOC by these
routes usually demonstrate signs of increased metabolic rate. Symptoms of
poisoning include profuse sweating, malaise, thirst, lassitude, loss of
weight, headache, a sensation of heat, and yellow staining of the skin,
hair, sclera, and conjunctiva.
In additon to the effects associated with increased metabolism, other
effects occasionally reported in humans poisoned by DNOC included kidney
damage, diarrhea, unspecified changes in the gastrointestinal tract, in the
cardiovascular system, and in the peripheral vascular and central nervous
systems.
C-77
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TABLE 12
Acute Toxicity of 4,6-Dinitro-o-cresol
Dose
Species (-rig/kg)
Mouse 187
Ratbit 1000
Guinea Pig 500
Rat 85
Rat 30
Rat 40
Rat 30
Mouse 47
Mouse 16.4
Hare 24.8
Cat 50
Pheasant 8.4
Partridge 8.3
Rat 26-39
Rat 20
Mouse 24.2
Rat 24.6
Goat 50
Ooq 15
Dog 5
Ooq 10
Pigeon 5
MLD = Minimum Lethal Dose
s.c. = subcutaneous
i.m. = intramuscular
i.v. = intravenous
i .p. = intraperitoneal
Route of
Administration
Dermal
Dermal
Dermal
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
s.c.
s.c.
s.c.
s.c.
s.c.
i.v.
i.m.
i.p.
i.m.
Effects
LD50
LD50
100% Lethal
LD50
MLD
100% Lethal
I-DSO
LD50
LD50
L050
L050
LD50
LD50
LD50
MLD
LD50
LD50
LD50
LD
LD
LD
LD
References
Arustamyan, 1972
Burkatskaya, 1965
Spencer, et al . 1948
Burkatskay, 1965
Ambrose, 1942
Ambrose, 1942
Spencer, et al. 1948
Burkatsukaya, 1965
Arustamyan, 1972
Janda, 1970
Burkatsukaya, 1965
Janda, 1970
Janda, 1970
Harvey, 1952
Ambrose, 1942
Parker, et al. 1951
Spector, 1956
Ambrose, 1942
Soector, 1956
Spector, 1956
Soector, 1956
Spector, 1956
-------
It is gene^i1;/ relieved that the toxic effects of D'.CC ^esjlt f'-om its
ability to uncouple the oxidative phosphorylat ion process. DNOC is an ex-
tre^ely potent uncoupler of oxidative phosphorylation. This effect results
in tie decreased for-nation of adenosine triphosphate (ATP) and a resulting
inhibitory effect of enzyme reactions requiring ATP. Such a toxicant is ex-
pected to have extreme and profound effects on all tissues where the concen-
tration of the chemical is high enough to severely affect oxidative phos-
ohorylation. Since energy generated in the body cannot be converted to its
usual form (ATP) in the presence of DNOC, it is released as heat instead,
causing many of the commonly observed signs and symptoms of DNOC toxicity.
Several investigators have correlated blood DNOC levels with the sever-
ity of toxic effects in humans (Harvey, et al. 1951; Bidstrup, et al. 1952;
Pollard and Filbee, 1951) and have shown that, unlike the situation in ani-
mals, DNOC accumulates in the blood of humans. Accumulation is believed to
occur as a result of DNOC binding to albumin in the blood (Harvey, et al.
1965). In one of the few cases where DNOC in the blood of a poisoned human
was monitored throughout his recovery period, (Pollard and Filbee, 1951) the
severity of the symptoms decreased as blood levels of DNOC decreased. Data
on blood DNOC levels in humans and the accompanying effects are compiled and
presented in Table 13. The data show that workers with DNOC concentrations
of 40 mg/kq of whole blood (approximately 80 mg/1 of serum) or greater will
Tiost likely develop toxic effects. In the concentration range between 20
and 40 mg/kg of whole blood, some workers are affected and others show no
adverse effects (probably because of differences in individual susceptibil-
ity). fj1ost individuals with blood levels of DNOC below 20 mg/kg were not
affected, although some exhibited mild effects. As the data in Table 4 sug-
gest, most investigators have concluded that blood DNOC levels are associ-
ated with the severity of intoxication in humans (NIOSH, 1978).
