NITROPHENOLS
Ambient Water Quality Criteria
Criteria and Standards Division
Office of Water Planning and Standards
U.S. Environmental Protection Agency
Washington, D.C.
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CRITERION DOCUMENT
NITROPHENOLS
CRITERIA
Aquatic Life
2-nitrophenol
The data base for freshwater aquatic life is insuffi-
cient to allow use of the Guidelines. The following recommenda-
tion is inferred from toxicity data on 4-nitrophenol and salt-
water organisms.
For 2-nitrophenol the criterion to protect freshwater
aquatic life as derived using procedures other than the Guide-
lines is 2,700 ug/1 as a 24-hour average and the concentration
should not exceed 6,200 ug/1 at any time.
For saltwater aquatic life, no criterion for 2-nitro-
phenol can be derived using the Guidelines, and there are insuf-
ficient data to estimate a criterion using other procedures.
4-nitrophenol
The data base for freshwater aquatic life is insuffi-
cient to allow use of the Guidelines. The following recommenda-
tion is inferred from toxicity data on 4-nitrophenol and salt-
water organisms.
For 4-nitrophenol the criterion to protect freshwater
aquatic life as derived using procedures other than the Guide-
lines is 240 ug/1 as a 24-hour average and the concentration
should not exceed 550 ug/1 at any time.
For 4-nitrophenol the criterion to protect saltwater
aquatic life as derived using the Guidelines is 53 ug/1 as a
24-hour average and the concentration should not exceed 120 ug/1
at any time.
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2,4-d initrophenol
The data base for freshwater aquatic life is insuffi-
cient to allow use of the Guidelines. The following recommenda-
tion is inferred from toxicity data on 4-nitrophenol and salt-
water organisms.
For 2,4-dinitrophenol the criterion to protect fresh-
water aquatic life as derived using procedures other than the
Guidelines is 79 ug/1 as a 24-hour average and the concentration
should not exceed 180 ug/1 at any time.
The data base for saltwater aquatic life is insuffi-
cient to allow use of the Guidelines. The following recommenda-
tion is inferred from toxicity data on 4-nitrophenol and salt-
water organisms.
For 2,4-dinitrophenol the criterion to protect salt-
water aquatic life as derived using procedures other than the
Guidelines is 37 ug/1 as a 24-hour average and the concentration
should not exceed 84 ug/1 at any time.
2,4-d initro-6-methyIphenol
The data base for freshwater aquatic life is insuffi-
cient to allow use of the Guidelines. The following recommenda-
tion is inferred from toxicity data on 4-nitrophenol and salt-
water organisms.
For 2,4-dinitro-6-methylphenol the criterion to protect
freshwater aquatic life as derived using procedures other than
the Guidelines is 57 ug/1 as a 24-hour average and the concentra-
tion should not exceed 130 ug/1 at any time.
For saltwater aquatic life, no criterion for 2,4-di-
nitro-6-methylphenol can be derived using the Guidelines, and
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there are insufficient data to estimate a criterion using other
procedures.
2,4,6-trinitrophenol
The data base for freshwater aquatic life is insuffi-
cient to allow use of the Guidelines. The following recommenda-
tion is inferred from toxicity data on 4-nitrophenol and salt-
water organisms.
For 2,4,6-trinitrophenol the criterion to protect freshwater
aquatic life as derived using procedures other than the Guide-
lines is 1,500 ug/1 as a 24-hour average and the concentration
should not exceed 3,400 ug/1 at any time.
The data base for saltwater aquatic life is insuffi-
cient to allow use of the Guidelines. The following recommenda-
tion is inferred from toxicity data on 4-nitrophenol and salt-
water organisms.
For 2,4,6-trinitrophenol the criterion to protect salt-
water aquatic life as derived using procedures other than the
Guidelines is 150 ug/1 as a 24-hour average and the concentration
should not exceed 340 ug/1 at any time.
Human Health
To protect human health from the adverse effects of various
nitrophenols ingested in contaminated water and fish, suggested
criteria are as follows:
Mononitrophenols no criterion
Dinitrophenols 68.6 ug/1
Trinitrophenols 10 ug/1
Dinitrocresols 12.8 ug/1
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MONONITROPHENOLS
Introduction
Mononitrophenol has three isomeric forms, distinguished
by the position of the nitro group 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-nitrophenol, respectively.
Commercial synthesis of 2-nitrophenol and 4-nitrophenol
is accomplished through the hydrolysis of the appropriate
chloronitrobenzene isomers with aqueous sodium hydroxide at
elevated temperatures (Howard, et al. 1976). Production of
3-nitrophenol is achieved through the diazotization and hy-
drolysis of m-nitroaniline (Matsuguma, 1967). The mononitro-
phenol isomers are used in the United States primarily as in-
termediates for the production of dyes, pigments, Pharmaceu-
ticals, rubber chemicals, lumber preservatives, photographic
chemicals, and pesticidal and fungicidal agents (U.S. Int.
Trade Comm., 1976). As a result of this use pattern, the
major source for environmental release of mononitrophenols
is likely from production plants and chemical firms where the
compounds are used as intermediates. The mononitrophenols
may also be inadvertently produced via microbial or photodeg-
radation of pesticides which contain mononitrophenol moie-
ties. Approximately 10 to 15 million pounds of 2-nitrophenol
are produced annually (Howard, et al. 1976) for uses includ-
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ing synthesis of o-aminophenol, o-nitroanisole, and other dye
stuffs (Matsuguma, 1967; Howard, et al. 1976). Although pro-
duction figures for 3-nitrophenol are not available, Hoecker,
et al. (1977) estimate that production is less than one mil-
lion pounds annually. 3-Nitrophenol is used in the manufac-
ture of dye intermediates such as anisidine and m-aminophenol
(Kouris and Northcott, 1963; Matsuguma, 1967). 4-Nitrophenol
is probably the most important of the mononitrophenols in
terms of quantities used and potential environmental contam-
ination. Demand for 4-nitrophenol was 35,000,000 pounds in
1976 and production is projected to increase to 41,000,000
pounds by 1980 (Chem. Market. Reporter, 1976). Most of the
4-nitrophenol produced (87 percent) is used in the manufac-
ture of ethyl and methyl parathions. Other uses (13 percent)
include the manufacture of dye-stuffs and n-acetyl-p-amino-
phenol (APAP) and leather treatments. A possible source of
human exposure to 4-nitrophenol is as a result of microbial
or photodegradation of the parathions. In vivo production of
4-nitrophenol following absorption of parathion or other
pesticides by humans is another possible source of human
exposure.
Physical and chemical properties of the mononitrophenols
are summarized in Table 1.
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TABLE 1
Properties of Mononitrophenols
Formula
Molecular Weight
Melting Point (°C)
Boiling Point
Density
Water Solubility
(g/D
Vapor Pressure
Ka
2-Nitrophenol
C6H5N03
139.11
44-45
214-216
1.485
0x3.2 at 38°C
1x0.8 at 100°C
1 mm Hg at
49.3°C
7.5xlO-8
3-Nitrophenol
C6H5N03
139.11
97
194
1.485
1x3.5 at 25°C
13x3.0 at 90°C
5.3x10-9
4-Nitrophenol
C6H5N03
139.11
113-114
279
1.479
0x8.04 at 15°C
1x6.0 at 25°C
7xlO-8
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DINITROPHENOLS
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-dinitrophenol is by far the most important. The most
recent production figure for 2,4-dinitrophenol is 863,000 Ib
reported by the U.S. International Trade Commission (1968).
Approximate consumption per year is estimated at 1,000,000 Ib
(Howard, et al, 1976). 2, 4-dinitrophenol is used primarily
as a chemical intermediate for the production of sulfur dyes,
azo dyes, photochemicals, pest control agents, wood
preservatives, and explosives (Matsuguma, 1967; Perkins,
1919; Springer, et al. 1977a,b).
Production figures and usage data for the remaining five
dinitrophenol isomers are not available. It is reasonable to
assume that production and usage of these compounds are ex-
tremely limited in the United States.
Commercial synthesis of 2,4-dinitrophenol is accom-
plished by the hydrolysis 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
from production plants and chemical firms where th% compound
is used as an intermediate. It is possible that 2,4-DNP may
also be produced via microbial or photodegradation of com-
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pounds which contain the dinitrophenol moiety, such as Para-
thion (Gomaa and Faust, 1972). 2,4-DNP has also been identi-
fied as an impurity in technical preparations of the herbi-
cide DNPP (2-isopropyl-4,6-dinitrophenol) by Mosinska and
Kotarski (1972).
The physical and chemical properties of the dinitro-
phenol isomers are summarized in Table 2.
TABLE 2
Properties of Dinitrophenol Isomers3
Isomer
m. p .
(°C)
K
(at 25°C)
Water
Solubility
Density
(g/D
2
2
2
2
3
3
, 3-Dinitrophenol
, 4-Dinitrophenol
, 5-Dinitrophenol
, 6-Dinitrophenol
, 4-Dinitrophenol
, 5-Dinitrophenol
144
114-115
(sublimes )
104
63.5
134
122-123
1.
1.
2.
4.
2.
3 x
0 X
7 x
7 x
3 x
1 x
10-5
10-4
10~6
10-4
10-5
10-4
2.
0.
0.
0.
2.
1.
2
79
68
42
3
6
1.681
1.683
1.672
1.702
a Source: Harvey, 1959; Windholz, 1976; Weast, 1975.
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TRINITROPHENOLS
Six isomeric forms of trinitrophenol are possible,
distinguished by the position of the nitro groups relative to
the hydroxy group on the six carbon benzene ring. The five
isoraers are: 2,3,4-, 2,3,5-, 2,3,6-, 2,4,5-, 2,4,6- and 3,4,
5-trinitrophenol. Production volumes for the trinitrophenols
are not available. Usage of the trinitrophenol isomers is
apparently limited to 2,4,6-trinitrophenol, otherwise known
as picric acid. In fact, a comprehensive 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 is the chemical and physical properties
found in Table 3.
According to Matsuguma (1967) picric acid has found
usage as: a dye intermediate, 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, steel and
copper surfaces. The extent to which picric acid finds usage
in any of these applications at the present time is unknown.
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TABLE 3
Properties of Trinitrophenols
2,3,4-Trinitrophenol
Molecular Weight
229.11
2 ,3 , 5-Trinitrophenol
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-Trinitrophenol
Molecular Weight
Melting Point
Water Solubility
Room Temperature
Hot Water
229.11
96°C
Slightly Soluble
Soluble
2 , 4,6-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
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DINITROCRESOLS
Dinitro-ortho-cresol is a yellow crystalline solid
derived from o-cresol. 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 dinitro cresol isomers.
4,6-dinitro-o-cresol (hereafter referred to as DNOC) is
produced either by sulfonation of o-cresol followed by treat-
ment with nitric acid or by treatment of o-cresol in glacial
acetic acid with nitric acid at low temperature. Some impor-
tant chemical and physical properties of DNOC are shown in
Table 4.
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
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An excellent review of the toxicological effects of DNOC
on human and laboratory animals has recently been published
by the National Institute for Occupational Safety and Health
(1978). In view of the comprehensive coverage of both Eng-
lish and foreign language literature, no attempt will be made
to duplicate this impressive effort within this criterion
document. Key papers used for criterion formulation will be
cited, where appropriate, and frequent reference to the NIOSH
review will be used where the available literature does not
contain information directly relevant to criteria formulation.
DNOC usage in the U.S. has declined in recent years be-
cause the compound is highly toxic to plants in the growing
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 agricul-
tural pesticides.
The Environmental Protection Agency has no record of
DNOC being currently 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 (Natl.
Inst. Occup. Safety Health, 1978). Since DNOC is not manu-
factured in the U.S., pesticide formulators and sprayers 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 season. NIOSH (1978) esti-
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mates that 3,000 workers in the U«So are potentially 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.
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
significance of such studies as related to the stability of
nitrophenols in the environment is not known.
Several investigators have shown that individual species
of aerobic and anaerobic bacteria, including Azotabacter
chroococcum and Clostridium butyrium, and the fungus Fusar-
ium, are capable of reducing 2,4-dinitrophenol in culture
(Radler, 1955; Lehmber, 1956; Madhosingh, 1961). However,
the precise pathway for metabolic degradation is not known.
Jensen and Lautrup-Larson (1967) found that Arthrobacter sim-
plex, 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 reported 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 sta-
bility of nitrophenols in the environment is not known. Cer-
;
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tain investigators have postulated that ambient nitrophenol
concentrations may be too low to induce the appropriate mi-
crobial enzymes necessary to facilitate population growth and
metabolism of the compounds (U.S. EPA).
Information regarding the mobility and persistence of
nitrophenols in natural soil and water environments is limit-
ed. Based upon experimentally determined solubilities and
sorption characteristics, the persistence of some of the ni-
trophenols might be estimated. For example, although 2-ni-
trophenol is soluble in water, it has also been shown to be
strongly attracted through hydrogen bonding to montmorillon-
ite clays, perhaps reducing its movement through the ground-
water regime (Saltzman and Yariv, 1975; Yariv, et al. 1966).
However, these estimates do not consider the data abailable
on microbial, photolytic, and oxidative degradation available
in the literature.
No measured steady-state data are available regarding
the bioconcentration of nitrophenols. However, BCF's are es-
timated in this document using the octanol-water partition
coefficients. Only limited no data are available on the
levels of nitrophenols in municipal effluents or treated
drinking waters.
None of the nitrophenols addressed in this document is
found to be carcinogenic, mutagenic, or teratogenic; however,
because of their widespread use as agricultural chemicals,
their toxicity to microorganisms, fish, and mammals, the ni-
trophenols pose a potential threat to aquatic and terrestrial
life, including man.
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REFERENCES
Chambers, C.W., et al. 1963. Degradation of aromatic com-
pounds by phenol-adapted bacteria. Jour. Water Pollut. Con-
trol Fed. 35: 1517.
Gomaa, H.M., 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 Aqua-
tic Environ.
Guillaume, J., et al. 1963. Oxidation of p-nitrophenol by
certain Mycobacteria. Compt. Rend. 256: 1634.
Harvey, D.G. 1959. On the metabolism of some aromatic nitrb
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. Pharmacol.
11: 462.
Hoecker, J.E., et al. 1977. Information profiles on poten-
tial occupational hazards. Nat. Inst. Occup. Safety Health,
Cincinnati, Ohio.
Howard, H., et al. 1976. Investigation of selected potential
environmental contamination: Nitroaromatics. Off. Tox. Subs.
U.S. Environ. Prot. Agency, Washington, D.C.
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Jensen, H.L., and G. Lautrup-Larson. 1967. Microorganisms
that decompose nitro-aromatic compounds, with special refer-
ence to dinitro-o-cresol. Acta Agric. Scand. 17: 115.
Kouris, C.S., and J. Northcott. 1963. Aniline and its deriva-
tives. Page 411 rn Kirk-Othmer Encyclopedia of Chemical Tech-
nology. 2nd ed. Vol 2.
Lehmber, C. 1956« Untersuchungen uber die Winbung von Ascor-
bunsaure, Stoffwechselgifren and Anderen Faktoren auf den
Staffwechsel von Clostridium butyrium. Beif. Arch. Mikrobiol
24: 323.
Madhosingh, C. 1961. The metabolic detoxification of 2,4-di-
nitrophenol by Fusarium oxysporum. Can. Jour. Microbiol. 7:
553.
Matsuguma, H.J. 1967. Nitrophenols. Page 888 in. Kirk-Othmer
Encyclopedia of Chemical Technology. 2nd. ed. Vol. 13.
Mosinska, K., and A. Kotarski. 1972. Determination of 2-iso-
propyl-4,6-dinitrophenol and 2,4-DNP in herbicides and in
technical 2-isopropyl-4,6-DNP by TCL. Chemia Analityezma 17:
327.
National Institute for Occupational Safety and Health. 1978.
Criteria for a recommended standard: Occupational exposure to
dinitro-ortho-cresol. Dep. Health Edu. Welfare, Washington,
B.C.
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Perkins, R.G. 1919. A study of the munitions intoxications in
France. Pub. Health Rep. 34: 2335.
Radler, F. 1955. Untersuchunger uber den verlauf der stoff-
wech Selvorgangebei Azotobacter chroococcum. Beig. Arch.
Microbiol. 22: 335.
Raymond, D.G.M., and M. Alexander. 1971. Microbial metabolism
and cometabolism of nitrophenols. Pestic. Biochem. Physiol.
1: 123.
Saltzman, S., and S. Yariv. 1975. Infrared study of the sorb-
tion of phenol and p-nitrophenol by montmorillonite. Soil
Sci. Soc. Am. Proc. 39: 474.
Simpson, J.R., and W.C. Evans. 1953. The metabolism of
nitrophenols by certain bacteria. Biochem. 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-dinitrophenol treatment. Tapi 60: 88.
Springer, E.L. et al. 1977b. Evaluation of chemical treat-
ments to prevent deterioration of wood chips during storage.
Tapi 60: 93.
Tewfik, M.S., and W.C. Evans. 1966. The metabolism of 3,5-di-
nitro-o-cresol (DNOC) by soil microorganisms. Biochem. Jour.
99: 31. /
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U.S. EPA. 1976. Investigation of selected potential environ-
mental contaminants: nitroaromatics. Final Rep. Off. Tox.
Subst. Washington, D.C.
U.S. International Trade Commission. 1967-73. Synthetic or-
ganic chemicals: U.S. production and sales. Washington, D.C.
U.S. International Trade Commission. 1976. Imports of benzen-
oid chemicals and products, 1974. Publ. No. 762. Washington,
D.C.
Weast, R.C., ed. 1975. Handbook of chemistry and physics.
57th ed. CRC Press.
Windholz, M., ed. 1976. The Merck Index. 9th ed. Merck and
Co., Rahway, N.J.
Yariv, S., et al. 1966. Infrared study of the absorption of
benzoic acid and nitrobenzene in montomorillonite. Israel
Jour. Chera. 4: 201.
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AQUATIC LIFE TOXICOLOGY*
FRESHWATER ORGANISMS
Introduction
Although fish apd 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 each
individual nitrophenol. There are no data available dealing with
chronic effects of any nitrophenol on freshwater aquatic
organisms, and no suitable substitute chronic value can be
determined from available toxicity information. The derivation
of a single criterion which would protect freshwater aquatic
organisms from all nitrophenols is impractical because of the
limited toxicity data for each compound and because of the wide
differences in toxicity results obtained for individual nitro-
phenols.
*The reader is referred to the Guidelines for Deriving Water
Quality Criteria for the Protection of Aquatic Life [43 FR 21506
(May 18, 1978) and 43 FR 29028 (July 5, 1978)] in order to better
understand 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 the calcula-
tions for deriving various measures of toxicity as described in
the Guidelines.
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Acute Toxicity
The fish acute toxicity data base (Table 1) consists of
eight LC50 values for five nitrophenols and two species of fresh-
water fish. Using adjusted values for the three nitrophenols for
which data are available for both fish species, bluegills were
found to be at least 13 times more sensitive than were fathead
minnows although the tests with fathead minnows were flow-through
with measured concentrations and the tests with bluegills were
static without measured concentrations. Comparisons of adjusted
LC50 values (Table 1) indicate that 2,4-dinitro-6-methylphenol is
the most toxic nitrophenol with 96-hour LC50 values of 126 ug/1
(U.S. EPA, 1978) and 2,040 ug/1 (Phipps, et al. manuscript) for
bluegills and fathead minnows, respectively. 2,4-dinitro-
6-methylphenol is followed in order of decreasing toxicity by
2,4-dinitrophenol, 4-nitrophenol, 2-nitrophenolf and 2,4,6-tri-
nitrophenol. The adjusted 96-hour LC50 values for 2,4-dinitro-
phenol are 339 ug/1 for bluegills (U.S. EPA, 1978) and 16,720
ug/1 for fathead minnows (Phipps, et al. manuscript). Toxicity
differences between the various mononitrophenol compounds are in-
dicated by the adjusted bluegill LC50 values of 4,527 ug/1 for
4-nitrophenol (U.S. EPA, 1978) and 24,139 ug/1 for 2-nitrophenol
(Lammering and Burbank, 1960). The high 2,4,6-trinitrophenol ad-
justed LC50 value of 91,299 ug/1 for bluegills (U.S. EPA, 1978)
indicates the toxicity of nitrophenols does not increase directly
with increasing nitro-group substitution. The Final Fish Acute
Values for 2-nitrophenol, 4-nitrophenol, 2,4-dinitrophenol, 2,4-
dinitro-6-methylphenol, and 2,4,6-trinitrophenol are 6,200, 4,200,
610, 130, and 23,000 ug/1, respectively.
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The data base for invertebrate species (Table 2) contains
seven data points for four nitrophenol compounds with two inver-
tebrate species. The order of toxicity of these four nitro-
phenols is the same for invertebrate species as observed with
fish. An unspecified dinitromethylphenol is the most toxic and
is followed in order of decreasing toxicity by 2,4-dinitrophenol,
4-nitrophenol, and 2,4,6-trinitrophenol. For 2,4-dinitrophenol,
the two adjusted LC50 values for daphnids are quite close and are
3,989 ug/1 (Kopperman, et al. 1974) and 3,464 ug/1 (U.S. EPA,
1978). The toxicity of 4-nitrophenol to daphnids showed greater
variation and adjusted LC50 values are 7,111 ug/1 (Kopperman, et
al. 1974) and 18,549 ug/1 (U.S. EPA, 1978). As previously noted
with fish, 2,4,6-trinitrophenol, with an LC50 value of 71,741
ug/1 for daphnids (U.S. EPA, 1978), is much less toxic than other
nitrophenols. The Final Invertebrate Acute Values for 4-nitro-
phenol, 2,4-dinitrophenol, 2,4-dinitro-6-methylphenol, and 2,4,6-
trinitrophenol are 550, 180, 130, and 3,400 ug/1/ respectively.
The data indicate that the Final Invertebrate Acute Values
for nitrophenols are lower than or equivalent to, as in the case
of 2,4-dinitro-6-methylphenol, comparable values for fish. Thus,
when a Final Invertebrate Acute Value exists, it becomes the
Final Acute Value. The Final Fish Acute Value of 6,200 ug/1 for
2-nitrophenol is the Final Acute Value for that compound.
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Chronic Toxicity
There are no data available on the chronic effects of any of
the various nitrophenols on freshwater aquatic life.
Plant Effects
Plant toxicity values (Table 3) are lower, in certain
instances, than adjusted acute toxicity values for fish and
invertebrate species. However, no plant toxicity effects are
lower than the Final Fish or Final Invertebrate Acute Values.
Tests which elicited the relative toxicity of the three isomeric
forms of mononitrophenols to plants (Huang and Gloyna, 1967)
indicated that chlorophyll synthesis in the alga, Chlorella
pyrenoidosa, was inhibited at 25,000 u9/l by 4-nitrophenol, at
35,000 jig/1 by 2-nitrophenol, and at 50,000 wg/1 by 3-nitro-
phenol. Studies with.three species of algae (Table 3) indicate
that 4-nitrophenol is more toxic to plants than is 2,4-dinitro-
phenol. The one exception to this toxicity trend was determined
by Simon and Blackman (1953), who found that 50 percent growth
reduction in duckweed, Lemna minor, occurred at 2,4-dinitrophenol
and 4-nitrophenol concentrations of 1,472 ug/1 and 9,452 ug/lf
respectively. As observed with fish and invertebrate species,
2, 4,6-trinitrophenol was less toxic to the alga, Selenastrum
capricornutum than was either 4-nitrophenol or 2,4-dinitrophenol
(U.S. EPA, 1978).
B-4
-------
Residues
No measured steady-state bioconcentration factors (BCFs) are
available for any nitrophenol. BCFs can be estimated using the
octanol-water partition coefficients of 32, 150 and 110 for
2,4-dinitrophenol, 2,4-dinitro-6-methylphenol, and 2,4,6-tri-
nitrophenol, respectively. These coefficients are used to derive
estimated BCFs of 8.1, 26, and 21 for 2,4-dinitrophenol,
2,4-dinitro-6-methylphenol and 2,4,6-trinitrophenol, respec-
tively, for aquatic organisms that contain about 8 percent
lipids. If it is known that the diet of the wildlife of concern
contains a significantly different lipid content, appropriate
adjustments in the estimated BCFs should be made. No estimates
can be made for 2-nitrophenol and 4-nitrophenol.
Miscellaneous
Table 4 contains no data that would be a suitable substitute
for a Final Chronic Value for any nitrophenol compound. All data
are for short duration (less than 96'hours) and none of the
toxicity values are below the lowest adjusted acute toxicity
values for fish or invertebrate species. One set of data (Table
4) indicates the relative toxicity of the three i-someric forms of
mononitrophenol to fish. Gersdorff (1939) found that 8,000 y.g/1
*
of 4-nitrophenol, 24,000 u.g/1 of 3-nitrophenol, and 33,300 ug/1
of 2-nitrophenol produced 42 percent, 53 percent, and 38 percent
mortality, respectively, in goldfish after 8 hours.
B-5
-------
CRITERION FORMULATION
Freshwater-Aquatic Life
Summary of Available Data
The concentrations below have been rounded to two significant
figures.
2-nitrophenol
Final Fish Acute Value = 6,200 ug/1
Final Invertebrate Acute Value = not available
Final Acute Value = 6,200 ug/1
Final Fish Chronic Value = not available
Final Invertebrate Chronic Value = not available
Final Plant Value = 35,000 ug/1
Residue Limited Toxicant Concentration = not available
Final Chronic Value = 35,000 ug/1
0.44 x Final Acute Value = 2,700 ug/1
4-nitrophenol
Final Fish Acute Value = 4,200 ug/1
Final Invertebrate Acute Value = 550 ug/1
Final Acute Value = 550 ug/1
Final Fish Chronic Value = not available
Final Invertebrate,Chronic Value = not available
Final Plant Value = 4,900 ug/1
Residue Limited Toxicant Concentration = not available
Final Chronic Value = 4,900 ug/1
0.44 x Final Acute Value = 240 ug/1
2,4-dinitrophenol.
Final Fish Acute Value = 610 ug/1
Final Invertebrate Acute Value = 180 ug/1
Final Acute Value! = 180 ug/1
B-6
-------
' Final Fish Chronic Value = not available
Final Invertebrate Chronic Value = not available
Final Plant Value = 1,500 ug/1
Residue Limited Toxicant Concentration = not available
Final Chronic Value = 1,500 ug/1
0.44 x Final Acute Value = 79 ug/1
2,4-dinitro-6-methylphenol
Final Fish Acute Value = 130 ug/1
Final Invertebrate Acute Value = 130 ug/1
Final Acute Value = 130 ug/1
Final Fish Chronic Value = not available
Final Invertebrate Chronic Value = not available
Final Plant Value = 50,000 ug/1
Residue Limited Toxicant Concentration = not available
Final Chronic Value = 50,000 ug/1
0.44 x Final Acute Value = 57 ug/1
2,4,6-trinitrophenol
Final Fish Acute Value = 23,000 ug/1
Final Invertebrate Acute Value = 3,400 ug/1
Final Acute Value = 3,400 ug/1
Final Fish Chronic Value = not available
Final Invertebrate Chronic Value = not available
Final Plant Value = 62,000 ug/1
Residue Limited Toxicant Concentration = not available
Final Chronic Value = 62,000 ug/1
0.44 x Final Acute Value = 1,500 ug/1
B-7
-------
No freshwater criterion can be derived for any nitrophenol
using the Guidelines because no Final Chronic Value for either
fish or invertebrate species or a good substitute for either value
is available.