C-79
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TABLE 13
Relationship to Blood DNOC Levels and Ejects in Humans*
Route
Exposure
Inhalation, Dermal
Inhalation, Dermal
Inhalation, Dermal
Inhalation, Dermal
Inhalation, Dermal
Inhalation, Dermal
Inhalation, Dermal
Oral
Inhalation, Dermal
Inhalation, Dermal
Inhalation, Dermal
Inhalation, Dermal
Inhalation, Dermal
Inhalation, Dermal
Inhalation, Dermal
Inhalation, Dermal
Inhalation, Dermal
Inhalation, Dermal
Inhalation, Dermal
Inhalation, Dermal
Inhalation, Dermal
Oral
No. of Individuals
and Occupation
1 Agricultural Worker
1 Agricultural Worker
1 Agricultural Worker
1 Agricultural Worker
1 Agricultural Worker
1 Agricultural Worker
2 Agricultural Workers
5 Experimental Subjects
4 Agricultural Workers
5 Agricultural Workers
6 Agricultural Workers
32 Agricultural Workers
1 Agricultural Worker
16 Agricultural Workers
1 Agricultural Worker
21 Agricultural Workers
149 Agricultural Workers
4 Agricultural Workers
23 Agricultural Workers
1 Agricultural Worker
2 Manufacturing Workers
5 Experimental Subjects
Blood DNOC
Level (mg/kg)
10003, b
2003, b
75
60
60a»b
55
44-55
40-48
20-403
30-40
21-403
7-3?a
303
20-30
253
10-20
<10
4_ga,b
l-8a»b
<53,b
10-20
20
Effects
Death
Sweating, labored
breathing, vomiting
Death
Headache, lassitude,
BMR 275%
Sweating, headache,
labored breathing, fatigue
Unconsciousness
Acute Poisoning
Headache, lassitude,
malaise
Liver damage
No effects
Moderate poisoning;
recovery period longer
than 8 days
Mild poisoning; recovery
within 8 days
Fever
No effects
Kidney damage
No effects
No effects
Sweating, thirst
No effects
Fatigue
No effects
Exaggerated feeling of
well-being
*Source: NIOSH, 1978
Reported as mg/1
bSerum or Plasma DNOC Level
BUR = Basal Metabolic Rate
:-ao
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In conparing studies on blood DNOC levels, certain orecautions must 3e
taken when correlating the results. It has been reported that over 90 per-
cent of the DNOC detected in the blood is found in serum (Parker, et al.
1951) and that most of this amount is bound to albumin in humans (Harvey, et
al. 1951). A comparison of numerically similar blood DNOC levels expressed
as weight/volume of serum with those expressed as weight/weight of whole
blood can therefore only be done by approximate conversions. Any given O.NOC
serum level will have a lower value when expressed per unit of whole blood.
It is impossible to develop a dose-response relationship for occupa-
tional DNOC poisoning in humans since air concentrations of DNOC are rarely
reported and the exposure time of poisoned individuals is highly variable.
In most cases of human poisoning total exposure amounts can only be esti-
mated. This lack of data make's assessment of a minimum toxic dose for
humans extremely difficult. Several studies however, where the oral tox-
icity of ONOC has been assessed in humans, shed some light on this question.
Harvey, et al. (1951) orally administered DNOC to five male volunteers
and studied both the resulting blood, levels and toxic effects. Each man was
given capsules containing 75 mg of pure DNOC daily for five consecutive
days, amounting to a total dose of 0.92 to 1.27 mg/kg/day. The men exper-
ienced an exaggerated sense of well-being when blood levels were about 20
mg/kg. Headache, lassitude, and malaise were associated with DNOC blood
levels of 40 to 48 mg/kg. Although individual variation was evident in
these experiments, it is obvious that chronic administration of 1 mg/kg/day
DNOC to healthy humans may result in signs of toxicity. The exaggerated
sense of well-being described by Harvey, et al. (1951) is a typical sign of
impending toxic effects among agricultural workers exposed to DNOC.