Results obtained with 4-nitrophenol and saltwater organisms
indicate how criteria may be estimated for nitrophenols and fresh-
water organisms.
For 4-nitrophenol and saltwater organisms 0.44 times the
Final Acute Value is less than the Final Chronic Value which is
derived from results of an embryo-larval test with the sheepshead
minnow. Therefore, it seems reasonable to estimate criteria for
nitrophenols and freshwater organisms using 0.44 times the Final
Acute Valueo
2-nitrophenol
The maximum concentration of 2-nitrophenol is the Final Acute
Value of 6,200 ug/1 and the estimated 24-hour average concentra-
tion is 0.44 times the Final Acute Value. No important adverse
effects on freshwater aquatic organisms have been reported to be
caused by concentrations lower than the 24-hour average concentra-
tion.
CRITERION: For 2-nitrophenol the criterion to protect
freshwater aquatic life as derived using procedures other than the
Guidelines is 2,700 ug/1 as a 24-hour average and the concentra-
tion should not exceed 6,200 ug/1 at any time.
4-nitrophenol
The maximum concentration of 4-nitrophenol is the Final Acute
Value of 550 ug/1 and the estimated 24-hour average concentration
is 0.44 times the Final Acute Value. No important adverse effects
B-8
-------
on freshwater aquatic organisms have been reported to be caused by
concentrations lower than the 24-hour average concentration.
CRITERION: For 4-nitrophenol the criterion to protect fresh-
water aquatic life as derived using procedures other than the
Guidelines is 240 ug/1 as a 24-hour average and the concentration
should not exceed 550 ug/1 at any time.
2 ,4-dinitrophenol
The maximum concentration of 2,4-dinitrophenol is the Final
Acute Value of 180 ug/1 and the estimated 24-hour average concen-
tration is 0.44 times the Final Acute Value. No important adverse
effects on freshwater aquatic organisms have been reported to be
caused by concentrations lower than the 24-hour average concentra-
tion.
CRITERION: For 2,4-dinitrophenol the criterion to protect
freshwater aquatic life as derived using procedures other than the
Guidelines is 79 ug/1 as a 24-hour average and the concentration
should not exceed 180 ug/1 at any time.
2 / 4-d initro-6-methylphenol
The maximum concentration of 2,4-dinitro-6-methylphenol is
the Final Acute Value of 130 ug/1 and the estimated 24-hour
average concentration is 0.44 times the Final Acute Value. No
important adverse effects on freshwater aquatic organisms have
been reported to be caused by concentrations lower than the
24-hour average concentration.
CRITERION: For 2,4-dinitro-6-methylphenol the criterion to
protect freshwater aquatic life as derived using procedures other
than the Guidelines is 57 ug/1 as a 24-hour average and the
concentration should not exceed 130 ug/1 at any time.
B-9
-------
2,4,6-trinitrophenol
The maximum concentration of 2,4,6-trinitrophenol is the
Final Acute Value of 3,400 ug/1 and the estimated 24-hour average
concentration is 0.44 times the Final Acute Value. No important
adverse effects on freshwater aquatic organisms have been reported
to be caused by concentrations lower than the 24-hour average
concentration.
CRITERION: For 2,4,6-trinitrophenol the criterion to protect
freshwater aquatic life as derived using procedures other than the
Guidelines is 1,500 ug/1 as a 24-hour average and the concentra-
tion should not exceed 3,400 ug/1 at any time.
j-10
-------
Table I. Freshwater fish acute values for nitrophenols
Biodeeay Test Chemical Tine
!_ £20£*** pe script ton
Adjusted
LCbu U-'bO
iu
-------
Table 2. Freshwater invertebrate acute values for nltrophenola
Biotaaay Test
Adjuated
Chenical Time LCbO LOU
Heterence
**4
Do
K-1
N)
Cladoceran,
Daphnta magna
Cladoceran.
Daphnia magna
Cladoceran,
Daphnia magna
Cladoceran.
Daphnia magna
Cladoceran,
Daphnia roap.na
Cladoceran,
Daplmia magna
Stonefly (naiad),
Pteranarcya californica
irw ~ii~
s
s
s
s
s
s
s
u
u
u
u
u
u
u
4-Nitrophenol
i
4-Nltrophenol
2,4-
Dinitrophenol
2,4-
Dtnitrophenol
2,4,6-
Trinitrophenol
2,4-Dinttro-6-
methylphenol
Dinitromethyl-
phenol***
48
48
48
48
48
48
96
T ii^T- ' ~_
8.396
21.900
4.710
4,090
84,700
3.120
320
•
7.111
18.549
3.989
3.464
71,741
2.643
271
Kopperman. et
al. 1974
U.S. EPA, 1978
Kopperman, et
al. 1974
U.S. EPA, 1978
U.S. EPA, 1978
U.S.4 EPA, 1978
Sanders &
Cope, 1968
*S - static
.** U - unmeasured
***Thia LC50 value was not used in calculating any geometric mean because the dinitromethylphenol tested
was not specified. Authors reported results as dinitrocresol.
Geometric mean of adjusted values: 4-nitrophenol - 11,485 Mg/l ~2*T^~ * 55° MB/1
2,4-dinitrophenol - 3,717 Mg/l i'l~ " l*° »*E/1
2,4,6-trinltrophenol - 71,741 ng/l ~ll" " ^•e>QQ »'B/1
2,4-dinltro-6-methylphenol.- 2,643 Mg/l %f^ " ^30 »*B/l
-------
Tdbla 3. Freshwater plant effecta for nitrophenols
Concentration
Alga.
Chlorella
pyrenoidoaa
Alga.
Chlorella
pyfenoldoaa
Alga.
Chlorella
pyrenoidoaa
Alga.
Chlorella
03 Pyrenordoaa
1
W Alga.
Chlorella
pyrenaldoaa
Alga.
Chlorella vulgarla
Alga.
Chlorella vulgaria
Alga.
Selenastrum
caprlcornutum
Alga.
Selenastrum
caprlcornutum
Alga.
Selenaatruro
caprlcornutum
Inhibition of 35.000 Huang & Gloyna. 1967
chlorophyll 2-Nltrophenol
synthesis (
after 3 days
Inhibition of 50,000 Huang & Gloyna. 1967
chlorophyll 3-Nitrophenol
synthesis
after 3 days
Inhibition of 25.000 Huang «« Gloyna, 1967
chlorophyll 4-Nitrophenol
synthesis
after 3 days
Inhibition of 50,000 Huang & Gloyna. 1967
chlorophyll 2,4-
synthesis Dlnitrophenol
after 3 days
Inhibition of 50,000 Huang & Gloyna, 1967
chlorophyll 2.4-dlnltro-6-
aynthesis methylphenol*
after 3 days
50% growth 6,950 Dedonder & Van Sumere. 1971
inhibition 4-Nitrophenol
in 80 hrs
70% growth 9,200 Dedonder :& VaarSumere. 1971
inhibition 2.4-
in 60 hrs Dlnitrophenol
50% reduction 4,190 U.S. EPA. 1978
in chlorophyll 4-Nitrophenol
a in 96 hrs
50% reduction 9,200 U.S. EPA, 1978
in chlorophyll 2.4-
a in 96 hra Dlnitrophenol
50% reduction 41 700 U.S. EPA. 1978
in chlorophyll 2.4,6-
a In 96 hrs Trlnttrophenol
-------
Table 3, (Continued)
Concentration
Organism
Duckweed,
Lemna minor
Duckweed,
Lemna minor
Duckweed,
Lemna minor
Effect
501 growth
reduction
50% growth
reduction
501 growth
reduction
fuq/l|
62,550
2-Nitrophenol
9,452
4-Nitrophenol
1,472
2,4-
Dinltrophenol
petertnce
Simon & Blackman.
Simon & Blackman,
Simon & Blackman, •
1953
1953
1953
^Authors reported results as 4,6-dinitro-o-creaol.
CD
-------
Table A. Other freshwater data for nltrophenols
Organ!cm
Teat
QiLElilQQ £!££££.
Result
CO
I
t-1
en
Alga.
Chlamydomonaa
Amoeba ,
Amoeba proteus
30 sec
24 hrs
Amoeba,
Amoeba proteua
Southern bullfrog
(tadpole),
Rana grylip
Goldfish.
Carasslua auratua
Goldfish.
Carasslua auratus
Goldfish,
Carasslua auratua
50% Inhibition of
flagellar motlllty
46% reduction In
ameba containing
golgl bodies
48 hra 16% mortality
7 hra Increased
respiration
8 hra 38% mortality
8 hrs 53% mortality
6 hra 42% mortality
18.400 Marcus & Mayer, 1963
Dlnltrophenol
92,000 Fllcklnger, 1972
Dlnltrophenol
92,000 Fllcklnger, 1972
Dlnltrophenol
5.520 Lewia & Frleden, 1959
2,4-
Dlnltrophenol
33,300 Geradorff, 1939
2-
Nltrophenol
24,000 Gersdorff, 1939
3-
Nltrophenol
8,000 Geradorff, 1939
4-
Nltrophenol
-------
SALTWATER ORGANISMS
Introduction
The three nitrophenols having saltwater data are 4-nitro-
phenol, 2,4-dinitrophenol and 2,4,6-trinitrophenol. Since
2,4-dinitrophenol is known to uncouple oxidative-phosphosylation,
it is not surprising that it is the most toxic compound for both
invertebrate and fish species. No invertebrate chronic infor-
mation could be found and only one study of fish chronic toxicity
(4-nitrophenol) is available.
Acute Toxicity
The sheepshead minnow has been exposed for 96 hours (U.S.
EPA, 1978) to 4-nitrophenol, 2,4-dinitrophenol, and 2,4,6-tri-
nitrophenol; the adjusted LC50 values are 14,816, 16,073, and
73,258 ug/lf respectively (Table 5). As with freshwater fish
(Table 1), 2,4,6-trinitrophenol was less toxic than the other two
compounds. A test with embryos of the herring, Clupea harengus,
and 2,4-dinitrophenol (Rosenthal and Stelzer, 1970) provided an
adjusted LC50 value of 3,007 ug/1 as compared to the value for the
sheepshead minnow and the same chemical of 16,073 ug/l« The Final
Fish Acute Values for these nitrophenols after adjustment for test
methods and species sensitivity are 4,000 ug/1 (4-nitrophenol') ,
1,900 ug/1 (2,4-dinitrophenol) and 20,000 ug/1 (2,4,6-trinitro-
phenol) . ' •• •
The mysid shrimp, Mysidopsis bahia, has also been exposed
to the same nitrophenols (U.S. EPA, 1978) and, again, 2,4,6-=tri-
nitrophenol (96-hour LC50 of 16,686 ug/D was less toxic than
B-16
-------
4-nitrophenol (96-hour LC50 of 6,073 ug/D and 2,4-dinitrophenol
(96-hour LC50 of 4,108 ug/D (Table 6). In general, the LC50
values for the mysid shrimp were about 2 to 4 times lower than
comparable values for the sheepshead minnow. The Final Inverte-
brate Acute Values, and Final Acute Values since they are lower
than those for fish, are 120, 84, and 340 ug/1 for 4-nitrophenol,
2,4-dinitrophenol, and 2,4,6-trinitrophenol, respectively.
Chronic Toxicity
An embryo-larval test with the sheepshead minnow and 4-nitro-
phenol (U.S. EPA, 1978) is the only test with any nitrophenol that
provides a chronic value. This concentration is 6,325 ug/1 (Table
7) and is obtained by dividing the geometric mean of the highest
no observed effect and lowest observed effect concentrations by
two. The adverse effects observed were on hatching and survival.
These results are not much lower than the unadjusted 96-hour LC50
value of 27,100 ug/1 (Table 5) from the same study. The Final
Fish Chronic Value derived after use of the species sensitivity
factor (6.7) is 940 ug/1. This concentration is higher than the
Final Acute Value (120 ug/D » because the latter is based on the
more sensitive invertebrate species.
Plant Effects
The saltwater alga, Skeletonema costaturn, is more sensitive
to 4-nitrophenol with 96-hour EC50 values of 7,370 and 7,570 ug/1
for inhibition of chlorophyll a_ and cell number production,
respectively, than to 2,4-dinitrophenol and 2,4,6-trinitrophenol
B-17
-------
(Table 8). The Final Plant Values for 4-nitrophenol, 2,4-dinitro-
phenol, and 2,4,6-trinitrophenol are 7,400, 93,000, and 63,000
ug/1, respectively.
Residues
No measured steady-state bioconcentration factors (BCFs) are
available for any nitrophenol. BCFs can be estimated using the
octanol-water partition coefficients of 32, 150, and 110 for
2,4-dinitrophenol, 2,4-dinitro-6-methylphenol, and 2,4,6-trinitro-
phenol, respectively. These coefficients are used to derive
estimated BCFs of 8.1, 26, and 21 for 2,4-dinitrophenol,
2,4-dinitro-6-methylphenol and 2,4,6-trinitrophenol, respectively,
for aquatic organisms that contain about 8 percent lipids. If it
is known that the diet of the wildlife of concern contains a
significantly different lipid content, appropriate adjustments in
the estimated BCFs should be made. No estimates can be made for
2-nitrophenol and 4-nitrophenol.
Miscellaneous
The lethal threshold value after a 96-hour exposure of
Atlantic salmon to 2,4-dinitrophenol (Zitko, 1976) is 700 ug/1
(Table 9) which is lower than the Final Fish Acute Value (1,900
ug/1) but not the Final Acute Value (84 ug/D-
B-18
-------
CRITERION FORMULATION
Saltwater-Aquatic Life
Summary of Available Data
The concentrations below have been rounded to two significant
figures.
4-nitrophenol
Final Fish Acute Value = 4,000 ug/1
Final Invertebrate Acute Value = 120 ug/1
Final Acute Value = 120 ug/1
Final Fish Chronic Value = 940 ug/1
Final Invertebrate Chronic Value = not available
Final Plant Value = 7,400 ug/1
Residue Limited Toxicant Concentration = not available
Final Chronic Value = 940 ug/1
0.44 x Final Acute Value = 53 ug/1
2,4-dinitrophenol
Final Fish Acute Value = 1,900 ug/1
Final Invertebrate Acute Value = 84 ug/1
Final Acute Value = 84 ug/1
Final Fish Chronic Value = not available
Final Invertebrate Chronic Value = not available
Final Plant. Value = 93,000 ug/1
Residue Limited Toxicant Concentration = not available
Final Chronic Value = 93,000 ug/1
0.44 x Final Acute Value = 37 ug/1
2,4,6-trinitrophenol
Final Fish Acute Value = 20,000 ug/1
Final Invertebrate Acute Value = 340 ug/1
B-19
-------
Final Acute Value = 340 ug/1
Final Fish Chronic Value = not available
Final Invertebrate Chronic Value = not available
Final Plant Value = 63,000 ug/1
Residue Limited Toxicant Concentration = not available
Final Fish Chronic Value = 63,000 ug/1
0.44 x Final Acute Value = 150 ug/1
No saltwater criterion can be derived for most nitrophenols
using the Guidelines because no Final Chronic Value for either
fish or invertebrate species or a good substitute for either value
is available.
Results obtained with 4-nitrophenol and saltwater organisms
indicate how criteria may be estimated for other nitrophenols and
saltwater organisms.
For 4-nitrophenol and saltwater organisms 0.44 times the
Final Acute Value is less than the Final Chronic Value which is
derived from results of an embryo-larval test with the sheepshead
minnow. Therefore, it seems reasonable to estimate criteria for
other nitrophenols and saltwater organisms using 0.44 times the
Final Acute Value.
4-nitrophenol
The maximum concentration of 4-nitrophenol is the Final Acute
Value of 120 ug/1 and the estimated 24-hour average concentration
is 0.44 times the Final Acute Value. No important adverse effects
on saltwater aquatic organisms have been reported to be caused by
concentrations lower than the 24-hour average concentration.
CRITERION: For 4-nitrophenol the criterion to protect salt-
water aquatic life as derived using the Guidelines is 53 ug/1 as a
B-20
-------
24-hour average and the concentration should not exceed 120 ug/1
at any time.
2,4-dinitrophenol
The maximum concentration of 2r4-dinitrophenol is the Final
Acute Value of 84 ug/1 and the estimated 24-hour average concen-
tration is 0.44 times the Final Acute Value. No important adverse
effects on saltwater aquatic organisms have been reported to be
caused by concentrations lower than the 24-hour average concentra-
tion.
CRITERION: For 2,4-dinitrophenol the criterion to protect
saltwater aquatic life as derived using procedures other than the
Guidelines is 37 ug/1 as a 24-hour average and the concentration
should not exceed 84 ug/1 at any time.
2,4,6-trinitrophenol
The maximum concentration of 2,4,6-trinitrophenol is the
Final Acute Value of_J40 ug/1 and the estimated 24-hour average
concentration is 0.44 times the Final Acute Value. No important
adverse effects on saltwater aquatic organisms have been reported
to be caused by concentrations lower than the 24-hour average
concentration.
CRITERION: For 2,4,6-trinitrophenol the criterion to protect
saltwater aquatic life as derived using procedures other than the
Guidelines is 150 ug/1 as a 24-hour average and the concentration
should not exceed 340 ug/1 at any time.
B-21
-------
Table 5. Marine fish acute values for nitrophenols
to
Adjusted
Bioaeaay Test Chemical Tine
CD
1
y ft jgU * "Bf TJJ^ iTi"" i «T "T4?« f."
Sheepshead minnow. S U
Cyprinodon variegatua
Sheepshead minnow, S U
Cyprinodon varlegatys
Herring (embryo) , S U
Clupea harengua
Sheepshead minnow. S U
Cyprinodon variegatua
* S • static
ifci II H • mm A a at»rt>A
4-nitrophenol 96
2,4- 96
dinitrophenol
2,4- 96
dinitrophenol
2,4.6- 96
trinltrophenol
27,100 14,816 U.S. EPA.
29,400 16,073 U.S. EPA,
5,500 3,007 Rosenthal
Stelzer,
134.000 73.258 U.S. EPA.
1978
1978
&
1970
1978
Geometric mean of adjusted values: 4-nltrophenol - 14,816 wg/1 —^
2,4-dinitrophenol - 6,928 wg/1
2,4,6-trinltrophenol > 73,258 ug/l
- 4,000 wg/1
• 1,900
• 20,000 pg/l
-------
Table 6. Marine invertebrate acute values for nttroplienols (U.S. EPA, 1978)
Uioabsay Teat Chemical Tine LCSn.
CO
1
W
CO
— •
Myald shrimp,
Mystdopsia bahia
Mysid shrimp,
Mysidopsia bahia
Mysid shrimp,
Hysidopaia bahta
* S - static
** U - unmeasured
Geometric mean of
Method* Cone , ** Qaiicri ut xpn ^|
S U 4-nltrophenol
i
S U 2,4-
dinitrophenol
S U 2,4,6-
trinitrophenol
adjusted values; 4-nltrophenol - 6,073
2,4-dinltrophenol - 4
n t A |Uj/ AI^
96 7,170
96 4,850
96 19,700
Mg/1 ~fa — •
,108 Mg/i ^J
Adjusted
LCbO
lUCj/ ^|
6,073
4,108
16.686
120 iig/l
— - 84 Mg/1
2,4,6-trinitrophenol - 16.686 Mg/l to°° - 340 pg/1
-------
Table 7. Marine flail chronic values for nltrophenols (U.S. EPA, 1978)
Chronic
Limits Value
Organism . £§££* luq/l> fug/i>
Sheepahead minnow, E-L 10.000- 6,325"
Cyprinodon varleqatua 16,000
* E-L • embryo-larval
** A-nitrophenol
£ «AC
Geometric mean of chronic values - 6,325 ug/1 °tjtj - 940 ug/1
Lowest chronic value - 6,325 ug/1
CO
to
-------
Table 8. Marine plant effects for nltrophenoia (U.S. El'A. 1978)
Organism
Alga,
Skeletonema coacatum
Alga.
Skeletonema costatum
Alga,
Skeletonema coatatum
Alga,
Skeletonema coatatum
Alga,
Skeletonema costatum
Alga,
Skeletonema costatum
Effect
96-hr ECSO
Chlorophyll £
96-hr ECSO
cell number
96-hr ECSO
Chlorophyll a
96-hr ECSO
cell number
96-hr ECSO
Chlorophyll a
96-hr ECSO
cell number
Concentration
7,370
4-nitrophenol
7,570
4-nitrophenol
93,200
2,4-dinltrophenol
98,700
2,4-dinitrophenol
62.700
2,4,6-trinitrophenol
141,000
2,4,6-trinltrophenol
DO
I
en
•Lowest plant value: 4-nitrophenol •> 7,370 pg/l
2,4-dinitrophenol - 93,200 ng/1
2,4,6-trinitrophenol - 62,700
-------
CO
I
K>
en
Table 9. Other marine data for nltrophenola
Organism
Teat
Result
Jti&lil
fi£i.SI.£U££
2,A-Dtnttrophenol
Atlantic salmon
(Juvenile), 96 hrs
Salmo salar
Sea urchin (sperm), 1+ hra
Strongylocentrotua
purpuratus
Sea urchin (embryo), 2 hrs
Pseudocentrotua
depreasua
Lethal threshold
value
Inhibit respiration.
mobility
Abnormal cleavage
700 Zltko, 1976
92,000 Bernstein, 1955
46,000 Kojina, 1960
-------
NITROPHENOLS
REFERENCES
Bernstein, G.S. 1955. Effect of 2,4-dinitrophenol on sea
urchin sperm. Proc. Soc. Exp. Biol. Med. 90: 28
Dedonder, A., and C.F. Van Sumere. 1971. The effect of
phenolics and related compounds on the growth and respiration
of Chlorella vulgaris. Z. Pflanzen. Physiol. 65: 70.
Flickinger, 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.
Kojima, M.K. 1960. The effect of DNP and NaN3 on fertilized
eggs of the sea urchin with special reference to the induc-
tion of the abnormal cleavage. Embryologia 5: 71.
B-27
-------
Kopperman, H.L., et al. 1974. Aqueous chlorination and
ozonation studies. I. Structure-toxicity correlations of
phenolic compounds to Daphnia magna. Chem. Biol. Interact.
9: 245.
Lammering, M.W., and N.C. Burbank. 1960. The toxicity
of phenol, o-chlorophenol and o-nitrophenol to bluegill
sunfish. Eng. Bull. Purdue Univ. Engin Ext. Serv.
106: 541.
Lewis, E.J.C., and E. Frieden. 1959. Biochemistry of amphib-
ian metamorphosis: effect of triiodothyronine, thyroxin,
and dinitrophenol on the respiration of the tadpole. Endocri-
nology 65: 273.
Marcus, M., and A.M. Mayer. 1963. Flagellar movement in
Chlamydomonas snowiae and its inhibition by ATP and dinitro-
phenol. In Studies on microalgae and photosynthetic bacteria.
Jap. Soc. Plant Physiol. University of Tokyo Press, Tokyo. Japan.
Phipps, G.L., et al. The acute toxicity of phenol and substi-
tuted phenols to the fathead minnow. (Manuscript).
Rosenthal, H., and R. Stelzer. 1970. Wirkungen von 2,4-
und 2,5-dinitrophenol auf die Embryonalentwicklung des Herings
Clupea harengus. Mar. Biol. 5: 325.
B-28
-------
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 prin-
ciples of phytotoxicity. IV. The effects of the degree
of nitration on the toxicity of phenol and other substituted
benzenes. Jour. Exp. Bot. 4: 235.
U.S. Environmental Protection Agency. 1978. In-depth studies
on health and environmental impacts of selected water pollut-
ants. Contract No. 68-01-4646.
Zitko, V., et al. 1976. Toxicity of alkyldinitrophenols
to some aquatic organisms. Bull. Environ. Contam. Toxicol.
16: 508.
B-29
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MONONITROPHENOLS
Mammalian Toxicology and Human Health Effects
EXPOSURE
Introduction
Mononitrophenol has three isomeric forms, distinguished
by the position of the nitro group 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-nitrophenol, respectively.
Commercial synthesis of 2-nitrophenol and 4-nitrophenol
is accomplished through the hydrolysis of the appropriate
chloronitrobenzene isomers with aqueous sodium hydroxide at
elevated temperatures (Howard, et al. 1976). Production of
3-nitrophenol is achieved through the diazotization and hy-
drolysis of m-nitroaniline (Matsuguma, 1967). The mononitro-
phenol isomers are used in the United States primarily as in-
termediates for the production of dyes, pigments, Pharmaceu-
ticals, rubber chemicals, lumber preservatives, photographic,
chemicals and pesticidal and fungicidal agents (U.S. Int.
Trade Comm. 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 mononitrophe-
nols may also be inadvertently produced via microbial or pho-
todegradation of pesticides which contain mononitrophenol
moieties. Aproximately 10 to 15 million pounds of 2-nitro-
phenol are produced annually (Howard, et al. 1976) for uses
C-l
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including synthesis of o-aminophenol, o-nitroanisole, and
other dye stuffs (Matsuguma, 1967; Howard, et al. 1976). Al-
though production figures for 3-nitrophenol are not avail-
able, Hoecker, et al. (1977) estimate that production is less
than one million pounds annually. 3-nitrophenol is used in
the manufacture of dye intermediates such as anisidine and
m-aminophenol (Kouris and Northcott, 1963; Matsuguma, 1967).
4-nitrophenol is probably the most important of the mononi-
trophenols in terms of quantities used and potential environ-
mental contamination. Demand for 4-nitrophenol was 35,000,000
pounds in 1976 and production is projected to increase to
41,000,000 pounds by 1980 (Chem. Market. Reporter, 1976).
Most of the 4-nitrophenol produced (87 percent) is used in
the manufacture of ethyl and methyl parathions. Other uses
(13 percent) include the manufacture of dye-stuffs and n-ace-
tyl-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 parathions. In vivo
production of 4-nitrophenol following absorption of parathion
or other pesticides by humans is another possible source of
human exposure.
Physical and chemical properties of the mononitrophenols
are summarized in Table 1.