C-81
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ONOC was introduced in 1933 as an alternative to dinitrophenol for tie
treatment of obesity (NIOSH, 1978). Many poisonings, and some deaths, re-
sulting from overdoses were reported, as well as the development of catar-
acts in some patients, months after they had stopped taking ONOC. Some
patients developed symptoms of DNOC poisoning at the accepted theraputic
dose level. Signs and symptoms of DNOC intoxication including thirst,
fatigue, excessive sweating, decreased appetite, and elevated basal metabol-
ic rates, appeared in three persons who had taken as little as 0.35 to 1.5
mg/kg/day of ONOC for up to 9 weeks (Plotz, 1936). Hunter (1950) noted
that, although, less than one percent of those individuals treated with ONOC
developed complications, he considered the difficulty of setting a safe dose
for each individual to be the reason that its use as an aid to weight loss
was discontinued.
Although ONOC is considered a cumulative poison in humans, probabl-y as a
result of slow metabolism and inefficient excretion, true chronic or sub-
acute effects (with the possible exception of cataract formation) have never
been reported in either human or experimental animals. Signs and symptoms
of toxicity occur when the total body burden exceeds a threshold level. The
toxic effects noted after either acute or chronic administration are similar
in quality and their severity appears to be correlated with DNOC blood
levels (and by inference, total body burden). It is generally agreed that
the toxic manifestations of DNOC result from its potent effects on metabol-
ism (NIOSH, 1978).
Several long-term studies designed to determine dietary levels of DNOC
necessary to cause toxic symptoms in experimental animals have been con-
ducted. Spencer, et al. (1948) maintained rats on a diet containing DNOC
for six months. Growth curves, periodic blood counts, analyses of urea-
C-82
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nitrogen, organ weights, and nistrcpathological examinations were performed
on a:l animals. 'Jo adverse effects on these parameters were detected among
rats fed diets containing 100 mg 3NOC/"
-------
health effects in humans. Exposure to airborne ONOC at concentrations that
averaged 0.9 mg/m produced unspecified changes in the cardiovascular sys-
tem, the central and autonomic nervous systems, the gastrointestinal tract,
and the cell pattern of the peripheral blood of workers involved in manufac-
turing and applying DNOC. In agricultural workers exposed to ONOC at an
average concentration of 0.7 mg/m , slight unspecified changes in the
blood and autonomic nervous system were observed.
Another study (Batchelor, et al. 1956) revealed that agricultural spray-
ers exposed to an airborne ONOC concentration about 0.23 mg/m failed to
demonstrate adverse effects of the compound. No symptoms of poisoning were
observed and blood ONOC levels were well below those associated with toxic
effects.
In the study by Burkatskaya (1965) the effect of airborne ONOC on cats
was examined. Cats exposed at 0.2 mg/m for two or three months had
slightly increased body temperatures and leucocyte counts and decreased hem-
oglobin concentrations, erythrocyte counts, and catalase and peroxidase ac-
tivities. The changes, which were characterized as slight and transient,
occurred after one to two weeks but further exposure produced no additional
effects.
The report by NIOSH (1978) concludes "since only slight effects were
seen in workers exposed to ONOC at an average concentration as low as 0.7
mg/m for an unspecified duration, and since short-term exposure at 0.2
mg/m had no lasting effect on cats." NIOSH recommends that the current
federal workplace environmental limit of 0.2 mg/m be retained.
It is possible to calculate the anticipated daily exposure of a 70 kg
human male exposed to ONOC at 0.2 mg/m for an 8-hour period. If one as-
sumed the average minute volume was 28.6 1 of air/minute (NIOSH, 1973) the
anticipated daily exposure is 39 ug/kg/day.