C-2
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TABLE 1
Properties of Mononitrophenols
Formula
Molecular Weight
Melting Point (°C)
Boiling Point
Density
Water Solubility (g/1)
Vapor Pressure
Ka
2-Nitrophenol 3-Nitrophenol
C6H5N03 C6H5N03
139.11 139.11
44-45 97
214-216 194
1.485 1.485
0x3.2 at 38°C 1x3.5 at 25°C
1x0.8 at 100°C 13x3.0 at 90°C
1 mm Hg at
49.3°C
7.5xlO-8
5.3x10-9
4-Nitrophenol
C6H5N03
139.11
113-114
279
1.479
0x8.04 at 15°C
1x6.0 at 25°C
7x10-8
Mononitrophenols
OH
2-nitrophenol
3-nitrophenol
4-nitrophenol
C-3
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Ingestion from Water
Monitoring data on the presence of mononitrophenols in
water are scant in the literature. Potential point sources
for mononitrophenol contamination of water include industrial
concerns engaged in the manufacture of these compounds or
their usage as intermediates in chemical synthesis.
Trifunovic, et al. (1971) detected unspecified levels of
4-nitrophenol in waste effluents from a parathion manufactur-
ing plant. Webb, et al. (1973) reported a 4-nitrophenol lev-
el of 1.4 mg/1 in the waste lagoon water of a chemical plant.
Burnham, et al. (1972) detected 4-nitrophenol at levels of
0.2 mg/1 in the potable water supply of Ames, Iowa. The
source of the contamination was believed to be residues from
a coal gas plant which ceased operation around 1930. 2-Ni-
trophenol was detected at unidentified levels in two river
water samples and in 4 samples of chemical plant effluent,
and 3-nitrophenol was found in one chemical plant effluent
sample (U.S. EPA, 1976). Systematic monitoring for mononi-
trophenols in the environment has not been done. It is rea-
sonable to assume that measureable (although perhaps trans-
ient) levels of the mononitrophenols may be present in local-
o-
ized areas where organophosphate pesticides are in use.
Little data is available regarding the breakdown of
mononitrophenols by natural communities of microorganisms.
Alexander and Lustigman (1966) studied the degradation of
mononitrophenols by a mixed population of soil microorga-
nisms. The inoculum was derived from a suspension of Niagra
silt loam soil. Their results indicated that 2-nitrophenol
was more resistant to degradation than either 3-nitrophenol
C-4
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or 4-nitrophenol. Utilizing the absorbancy of small soil
inoculums to estimate the loss of mononitrophenol, 3-nitro-
phenol was found to degrade completely within a 4-day period.
4-nitrophenol degraded fully within a 16-day period while
2-nitrophenol resisted degradation over a 64-day period.
Brebion, et al. (1967) examined the ability of
microorganisms derived from soil, water, or mud, and grown on
a porous mineral bed to attack 4-nitrophenol. The bacteria
were cultivated on a mineral nutrient solution to which
nitrophenols were added as the sole source of carbon. The
experimental 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 populations of microorganisms. Tabak,
et al. (1964) studied the ability of acclimated cultures de-
rived from garden soil, compost, and river mud to degrade the
mononitrophenols. Phenol-adapted bacteria derived from these
sources were found to readily degrade all three mononitro-
phenol isomers. Ninety-five percent degradation (measured
spectrophotometrically) occurred within three to six days.
Fitter (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 organ-
ic 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 fron the primary settling tank of the
city of Ithaca, N.Y. wastewater treatment plant, or a Windsor
C-5
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loamy fine sand soil were used as the source of the inoculum.
2-Nitro, 3-nitro, and 4-nitrophenol (16 mg/1) were completely
degraded in three to five days by the sludge system. Soil in-
ocula degraded 16 mg/1 of 3-nitrophenol in three to five days
while a similar concentration of 2-nitrophenol and 4-nitro-
phenol required 7 to 14 days for complete degradation.
Although definitive conclusions cannot be derived from
this limited number of studies, it appears that the mono-
nitrophenols are readily and rapidly degraded by microbial
population present in the environment.
Ingestion from Foods
No data were found demonstrating the presence of mono-
nitrophenols 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 con-
taining the nitrophenol moiety. The production of 4-nitro-
phenol by microbial metabolism of parathion is well docu-
mented (Munnecke and Hsieh, 1974, 1976; Siddaramappa, et al.
1973; Sethunathan and Yoshida, 1973; Katan and Lichtenstein,
1977; Sethunathan, 1973). Microbial metabolism of fluori-
difen (p-nitrophenyl, a, a, a-trifluoro-2-nitro-p-tolyl
ether) results in the intermediate formation of 4-nitrophenol
(Tewfik and Hamdi, 1975). The major degradation product of
fluoridifen following uptake by peanut seedling roots was
4-nitrophenol (Eastin, 1971). 4-nitrophenol was also de-
tected in soybean roots following absorption of fluoridifen
(Rogers, 1971). Photodecomposition of the herbicide nitrofen
(2,4-dichlorphenyl p-nitrophenyl ether) in aqueous suspen-
sions under sunlight or simulated sunlight is characterized
C-6
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by rapid cleavage of the ether linkage to form 2, 4-dichloro-
phenol and 4-nitrophenol (Nakagawa and Crosby, 1974).
El-Refai and Hopkins (1966) have investigated the metabolic
fate of parathion following foliar application or root ab-
sorption by bean plants, Phaseolus yulgaris. Detectable
amounts of 4-nitrophenol were found in chloroform rinses of
parathion treated leaves after four days.
In another experiment, analysis of nutrient solutions
containing parathion in which plants were grown for root ab-
sorption studies revealed 4-nitrophenol, paraoxon, and traces
of degradation products. Since these compounds were also
detected in control solutions which did not contain plants,
the authors concluded that possible photochemical oxidation
processes had occurred in the aqueous medium. The authors
believed that the 4-nitrophenol detected following foliar
application of parathion was due to photochemical processes.
4-nitrophenol was not detected in bean plants following in-
jection of parathion directly into the stems of bean plants
(El-Refai and Hopkins, 1966).
4-nitrophenol has also been detected as a photoaltera-
tion product of parathion following application to cotton
plants (Joiner and Baetcke, 1973).
Archer (1974) has examined the dissipation of parathion
and its metabolites from field spinach. Field plots were
sprayed with either 0.5 or one pound of active parathion/
acre. Application recommendations for parathion are: not
less than 14 days before harvest at the rate of 0.5 pounds of
active ingredient/acre. Spinach samples were analyzed daily
C-7
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for parathion residues and a number of known metabolites in-
cluding 4-nitrophenol. Levels of 4-nitrophenol in the treat-
ed spinach (calculated on a fresh weight basis) are presented
in Table 2. Unsprayed spinach control samples taken prior to
any spray treatments contained 95 ug/kg 4-nitrophenol. The
source of these residues was not determined. The effects of
washing or blanching following harvest on the levels of
4-nitrophenol in human food crops are unknown.
.4-nitrophenol has been detected in human urine. The
National Monitoring Program for Pesticides is collaborating
with the U.S.Public Health Service in a three-year study to
assess the exposure of the general population of the U.S.
through analysis of human urine for residues of selected pes-
ticides and their specific metabolites (Kutz, et al. 1978).
Based on the analysis of 416 samples collected from the gen-
eral population, 4-nitrophenol is detected in 1.7 percent of
the population. A mean urine level of 10.0 ug/1 with a maxi-
mum value of 113.0 ug/1 was reported. It is important to
note that 4-nitrophenol residues in the urine need not (and
probably do not) reflect exposure to 4-nitrophenol itself.
Mononitrophenols are readily formed in vivo following expo-
sure to a number of widely used pesticides.
Kutz, et al. (1978) considered exposure to methyl and
ethyl parathion as the origin of the urinary 4-nitrophenol
detected in their survey. However, if it is assumed that the
reported urinary residues of 4-nitrophenol reflect direct ex-
posure to the 4-nitrophenol, a pharmacokinetic estimate of
C-8
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TABLE 2
Levels of 4-Nitrophenol Following Aplication of Parathion to
Field Spinach at Two Different Application Rates3
SAMPLE DAY
4 Nitrophenol Residue (ug/kg)
0.5 Ib. Parathion/Acre
1.0 Ib. Parathion/Acre
n
1
2
3
4
5
6
7
8
9
10
11
12
13
14
172
88
73
76
73
72
35
40
34
34 ' fc
31
38
28
33
453
305
240
188
136
216
117
18
19
18
18
22
16
19
a Source: Modified from Archer, 1974.
b calculated on a fresh weight basis. Percent moisture from 86.4 to 89.2.
c Unsprayed spinach control samples taken prior to any spray treatments contained
95 ugAg.
-------
exposure can be made. Assuming that the exposure to nitro-
phenol is steady-state, that 100 percent of the absorbed
nitrophenol is excreted in the urine, and 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 1.7 ug/1
residue can be calculated as follows:
pvr,0_lirfa _ (10.0 ug nitrophenol/1) (1.4 1 of urine/day) _ n 9
Exposure - 70 kg man P /
y kg/day
A similar calculation using the maximum urine residue level
observed by Kutz, et al. (1978) (113 ug/D gives an exposure
of 2.26 ug/kg/day.
Knowles, et al. (1975) have demonstrated the production
of a wide number of mono-nitrophenols 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 employed approximated
those encountered during gastric digestion, their results in-
dicated 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 meta-
bolic degradation of pesticides such as parathion by humans.
C-10
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Excretion of 4-nitrophenol, a metabolite of the organophos-
phorus pesticides, parathion, methylparathion, 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-nitrophenol has
also been detected as a urinary metabolite of nitrobenzene in
humans (Myslak, et al. 1971).
A bioconcentration factor (BCF) relates the concentra-
tion of a chemical in water to the concentration in aquatic
organisms, but BCF's are not available for the edible por-
tions of all four major groups of aquatic organisms consumed
in the United States. Since data indicate that the BCF for
lipid-soluble compounds is proportional to percent lipids,
BCF's can be adjusted to edible portions using data on per-
cent lipids and the amounts of various species consumed by
Americans. A recent survey on fish and shellfish consumption
in the United States (Cordle, et al. 1978) found that the per
capita consumption is 18.7 g/day. From the data on the nine-
teen major species identified in the survey and data on the
fat content of the edible portion of these species (Sidwell,
et al. 1974), the relative consumption of the four major
groups and the weighted average percent lipids for each group
can be calculated:
Consumption Weighted Average
Group (Percent) Percent Lipids
Freshwater fishes 12 4.8
Saltwater fishes 61 2.3
Saltwater molluscs 9 1.2
Saltwater decapods 18 1.2
C-ll
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Using the percentages for consumption and lipids for each of
these groups, the weighted average percent lipids is 2.3 for
consumed fish and shellfish.
No measured steady-state bioconcentration factor (BCF)
is available for any nitrophenol, but the equation "Log BCF =
0.76 Log P - 0.23" can be used (Veith, et al., Manuscript) to
estimate the BCF for aquatic organisms that contain about
eight percent lipids from the octanol-water partition coeffi-
cient (P). An adjustment factor of 2.3/8.0 = 0.2875 can be
used to adjust the estimated BCF from the 8.0 percent lipids
on which the equation is based to the 2.3 percent lipids that
is the weighted average for consumed fish and shellfish.
Thus, the weighted average bioconcentration factor for edible
portion of all aquatic organisms consumed by Americans can be
calculated.
Table 2A
Compound
2-nitrophenol
4-nitrophenol
2,4-dinitrophenol
2, 4,6-trinitrophenol
4 , 6-dinitro-o-cresol
P
.62
81
32
110
150
BCF
14
17
8.2
21
26
Weighted BCF
4.0
4.9
2.4
6.0
7.5
C-12
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Inhalation
No quantitative data were found regarding the presence .
of mononitrophenols }.n air. Lao, et al. (1973) discussed the
application of a gas chromatograph quadrapole mass spectrqr
meter-data processor combination for routine analysis of air
pollutants. During a sample run of urban ambient particulate
matter (location not designated) these investigators identi'-
fied the presence of 4-nitrophenol as well as a large number
of other air pollutants. No quantitative data were provided,
however. Ambient air levels of 4-nitrophenol in a Boeing
plant where the compound was used for the preservation of the
cork surfaces of the Minuteman Missile were equal to or less
than 0.05 mg/m3 of air (Butler and Bodner, 1973).
4-nitrophenol may be produced in the atmosphere through
the photochemical reaction between benzene and nitrogen mon-
oxide. 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 pro-
ducts. The production of nitrobenzene, 2-nitrophenol, 4-ni-
trophenol, 2, 4-dinitrophenol and 2, 6-dinitrophenpl was
described by the authors. Identity of the compounds was con-
firmed using thin layer chromatography, gas chromatography,
gas-chromatography-mass spectrometry, and infrared spectro-
metry. The authors suggested that these nitro compounds may
C-13
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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 extremities.
In a second paper (Nojima, et al. 1976), the photochemi-
cal reaction of toluene with nitrogen monoxide was investi-
gated. It was felt that the products of photochemical reac-
tion of toluene with nitrogen monoxide might be more impor-
tant in the production of photochemical smog since the con-
centration of toluene in urban air is higher than that of
benzene. Compounds produced as a result of this reaction
included o-cresol, m-nitrotoluene, 4-nitrophenol, 2-methyl-
6-nitrophenol, 3-methyl-4-nitrophenol, 2-methyl-4-nitrophenol
and 2-methyl- 4,6-dinitrophenol. These compounds were iden-
tified by gas chromatography-mass spectrometry. In another
experiment, the investigators examined the organic compounds
present in rain. An analysis of rainwater yielded 4-nitro-
phenol, 2-methyl-6-nitrophenol, and 2-methyl-4-nitrophenol.
The authors suggested that the nitrophenols produced by the
photochemical 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 mono-
nitrophenols. However, it is impossible to estimate the
levels at which humans are exposed to these compounds via in-
halation, based on available data.
C-14
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Dermal
Roberts, et al. (1977) used human autopsy skin epidermal
membranes in an in vitro system to determine the permeability
of human skin to various compounds. 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 percent
(w/v), respectively. According to Patty (1963),
2-nitrophenol may be absorbed through the intact skin. No
information on possible human dermal exposure to the
mononitrophenols was found.
PHARMACOK II&V IC S
Absorption and Distribution
1 '"" ~^^•_•.«•m^^« * j
Data specific to the absorption and tissue distribution
of the mononitrophenols were not.available. It is reasonable
to assume, based on the rapid urinary elimination of the
3-
mononitrophenols, that the compounds may be resticted primar-
ily to the blood and urine following absorption by humans.
Metabolism
Metabolism of the mononitrophenols probably occurs via
one of three mechanisms in humans. The major route of mono-
nitrophenol metabolism is undoubtedly conjugation and the re-
sultant formation of either glucuronide or sulfate conju-
gates. Other possible routes of metabolism include reduction
to amino compounds or oxidation to dihydric-nitrophenols.
Sulfate and glucuronide conjugative processes are two of
the major detoxification mechanisms in many species, including
C-15
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mammals (Quebbemann and Anders, 1973). In recent years,-
4-nitrophenol has been used as a preferred substrate for bio-
chemical analysis of the glucuronidation reactions in a wide
number of species (Aitio, 1973; Sanchez and Tephly, 1974;
Ranklin, 1974; Heenan and Smith, 1974; Yang and Wilkinson,
1971). This usage reflects the simple techniques available
for quantitating the disappearance of 4-nitrophenol and the
synthesis of the glucuronide conjugate. The relevance of
many of these in vitro studies towards an assessment of the
metabolic fate of the mononitrophenols in humans is question-
able; thus only those in vivo studies with direct relevance
to the metabolic fate of the mononitrophenols in humans or
experimental animals are discussed here.
It has been known for some time that levels of the mixed
function oxidases and the enzymes responsible for conjugation
of many compounds are generally highest in the mammalian
liver. Litterst, et al. (1975) assayed liver, lung and kid-
ney from rat, mouse, rabbit, hamster and guinea pig for stan-
dard 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 percent of that found
in liver with kidney slightly more active than lung. UDP-
glucuronyl- transferase activity toward the acceptor 4-nitro-
phenol was higher in hamster and rabbit than other species.
C-16
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Conjugation activity need not be a constant even within
the same species, however. Pulkkinen (1966b) noted that sul-
fate conjugation of 4-nitrophenol is decreased during preg-
nancy in rabbits. The author suggested that large amounts of
estrogens may cause more protein binding, thus inhibiting the
reaction. 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 conjugates of
mononitrophenols or other compounds. As age increases, so
does conjugation capacity. In addition, Moldeus, et al.
(1976) noted that the relative rate of glucuronide versus
sulfate conjugation of 4-nitrophenol may depend on the levels
of substrate present. In in vitro tests utilizing isolated
rat liver cells, the investigators noted that at 4-nitro-
phenol concentrations of 25 uM the rate of glucuronide con-
jugation was low and over 75 percent of the conjugation pro-
ducts were found to be sulfates. The glucuronidation in-
creased more rapidly than did the sulfate conjugation with
increasing substrate conjugation. At 250 y.M 4-nitrophenol,
sulfate conjugation 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 detoxifi-
cation 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.
C-17
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Only small amounts (less than one percent) of the unchanged
free phenol were excreted. With all three of the mononitro-
phenol isomers, the major conjugation product was nitro-
phenyl-glucuronide, which accounted for about 70 percent of
the dose. The corresponding sulfate conjugates were also
excreted. Reduction of the nitrophenols occurred 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-nitrophenol yields traces of
nitroquinol; 3-nitrophenol yields nitroquinol and 4-nitro-
catechol; and 4-nitrophenol yields 4-nitrocatechol.
A summary of the metabolism of the mononitrophenols is
shown in Table 3. No data directly addressing the metabolic
fate of the mononitrophenols in humans are available. How-
ever, it is expected that following exposure to the mono-
nitrophenols humans will rapidly excrete both glucuronide and
sulfate conjugates in the urine.
Excretion
Data directly addressing the excretion of the mononitro-
phenols following exposure of humans were not found in the
literature. However, excretion patterns for 4-nitrophenol
following human exposure to parathion may shed some light on
their elimination kinetics. Arterberry, et al. (1961) .
studied the pharmaco-dynamics of 4-nitrophenol excretion fol-
lowing exposure to parathion. They noted that the excretion
of 4-nitrophenol in the urine was quite rapid "as might be
C-18
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TABLE 3
Urinary Metabolites of Mononitrophenols in Rabbits3
n
Nitrophenol
2-Nitrophenol
3-Nitrophenol
4-Nitrophenol
Nitro
Compounds
(N)
82
74
87
Amino
Compounds
(A)
3
10
14
Percentage of Dose Excreted
Glucuronides
(N + A)b (G)
85 71
84 78
101 65
as
Ethereal
Sulphates
(E) (G + E)b
11 82
19 98
16 81
a Source: Modified from Robinson, et al. 1951.
" (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 con-
jugates.
-------
expected in the case of a water-soluble metabolite of a sub-
stance which is quickly broken down by the animal organism."
4-nitrophenol usually had disappeared from the urine within
about 48 hours after cessation of exposure. In a similar
study of orchard spray-men involved in the application of
parathion, Wolfe, et al. (1970) noted that urinary levels of
4-nitrophenol rose promptly in response to parathion exposure
by spray-men and returned to the nondetectable level after
several days. Myslak, et al. (1971) reported on the excre-
tion of 4-nitrophenol from a 19-year-old female subject fol-
lowing a suicidal oral dose of nitrobenzene. Large quanti-
ties of 4-nitrophenol and 4-aminophenol were detected in the
urine. Elimination of 4-nitrophenol in the urine was
expressed by the equation Vt/Vo = e~°«008t where V° and Vfc
denote the excretion rate at the interval time O and t
measured in hours. The half-life for excretion corresponded
to about 84 hours„
Shafik, et al. (1973) studied the urinary excretion of
4-nitrophenol following administration of the pesticide EPN.
Following oral administration of the pesticide for three
days, animals were maintained and urine samples collected at
24-hour intervals. Three days were required for complete ex-
cretion of 4-nitrophenol under these conditions. The forego-
ing studies indicate that 4-nitrophenol is rapidly excreted
following its production in vivo from other organic com-
pounds .
C-20
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Only one study was found that examined excretion of
4-nitrophenol following direct administration of the com-
pound. Lawford, et al. (1954) studied the elimination of
various nitrophenolic compounds from the blood of experi-
mental animals. Elimination of 4-nitrophenol by the monkey
following oral and intraperitoneal doses of 20 mg/kg body
weight was complete within five hours. Elimination by mice/
rats, rabbits, and guinea pigs was also rapid. Most doses
were eliminated completely from the blood within two hours of
administration. Experimental animals eliminated 4-nitro-
phenol 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 mono-
nitrophenols are excreted rapidly via the urinary route and
that total elimination is likely not to exceed one week. The
mononitrophenols are highly water soluble and accumulation or
bioconcentration in various tissues is not expected to occur
to a large extent. However, much more data are needed to
precisely define the transport distribution and elimination
of these compounds in humans.
EFFECTS
Threshold concentrations for odor, taste, and color for
2-nitro, 3-nitro, and 4-nitrophenol in reservoir water have
been reported in an abstract of a paper from the Russian
literature (Makhinya, 1964). Reported threshold concentra-
tions for 2-nitrophenol were 3.83 mg/1 for odor, 8.6 mg/1 for
taste, and 0.6 mg/1 for color. Concentrations for 4-nitro-
phenol were 58.3, 43.4, and 0.24 mg/1 for odor, taste, and
C-21
-------
color, respectively. The values for 3-nitrophenol were given
as 389, 164.5 and 26.3 mg/1. Acceptability thresholds from
the standpoint of human consumption were not reported by
these investigators.
Acute, Sub-acute, and Chronic Toxicity
Known effects of 4-nitrophenol demonstrated in animal
\
experiments are methemoglobinemia, shortness of breath, and
initial stimulation followed by progressive depression (von
Oettingen, 1949).
Acute toxicity information for the mononitrophenol
isomers has been compiled and presented as Table 4. 4-nitro-
phenol is the most toxic of the mononitrophenols followed by
3-nitrophenol and 2-nitrophenol in relative toxicity. Toxi-
cological symptoms of mononitrophenol poisoning have not been
well described in the literature. Sax (1968) noted that
2-nitrophenol exposure produced kidney and liver injury in
experimental animals. Methemoglobin formation as a result of
mononitrophenol exposure has been reported (Patty, 1963).
Grant (1959), however, was unable to detect methemoglobin
formation after oral administration of 3-nitro and 4-nitro-
phenol to rats. Small inconstant amounts of methemoglobin
were formed with 3-nitrophenol administration. Smith, et al.
(1967) were able to show that the reduction products of mono-
nitrophenols, 2- and 4-aminophenol, would produce methemo-
globin in female mice. Methemoglobin formation, therefore,
may depend on the capacity of the organism to reduce the
mononitrophenolso As mentioned in the metabolism section of
C-22
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TABLE 4
Acute Toxicity of Mononitrophenol Isomers
ro
u>
Species
Frog
Mouse
Rabbit
Cat
Dog
Rat
Mouse
Guinea Pig
Dog
Rat
Mouse
Frog
Rabbit
Cat
Dog
Rat
Mouse
Rat
Dose
(mg/kg)
300
600
1700
600
100
2830
1300
900
83
930
1410
50
600
197
10
620
470
350
Route of
Administration
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
Effects
Lethal Dose
Lethal Dose
Lethal Dose
Lethal Dose
Lethal Dose
LD 50
LD 50
Lethal Dose
Minimum Lethal Dose
LD 50
LD 50
Minimum Lethal Dose
Minimum Lethal Dose
Minimum Lethal Dose
Lethal Dose
LD 50
LD 50
LD 50
References
Spector, 1956
Spector, 1956
Spector, 1956
Spector, 1956
Spector, 1956
Vernot, et al.
Vernot, et al.
Spector, 1956
Spector, 1956
Vernot, et al .
Vernot, et al.
Spector, 1956
Spector, 1956
Spector, 1956
Spector, 1956
Vernot, et al.
Vernot, et al.
Fairchild, 1977
1977
1977
1977
1977
1977
1977
-------
this document, reduction of the nitrophenols does not nor-
mally occur to any large extent.
Ogino and Yasukura (1957) reported the development of
cataracts in vitamin C deficient guinea pigs following admin-
istration of 4-nitrophenol. Cataracts developed in two of
three guinea pigs on days 7 and 11 following daily intra-
peritoneal administration of 8.3 to 12.5 mg 4-nitrophenol/kg
body weight. Subchronic administration of•4-nitrophenol over
a 20-day test period produced cataracts while 2- and
3-nitrophenol did not. The authors concluded that the para-
positioning of the hydroxyl and nitro groups is necessary for
cataract induction.
Several deficiencies in this study preclude definitive
conclusions on the cataractogenic properties of 4-nitro-
phenol. The investigators failed to report results on con-
trol animals, either totally untreated or treated with the
nitrophenols and a vitamin C supplement. Thus, it is pos-
sible, based on the results reported, to conclude that vita-
min C deficiency itself caused cataracts in some of the ani-
mals tested. The small size of the experimental groups
(three animals/test compound) also make definitive conclu-
sions difficult. 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-nitrophenol 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.
C-24
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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 for-
mation in humans following mononitrophenol exposure is un-
clear.
Both 2-nitro and 4-nitrophenol have been shown to in-
hibit porcine heart malate dehydrogenase in vitro (Wedding,
et al. 1967). The compounds acted as competitive inhibitors
for NAD in the forward direction of the enzymatic reaction.
The clinical significance of these findings is unknown.
The ventilatory effects of the mononitrophenols have
been examined in anesthetized rats (Grant/ 1959). Test com-
pounds were administered by stomach tube: 2-nitrophenol, 60
to 120 mg; 3-nitrophenol, 20 to 45 mg; 4-nitrophenol, 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 sublethal doses of 2-nitrophenol in rats
(Cameron, 1958). In contrast, oxygen uptake was decreased in
3-nitrophenol-treated rats while carbon dioxide output was
increased following 4-nitrophenol administration. Rectal
temperature was depressed in rats receiving any of the three
isomers. These results suggest that mononitrophenol isomers
are not potent uncouplers of oxidative phosphorylation, in
contrast to the chemically similar compound 2, 4-dinitro-
phenol.
C-25
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Although the mechanism of toxic action of the mononitro-
phenols is not well understood, the following studies suggest
that an action directly on cell membranes may occur. 3-ni-
trophenol binds readily to red blood cell (RBC) membranes.
Expansion of RBC ghosts occurs following nitrophenol treat-
ments, as measured by the resistance of such ghosts to hemol-
ysis (Machleidt, et al. 1972). 2-nitrophenol and 4-nitro-
phenol inhibit chloride transport in red blood cells (a
metabolism independent process) suggesting a direct action on
the cell membrane (Motais, et al. 1978). Further information
on the acute or chronic toxicity of the mononitrophenols to
humans was not found.