C-84
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If one assures that abso^ot'on of 3NGC across the respiratory tract, -s
identical to gastrointestinal absorption, and that a 70 kg human male con-
sumes 2.0 liters of water daily, the following calculation indicates the
maximum allowable levels of DNOC in drinking water based on the NIOSH
recommendation for workplace air.
39 ug/kg/day x 70 kg = 2.73 mg/day
2.75 mq/day m 1.33 mg/i
2 I/day
Although NIOSH (1978) states "the standard was not designed for the pop-
ulation-at-large, and any extrapolation beyond the occupational environment
is not warranted," development of a baseline level for chronic human effects
using the same data used by NIOSH appears to be a reasonable approach to the
development of a water criterion.
In summary, daily human exposure to 0.35 mg/kg ONOC may result in signs
of intoxication in humans. Some persons develop cataracts as a result of
chronic exposure to DNOC, but the no-effect-level for cataracts cannot be
calculated. Although true "chronic" effects of DNOC have never been docu-
mented, the compound accumulates in the human body and toxic symptoms may
develop when blood levels exceed 20 mg/kg. Such symptoms have been observed
in humans receiving as little as 0.35 mg/kg/day over a period of several
weeks. The no-observable-effeet-level for rats in long-term feeding studies
has been variously reported as 63 mg/kg food, 100 mg/kg food, and 200 mg/kg
food. Based on the available human and experimental animal data, NIOSH
(1973) has recommended a federal workplace limited of 0.2 DNOC/m air.
Based on an estimate of iman exposure for an eight-hour work shift, it was
calculated that a drinking ••ter level of 1.4 mg/1 would result in a similar
exposure to the general population.
C-85
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De*-tinent information could not be located in the available literature
describing synergistic or antagonistic effects associated with DNOC.
Ta>-atocem'ci ty
Pertinent information could not be located in the available literature
regarding the presence or absence of teratogenic properties of DNOC.
Mutagenicity
Andersen, et al. (1972) reported an evaluation of the ability of 110
herbicides, including DNOC, to produce point mutations in histidine-depen-
dent mutants of Salmonella typhimurium, bacteriophage T4, and in two RII
mutants of bacteriophage T4. The culture media were prepared by mixing
freshly grown cultures of the mutants with soft agar and pouring into petri
dishes. After the agar solidified, DNOC was applied to the surface of each
plate. They found that the mutation frequency rates produced by ONOC were
no greater than the spontaneous rates.
Nagy, et al. (1975) tested ONOC for its ability to induce back-mutations
of her* and her" derivatives of E_. col i WP2 Try- bacteria. DNOC failed to
induce reverse mutations in this system.
The difference in growth inhibitions of wild type Proteus mirabilis and
the corresponding repair-deficient strain has been used by Adler, et al.
(1976) as an indication of DMA damage. Evidence of DNA damage in the pres-
ence of ONOC was reported.
Information on the potential mutagenicity of DNOC for mammals is not
available.
C-86
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Carcinogenicity
Spencer, et al. (1948) failed to report tumor formation in rats main-
tained on diets containing ONOC for six moths. Similarly, no tumors were
reported in rats maintained on diets containing DNOC for 105 days (Ambrose,
1942) or 126 days (Parker, et al. 1951).
No further information was found regarding the presence or absence of
carcinogenic properties of DNOC.
C-87
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Existing 3ui3e1ines and Standards
U.S. standards for exoosure to the nitrophenols or dinitrocresols in
drinking or ambient water have not been set.
The following limits for toxic substances in drinking water have been
set in the USSR (Stofen, 1973):
2-nitrophenol 0.06 mg/1
3-nitrophenol 0.06 mg/1
4-nitrophenol 0.02 mg/1
2,4-dinitrophenol 0.03 mg/1
Based on organoleptic considerations, a limit of 0.5 mg/1 for 2,4,6-
trinitrophenol has been set by the USSR (Stofen, 1973).