The National Institute for Occupational Safety and
Health recently undertook a health hazard evaluation deter-
mination at the request of an employee of the Boeing Company
who had routinely handled 4-nitrophenol (Butler and Bodner,
1973). A 15 percent solution of 4-nitrophenol and methyl-
phenol is painted on the exposed cork surfaces of the Minute-
man Missile before arrival at the assembly plant. If the
surface is damaged in transit it is necessary to apply small
amounts of the 4-nitrophenol solution to the repaired areas
of cork. The worker in question was engaged in such touch-up
operations. Workers routinely wear an organic vapor cart-
ridge respirator, a face shield, cotton gloves, rubber
gloves, and are completely covered with protective clothing.
The employee complained of fatigue, joint pain, abdominal
cramps and diarrhea, and attributed these symptoms to his
exposure to both the treating solution and the dried cork
C-26
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impregnated with 4-nitrophenol during his work as a mechanic.
Medical examination failed to detect 4-nitrophenol in the
urine but revealed a complete absence of the immunoglobins
IgA and IgD in the employee. Based on medical judgement and
the existing literature, the study concluded that the employ-
ee's condition stemmed from the lack of IgA and IgD and that
this deficiency was not caused by exposure to 4-nitrophenol.
Gabor, et al. (1960; cited in Howard, et al. 1976)
reported a unique effect of 2-nitrophenol on blood platelet
levels. When 31 rats were administered 2-nitrophenol by
intraperitoneal injection at 1 mg/kg body weight, the
platelet count increased significantly. Even at doses of 0.1
mg/kg a similar effect was produced. Administration of
3-nitro or 4-nitrophenol did not produce a rise in platelet
levels. Additional data are not available to explain this
unique phenomenon nor is the clinical significance of these
findings known.
A report from the Russian literature (Makhinya, 1969)
reports that 2-nitro, 3-nitro, and 4-nitrophenol possess
distinct cumulative properties. Chronic administration of
any of the mononitrophenols to mammals caused alterations of
neurohumoral regulation and pathological changes including
colitis, enteritis, hepatitis, gastritis, hyperplasia of the
spleen, and neuritis. Limiting doses for the disruption of
conditioned reflex activity were established as .003 mg/kg
(equivalent to .006 mg/1 of water) for 2-nitrophenol and
3-nitrophenol and .00125 mg/kg (equivalent to .0025 mg/1 of
water) for 4-nitrophenol. Unfortunately a report of this
C-27
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study was available in abstract form only. Details of the
experiment including animal species, mode of administration,
duration of the treatment, and a good description of the ob-
served biological effects, were not reported. The results
must be considered questionable until an evaluation of the
experimental protocol becomes available.
Synergism and/or Antagonism
Only one report was found dealing with possible syner-
gistic effects of the mononitrophenols. Cairns, et al. (1976)
studied the effects of a sublethal exposure to zinc and sub-
sequent 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 .2 and .4 of the 48 hour LC$Q dose, respectively) followed
by an acutely lethal dose of 4-nitrophenol (1,000 mg/1). A
significantly reduced survival time following 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 decrease in the median survival time of the
snails during the temperature shock was noted. The applica-
bility of these data to humans or mammals is unknown. Data
regarding synergistic or antagonistic effects of the mononi-
trophenols in mammals were not found.
Teratogenicity
No information was found regarding the presence or ab-
sence of teratogenic properties of the mononitrophenols.
C-28
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Mutagenicity
Szybalski (1958) tested the three mononitrophenol iso-
mers for their ability to induce streptomycin-independence in
streptomycin-dependent E. coli. All three isomers gave nega-
tive results.
Buselmaier, et al. (1976) tested 4-nitrophenol for muta-
genic activity in mice with the host mediated assay and the
dominant lethal method, using Salmonella typhimurium G46 HIS",
Serratia marcescens a21, leu" and Serratia marcescens a31
HIS" as indicator organisms. Spot tests in vitro were also
carried out. Mutagenic activity was not demonstrated.
4-nitrophenol also failed to induce mutations in Salmo-
nella both with and without microsomal activation (McCann, et
al. 1975).
Fahrig (1974) demonstrated a weak mutagenic activity
when 4-nitrophenol was tested for mitotic gene conversion in
Saccharomyces cerevisiae. This test-system allows the detec-
tion 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 in-
hibition 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 mononitrophenol isomers in root
C-29
-------
tips of Allium 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 (1969) studied the effects of 2-nitro and
4-nitrophenol on the lateral root mitoses of Vicia faba
seedlings. The mitotic index was reduced at concentrations
of these compounds ranging from 0.025 percent to 0.1 percent.
Induction of anaphase bridges by both isomers was noted but
(in agreement with the work of Levin and Tjio (1948) with
Allium cepa) chromosome fragmentation was not detected. The
relationship of these changes in plants to alterations in
mammalian cells has not been established. Based on the
available data the mononitrophenols do not appear to pose a
mutagenic hazard to humans.
Carcinogenicity
Data on the possible Carcinogenicity of the mononitro-
phenols are scant in the literature. Boutwell and Bosch
(1959) have studied the ability of a number of phenolic com-
pounds to promote tumor formation on mouse skin following a
single initiating dose of dimethylbenzanthracene. Although
phenol itself has demonstrated a promoting capacity in this
system both 2- and 4-nitrophenol failed to promote tumor
development in mice. No other data on possible carcinogenic
potential of the mononitrophenols were found.
4-nitrophenol has been selected by the National Cancer
Institute for testing under the Carcinogenesis Bioassay
Program.
C-30
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DINITROPHENOLS
Mammalian Toxicology and Human Health Effects
EXPOSURE
Introduction
Six isomeric forms of dinitrophenol are possible, dis-
tinguished by the position of the nitro groups on the phe-
nolic 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 Ib
reported by the U.S. International Trade Commission (1968).
Approximate consumption per year is estimated at 1,000,000 Ib
(Howard, et al, 1976). 2, 4-dinitrophenol 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 remaining five
dinitrophenol isomers are not available. It is reasonable to
assume that production and usage of these compounds are ex-
tremely limited in the United States.
Commercial synthesis of 2,4-dinitrophenol is accom-
plished by the hydrolysis 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
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 com-
C-31
-------
pounds which contain the dinitrophenol moiety, such as Para-
thion (Gomaa and Faust, 1972). 2,4-DNP has also been identi-
fied as an impurity in technical preparations of the herbi-
cide DNPP (2-isopropyl-4,6-dinitrophenol) by Mosinska and
Kotarski (1972) .
The physical and chemical properties of the dinitro-
phenol isomers are summarized in Table 5.
TABLE 5
Properties of Dinitrophenol Isomers3
Isomer
2, 3-Dinitrophenol
2 , 4-Dinitrophenol
2 , 5-Dinitrophenol
2 , 6-Dinitrophenol
3 , 4-Dinitrophenol
3 , 5-Dinitrophenol
m.p .
144
114-115
(sublimes)
104
63.5
134
122-123
(at
1.3
1.0
7
2.7
4.3
2.1
K
25°C)
x 10~5
x 10-4
x 10~6
x ID"4
x ID"5
x 1Q-4
Water
Solubility
(9/D
2.2
0.79
0.68
0.42
2.3
1.6
Density
1.681
1.683
1.672
1.702
a Source: Harvey, 1959; Windholz, 1976; Weast, 1975.
C-32
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Dini trophe'io.ls
OH
NO
NO
2 , 3-dinitrophenol
2,4-dinitrophenol
OH
2, 5-dini tr
OH
NO.
NO,
[10
OH
MO
NO,
2,6-dinitrophenol
3,4-dinU.rophenol 3 , 5 —Un itroph-no 1
C-33
-------
Ingestion from Water
No data were available regarding human exposure via in-
gestion of dinitrophenols from water.
The enhancement of biological waste water treatment by
2,4-DNP has recently been examined (Shah, 1975; Shah, et al.
1975). Addition of 0.92 mg/1 2, 4-DNP 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/1) is undesirably high from the stand-
point of current Federal effluent regulations but that the
compound is completely eliminated by absorption 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-DNP contamination of surface
waters.
Games and Kites (1977) detected dinitrophenol (isomer
not identified) in the effluent waters of a dye manufacturing
plant. 400 to 3200 ug/1 dinitrophenol was detected in raw
waste water, prior to biological treatment. The final plant
effluent contained 42 to 270 ug/1 of dinitrophenol. Mud and
river water samples downstream from the effluent point were
analyzed by gas chromatography/mass spectromometry. Dinitro-
phenol was not detected in these samples.
The persistence of dinitrophenol isomers in ambient
waters has not been well studied. A number of investigators
have studied the bacterial degradation of the dinitrophenols
C-34
-------
utilizing acclimated populations of microorganisms. Phenol-
adapted bacteria obtained from garden soil, compost, and
river mud degraded 2,4-dinitrophenol in seven to ten days
(Tabak, et al. 1964). 2,6-dinitrophenol was degraded very
slowly in this system. 2,4-, 2,5-, and 2,6-dinitrophenol
were tested for biological degradability by an activated
sludge culture obtained from a sewage treatment plant
(Fitter, 1976). 2,5-dinitro and 2,6-dinitrophenol were not
degraded in this system although 85 percent removal of 2,4-
dinitrophenol was achieved within 20 days. Further degrada-
tion of 2,4-dinitrophenol did not occur in this system, how-
ever. 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 sus-
ceptible to partial degradation by certain microorganisms.
Of the dinitrophenol isomers, 2,4-DNP appears to be most
easily degraded. It may be speculated that dinitrophenols
will be subject to microbial attack in environmental situa-
tions where acclimated microbiological populations exist
(e.g. sewage treatment plants). The persistence of dinitro-
phenols in the environment where acclimated microbial popula-
tions do not exist is speculative.
Ingestion from Foods
No data were found demonstrating the presence of dini-
trophenols in food.
C-35
-------
No measured steady-state bioconcentration factor (BCF)
is available for any nitrophenol; however, an estimated value
can be derived by using a log equation (Veith, et al.. Manu-
script) based upon the octanol-water partition coefficient.
Thus, the weighted average BCF for 2,4-dinitrophenol and the
edible portion of all aquatic organisms consumed by Americans
is 2.4 (Table 2A).
Inhalation
Dinitrophenol isomers may be produced in the atmosphere
through a photochemical 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 photo-
chemical products: nitrobenzene, 2-nitrophenol, 4-nitro-
phenol, 2,4-dinitrophenol and 2,6-dinitrophenol. The authors
suggested that these nitrocompounds may be the cause of the
characteristic symptoms of seriously stricken victims of
photochemical smog in Japan, which include headache, breath-
ing difficulties, vomiting, rise in body temperature and
numbness in the extremities. In the absence of monitoring
data it is impossible to estimate the extent of human ex-
posure to dinitrophenols as a result of their photochemical
production in the atmosphere.
Dermal
2,4-DNP is rapidly absorbed through the intact skin
(Gleason, et al. 1969). Although no direct information on
the other dinitrophenol isomers is available, it is reason-
able to suppose that dermal absorption will readily occur
C-36
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with these compounds as well. Since 2,4-DNP is used pri-
marily as a chemical intermediate, dermal exposure is expect-
ed to occur most often in an industrial setting. 2, 4-DNP is
also used occasionally as a spray against aphids and mites,
as a fungicide for certain molds and mildews, as a weed kil-
ler, and as an ingredient in some wood preservative formula-
tions (Gleason, et al. 1969). Dermal exposure to humans may
occur among individuals handling 2,4-DNP in these applica-
tions. Direct data on the importance of the dermal exposure
route of dinitrophenols in humans are not available.
PHARMACOKINETICS
Absorption
The dinitrophenol isomers are readily absorbed from the
gastrointestinal tract based on the toxicological data to be
presented in a later section. In addition, absorption
through the skin and following inhalation readily occurs (von
Oettingen, 1949).
Gehring and Buerge (1969b) reported that 2,4-DNP is ab-
sorbed extremely rapidly by ducklings and rabbits following
intraperitoneal administration. In fact, immature rabbits
absorbed the administered DNP so rapidly that an absorption
constant could not be calculated from the data. DNP concen-
tration in serum peaked within five minutes of administration
Other quantitative information on the rate of absorption
of the dinitrophenol isomers was not found.
Distribution
Blood levels of the dinitrophenols rise rapidly follow-
ing absorption (Gehring and Buerge, 1969b; Harvey, 1959)
suggesting that the dinitrophenol isomers are transported by
C-37
-------
the blood regardless of the mode of absorption. 2,4-DNP
binds to serum proteins following intraperitoneal administra-
tion to rabbits and ducklings. Early after the administra-
tion of 2,4-DNP the concentration 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
are not stored to any significant extent in the tissues of
human or experimental animals following 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.
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 dinitrophenol (unspecified
isomer) and amino nitrophenol in liver, kidney, brain, blood,
and spinal fluid of dogs after fatal doses of dinitrophenol.
Recent work on the tissue distribution of the dinitrophenols
following absorption 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: amino-2-nitro-4-phenol, amino-4-nitro-
2-phenol, diamino-phenol, and a number of nitrogen compounds
resulting from a combination of two molecules of amino-
nitrophenol or of diamino-phenol. It has frequently been
reported that 2-amino-4-nitrophenol invariably exists in the
urine of persons suffering from serious intoxication by
C-38
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2,4-DNP. Williams (1959) stated that 2,4-DNP is excreted in
mammals in the following forms: partially unchanged; par-
tially conjugated with glucuronic acid; reduced to 2-amino-
4-nitrophenol, 2-nitro-4-aminophenol and probably 2,4-di-
aminophenol. Rats orally dosed with 1.5 to 12 mg/kg of 2,
4-DNP excreted both free dinitrophenol (78 percent) and
2-amino-4-nitrophenol (17 percent) (Senszuk, et al. 1971).