The maximum air concentration established by the American Conference of
Governmental Industrial Hygienists (ACGIH, 1971) is 0.1 mg/m for 2,4,6-
trinitrophenol and 0.2 mg/m for 4,6-dinitro-o-cresol for an eight-hour
exoosure (TLV).
The Code of Federal Regulations (40 CFR Part 180) establishes a toler-
ance of 0.02 rug/kg for residues of 4,6-dinitro-o-cresol and its sodium salt
in or on apples resulting from applications to apple trees at the blossom
stage as a fruit-thinning agent.
Current Levels of Exposure
Human exposure to the nitrophenols or dinitro-o-cresols has not been
monitored. Unspecified amounts of 4-nitrophenol have been detected in sam-
ples of urban ambient particulate matter.
The photochemical reaction between benzene vapor and nitrogen monoxide
results in the production of 2-nitrophenol, 4-nitrophenol, 2,4-nitrophenol,
and 2,6-dinitrophenol under laboratory conditions and 4-nitrophenol has been
:-88
-------
detected in rainwater in Japan. Available data indicate that the general
public may be exposed to nitrophenols in the atmosphere when severe photo-
chemical fog conditions develop. Quantitative estimates of such exposures
are not possible at the present time.
4-Nitrophenol has been detected in the urine of 1.0 percent of the gen-
eral population at levels as high as 0.1 mg/1 (with a mean urinary level of
10 ug/1).
If it is assumed that urinary residues of 4-nitrophenol reflect direct
exposure to the compound, a pharmacokinetic estimate of exposure based on
steady-state conditions can be made. The exposure level leading to the 1.0
ug/1 residue can be calculated as follows.
Exposure = (10 ug/1) (1.4 1 of urine/day) = o.02 ug/kg/day
(70 kg/man)
A similar calculation using the maximum urine residue level observed
(113. ug/1) gives an exposure of 2.26 ug/kg/day.
However, these urine levels are not oelieved to result from direct expo-
sure to 4-nitrophenol. A number of widely used pesticides, including para-
thion, are readily metabolized to 4-nitrophenol in the human body and are
believed to be the source of 4-nitrophenol residues in human urine.
Current levels of human exposure to the nitrophenols or dinitrophenols
(with the possible exception of 4-nitrophenol) are either very low, nonexis-
tent, or have gone undetected. In the absence of data any of the above
could be operative.
Special Groups at Risk
The only individuals expected to be at risk for high exposure to the
nitrophenols are industrial workers involved in the manufacture of compounds
for which the m'trophenols are intermediates. Since picric acid (2,4,6-tri-
C-89
-------
chloroohenol) ~nay find some jse as an explosive, germicide, tanni-ng agent,
fungicifle, tissue fixative, or industrial process material, a higher ris< of
exposure exists among personnel engaged in such operations.
Although 4,6-dinitro-o-cresol (DfJOC) is no longer manufactured in the
U.S., a limited quantity is imported and used as a blossom-thinning agent on
fruit trees and as a fungicide, insecticide, and miticide on fruit trees
during the dormant season. Hence, individuals formulating or spraying the
compound incur the highest risk of exposure to the compound.
3asis and Derivation of Criterion
The -ganoleptic thresholds for mononitrophenols in water range from
0.24 to 339 mg/1. These levels, extracted from the Russian literature, are
detection thresholds; acceptability thresholds from the standpoint of human
consumption are not available.
With the exception of a single study abstracted from the Russian litera-
ture, data on chronic mammalian effects of the mononitrophenols are absent
from the 1iterature.
The Russian investigation (Makhinya, 1969) was reported in abstract form
only. Attempts to obtain the full report proved fruitless. The investiga-
tors reported distinct cumulative toxic properties of the mononitrophenol
isomers in mammals. Threshold levels for effects of mononitrophenols on
conditioned reflexes were reported, but details of the experiment including
animal species, mode of administration, duration of the experiment, and the
exact parameters measured are not available. Hence, it does not seem pru-
dent to develop a criterion based on these results.
In the absence of data on chronic mammalian effects no water criterion
for human health can be established for any of th .• mononitrophenol isomers
at this time.
C-90
-------
Information on the dinitrophenol isomers is limited to 2,4-dinitro-
phenol. Spencer, et al. (1943), in a six-month feeding study with rats ad-
ministered 5 dietary levels of 2,4-dinitrophenol, demonstrated the no-ob-
served-effect-level (NOEL) to be between 5.4 mg/kg and 20 mg/kg. Using the
lower of the two figures and assuming a 70 kg man consumes 2 liters of water
daily and 6.5 grams of contaminated fish having a BCF of 1.51 the corre-
sponding NOEL for humans based on the results obtained in rats
may be calculated as follows:
5.4 mg/kg x 70 kg = 378 mg
378 mg = i88 mg/1
2 liters + (1.51 x 0.0065) x 1.0
Based on these calculations, no biological effect would be predicted in
a man drinking water containing 2,4-DNP at 188 mg/1.
Experience with the use of 2,4-DNP as an anti-obesity drug in the 1930's
indicates that adverse effects, including cataract formation, may occur in
utimans exposed to as little as 2 mg/kg/day. The drug was frequently used in
an uncontrolled manner and the available data do not allow the calculation
of a no-adverse-effect-level in humans. It is clear, however, that inges-
tion of 2,4-ONP at 2 mg/kg/day for a protracted period may result in adverse
effects, icluding cataracts, in a small proportion of the population. This
dietary intake level consitutes a low-observed-adverse-effect-level
(LOAEL). Assuming a 70 kg man consumes 2 1 of water daily and 6.5 grams of
contaminated fish having a BCF of 1.51 and assuming 100 percent
gastrointestinal absorption of 2,4-ONP, a 2 mg/kg dose of 2,4-ONP
would result if drinking water contained 2,4-ONP at 69.7 mg/1.
2mq/kg/day x 70 kg = 69.7 (or - 70 mg/1)
(2 liters + (1.51 x 0.0065) x 1.0
C-91
-------
According to current guidelines, extrapolation from a LOAEl requires ap-
plying an uncertainty factor of 10. Furthermore, in light of the demon-
strated bacterial mutagenicity of 2,4-DNP (Oemerec, et al . 1951) and the
suspected ability of the compound to induce chromosomal breaks in mammals
(Mitra, and Manna, 1971), an additional uncertainty factor of 100 must oe
used in the criterion formulation.
The suggested water criterion for 2,4-DNP is, therefore:
70 mg/1 =» 70 ug/1
10 x 100
If exposure is assumed to result from the consumption of contaminated fish
or shellfish only, the criterion is 14.3 mg/1.
The available data are insufficient to enable calculation of water cri-
terion levels for the remaining dinitrophenol isomers. For the present, it
seems reasonable to assume that the 2,4-dinitrophenol criterion would be ap-
propriate for the other isomers.
Chronic mammalian toxicilogy data for the trinitrophenols are absent
from the literature. An outbreak of microscopic hematuria among shipboard
U.S. Navy personnel exposed to 2,4,6-trinitrophenol in drinking water has
been reported, however. It is not possible to precisely estimate either the
2,4,6-trinitrophenol water level or duration of exposure required for the
development of hematuria. Consequently, criteria for trinitrophenol cannot
be derived.
Although 4,6-dinitro-o-cresol (DNOC) is considered a cumulative poison
in humans, probably as a result of slow metabolism and inefficient excre-
tion, true chronic or subacute effects have never been reported in either
humans or experimental animals. Since DNOC is not a cumulative poison in
experimental animals, extrapolation to humans from long-term animal studies
is of questionable value.
C-92
-------
The no-ODservab'e-effee'-level i'JCEL) for 3'
-------
Sufficient data is not available with which to derive
teria for other dinitro-o-cresol isomers.
C-94
-------
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