Although the in vitro metabolism of 2,4-dinitrophenol
has not been extensively studied in mammalian systems, Parker
(1952) examined the enzymatic reduction of 2,4-DNP by rat
liver homogenates and found 4-amino~2-nitrophenol to be the
major metabolite. The metabolite 2-amino-4-nitrophenol com-
prised less than 10 percent of the total metabolites formed;
2,4-diaminophenol was found in trace amounts. Presumably the
latter metabolite was formed from the reduction of the re-
maining nitro group of one of the two above compounds.
In contrast, Eiseman, et al. (1974) reported 2-amino-4-
nitrophenol was the major metabolite (75 percent of total
amine). In the latter report 4-amino-2-nitrophenol was found
to have been formed In considerably lesser amounts (23 per-
cent) when 2,4-DNP was enzymatically reduced in vitro by rat
liver homogenates. These investigators also detected only
traces of diaminophenol indicating that it may be a secondary
reduction product as suggested by Parker (1952). A precise
definition of the metabolic fate of the dinitrophenols in
humans awaits further investigation.
C-39
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Excretion
Data on the elimination kinetics of the dinitrophenols
or their metabolic products in humans were not found. Edsall
(1934) stated "judging from the metabolic 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 period." Information
on the elimination kinetics of the dinitrophenols from
experimental animals is also scant in the literature.
Gehring and Buerge (1969b) have developed equations
which describe the elimination of 2,4-DNP from the serum of
ducklings, mature rabbits, and immature rabbits following
intraperitoneal administration of the compound. Serum levels
of 2,4-DNP in the mature rabbit declined to less than one
percent of their original high values within seven hours.
Twenty- four hours were required before the serum levels in
the immature rabbit declined to two percent of their original
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 nitrophenolic compounds (including 2,4-dinitro-
phenol). Elimination from the blood of mice, rabbits, guinea
pigs, rats, and monkeys was complete within 30 hours. Harvey
(1959) calculated the elimination rates of all six dinitro-
phenol isomers from the blood of mice and rats following a
single large dose given intraperitonealy. His data are pre-
sented in Table 6. The data developed by these investigators
C-40
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TABLE 6
Elimination Rates of Dinitrophenol Isomers from the Blood 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-Dinitrophenol
2 , 5-Dinitrophenol
2 , 6-Dinitrophenol
3 , 4-Dinitrophenol
3 , 5-Dinitrophenol
Dose
(mgAg)
MICE
90
20
180
30
60
30
RATS
90
20
90
25
90
30
Half-time for Elimination
(min. )
2-7
54.0
3o3
238.0
3.5
2.7
12.5
225.0
13.0
210.0
11.5
2.1
Source: Modified from Harvey, 1959,
C-41
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must be taken with caution since the actual elimination of
the dinitrophenols or their metabolites in urine was not
directly measured. In view of the lack of data suggesting
concentration of the dinitrophenols in mammalian tissues and
their high water solubility, it is suggested that their elim-
ination via the urine may be a rapid process in humans.
EFFECTS
^^""^^"^^^^"••"^^ »
Acute, Sub-acute, 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 reviews on the
uses, chemistry, mode of action, and mammalian toxicity of 2,
4-dinitrophenol are available (Edsall, 1934; Metcalf, 1955;
Horner, 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-dinitrophenol is considered a classic uncoupler of
oxidative phosphorylation and is widely used by biochemists
to determine whether a given biochemical process is energy
dependent. Hence, an enormous body of literature has been
generated dealing with the biochemical effects of 2,4-dini-
trophenol on cellular and biochemical processes both in vivo
and in vitro. Only those studies with direct relevance to
the acute or chronic effects of the dinitrophenols on humans
are reviewed in this document.
C-42
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The toxic action of the dinitrophenols is generally
attributed to their ability to uncouple oxidative phosphory-
lation. These compounds prevent the utilization of the
energy provided by respiration and glycolysis by inhibiting
the formation of high energy phosphate bonds. All energy
dependent biochemical processes are therefore affected by the
action of the compounds (Metcalf, 1955). The large number of
clinical effects attributed to dinitrophenol toxicity result
essentially from the short-circuiting of metabolism in cells
which absorb sufficient dinitrophenol.
All six dinitrophenol isomers are potent uncouplers of
oxidative phosphorylation. 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-dinitrophenol as measured in this
system. The relative in vivo toxicities of the dinitrophenol
isomers have been determined by a number of investigators
(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
from the order developed by Burke and Whitehouse (1967).
Several explanations for these discrepancies are possible:
(1) differential tissue absorption of the isomers, (2) dif-
ferent metabolic detoxification mechanisms for the isomers or
(3) the presence of cellular or biochemical effects unrelated
C-43
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to the uncoupling of oxidative phosphorylation,, Resolution
of this question awaits further investigation.
At concentrations higher than those necessary to un-
couple oxidative phosphorylation, a number of inhibitory
effects of the dinitrophenol isomers on certain enzymatic
reactions may occur.
Both 2,4-dinitro and 3, 5-dinitrophenol inhibit porcine
heart malate dehydrogenase in vitro (Wedding, et al. 1967).
Inhibition of the reaction occurred at nitrophenol concentra-
tions 10 to 100 times those causing uncoupling, and resulted
from a competitive inhibition with NAD in the forward direc-
tion of the malate dehydrogenase reaction. In a similar
study Stockdale and Selwyn (1971) reported the in vitro inhi-
bition of both lactate dehydrogenase and hexokinase by
2,4-dinitro, 2,5-dinitro, and 2,6-dinitrophenol.
The dinitrophenols may also act directly on the cell
membrane, thus causing toxic effects on cells which do not
depend on oxidative phosphorylation for their energy require-
ments. 2,4-dinitro, 2,5-dinitro, and 2,6-dinitrophenol in-
hibit passive chloride permeability (a metabolic independent
process) in red blood cells (Motais, et al. 1978).
Acute toxicity information for the dinitrophenols has
been compiled and presented 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-DNP intoxication relate to its usage as a component of
explosives during World War !<, Thirty-six cases of fatal
C-44
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TABLE 7
Acute Toxicity of Dinitcophenol
Species
Rat
Rat
Rat
o
i Rat
en
Rat
Mouse
Mouse
Guinea Pig
Rabbit
Rabbit
Rabbit
Dog
Dog
Dog
Dog
Dose
(mg/kg)
25
35
30
28.5
31
36
26
700
30
200
100
30
20-30
22
20
Route of
Adminisration
2 ,4-Dinitrophenol
S.C.
I. P.
Oral
I. P.
I. P.
I. P.
I. P.
Dermal
S.C.
Oral
I. P.
UNK
Oral
S.C.
I.M.
Effects
LD 50
LD 50
LD 50 '
LD 50
LD 100
LD 50
LD 50
Lethal Dose
LD 50
LD 50
Lethal Dose
Minimum Lethal Dose
LD 50
LD 50
LD 50
References
von Oettingen,
Harvey, 1959
Spector, 1956
Lawford, et al
Gatz and Jones
Harvey, 1959
Lawford, et al
Spencer, et al
von Oettingen,
Spector, 1956
Spector, 1956
Harvey, 1959
Spector, 1956
Spector, 1956
Spector, 1956
1949
. 1954
, 1970
. 1954
. 1948
1949
-------
TABLE 7 (Continued)
Species
Dog
Pigeon
Pigeon
Human
Human
n Human
Rat
Mouse
Dog
Rat
Mouse
Dog
Dose
(mg/kg)
30
7
15-20
40 mg/m
1-3 g
4.3
190
200
1000
150
273
100
Route of
Administration
I.V.
I.M.
I.V.
Inhalation
Oral
Oral
2 , 3-Dinitrophenol
I. P.
I. P.
UNK
2,5-Dinitrophenol
I. P.
I. P.
UNK
Effects
LD 50
LethaL Dose
Lethal Dose
Lethal Concentration
Lethal Dose
Lethal Dose
LD 50
LD 50
MLD
LD 50
LD 50
MLD
References
Spector, 1956
Spector, 1956
Spector, 1956
MacBryde and Taussig, 193
Sax, 1968
Geiger, 1933
Harvey, 1959
Harvey, 1959
Harvey, 1959
Harvey, 1959
Harvey, 1959
Harvey, 1959
-------
TABLE 7 (Continued)
n
i
Dpse
Species (mg/kg)
Rat 38
Mouse 45
Dog 50
Rat 98
Mouse 112
Dog 500
Rat 45
Mouse 50
Dog 500
Route of
Administration Effects
2 , 6-Dinitrophenol
I. P. LD 50
I. P. LD 50
UNK MLD
3,4-Dinitrophenol
I. P. LD 50
I. P. LD 50
UNK MLD
3 , 5-Dinitrophenol
I. P. LD 50
I. P. LD 50
UNK MLD
References
Harvey,
Harvey,
Harvey,
Harvey,
Harvey,
Harvey,
Harvey,
Harvey,
Harvey,
1959
1959
1959
1959
1959
1959
1959
1959
1959
-------
occupational dinitrophenol poisoning occurred among employees
of the munitions industry in France between 1916 and 1918
(Perkins, 1919). A literature review by von Oettingen (1949)
revealed 27 reported cases of fatal occupational dinitro-
phenol 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 poisoning by 2,4-DNP.
Early in the 1930's, 2,4-dinitrophenol was widely recom-
mended as a treatment for obesity. Dinitrophenol was re-
ceived with overwhelinmg popularity (Homer, 1942) as a
slimming agent in spite of warnings of harmful side effects
caused by disruption of the metabolic rate. It was estimated
that during the first 15 months following its introduction,
100,000 persons took the drug for weight reduction (Horner,
1942). More than 1,200,000 capsules of 0.1 g each were dis-
pensed from a single clinic in San Francisco. More than 20
drug houses offered to supply both dinitrophenol and mixtures
containing the drug. Many of these remedies could be pro-
cured without prescription 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 com-
pound it is not surprising that both toxic side effects and
fatalities resulted. Horner (1942) reported a total of nine
deaths resulting from the use of dinitrophenol as a slimming
agent.
C-48
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Parascandola (1974) reviewed the history and public con-
cern which developed 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 disturbances (nausea, vomiting, colic, diar-
rhea, anorexia) profuse sweating, weakness, dizziness, head-
ache, and loss of weight. Acute poisoning has resulted in
the sudden onset of pallor, burning thirst, agitation, dy-
spnea, 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 (es-
timated at 2.5 to 5 g) was literally "cooked to death"
(Geiger, 1933). Rectal temperature at death exceeded 110°F.
Perkins (1919) made the interesting observation that
post-mortem examination of dinitrophenol victims demonstrated
no characteristic lesions. Acute edema of the lungs was men-
tioned but was believed to be secondary to toxic effects on
the vasomotor system. Microscopic lesions of the liver and
kidney cells were inconstant and typical changes were lacking
elsewhere.
C-49
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Spencer, et al. (1948) studied the chronic toxicity of
2,4-dinitrophenol in rats. Male rats were fed diets contain-
ing 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 and
pathological investigations on surviving animals were carried
out. Hematological 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 car-
ried out. Rats maintained on diets containing .02 percent
2,4-DNP (corresponding to 5.4 to 20 mg/kg body weight/day)
grew at a normal rate and the investigators failed to detect
discernible ill effects or pathological changes at autopsy.
Similary pathological changes were not found upon microscopic
examination of tissues from rats receiving diets containing
.05 percent 2,4-DNP (corresponding 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 ani-
mals were a very slight depletion 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 re-
vealed marked emaciation, an empty gastrointestinal tract, a
slightly enlarged dark spleen, and small testes. Microscopic
C-50
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examination showed slight congestion and cloudy swelling of
the liver, very slight parenchymatous degeneration of the
epithelium of the renal tubules, slight congestion and hemo-
siderosis of the spleen, and testicular atrophy. No signifi-
cant pathological changes were observed in the lung, heart,
adrenals, pancreas, or stomach of these animals. Based on
the work of Spencer, et al. (1948), the 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 dinitrophenol isomers in experimental animals was not
found. Langerspetz 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-dinitrophenol was administered via the subcutaneous injec-
tion of 10 mg, 2,4-DNP/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 L,D 50 was chosen (20 mg/kg) only small areas of cortical
tubular necrosis were observed in a few of the rats 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. Liver and kidney pathology were not detected
out an effect on spleen tissue was noted. Over large areas
of the spleen lymphocytes were replaced by a more or less
homogenous material containing "numerous large faintly stain-
ing cells with vesicular polyhedral nuclei."
C-51
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The widespread use of 2,4-dinitrophenol as a weight re-
ducing agent in humans during the 1930's provides some infor-
mation regarding the chronic effects of this compound in man.
Recommended theraputic doses of 2,4-DNP for weight control in
humans ranged from 2 to 5 mg/kg body weight/day (Dunlop,
1934; Homer, 1942; Tainter, et al. 1933). Tainter, et al.
(1933) administered 2,4-DNP to 113 obese patients for as long
as four months without demonstrating evidences of cumulative
or toxic effects. The most important side effect noted by
the investigator was a skin rash observed in about seven per-
cent of the patients treated. The rash was manifested usual-
ly after a one-day period of mild itching and consisted of a
maculopapular or urticarial type of rash. The itching was
intense and in some cases there was considerable swelling.
Symptoms subsided in two to five days following withdrawal
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 cleared up
following withdrawal from 2,4-DNP. The investigators failed
to detect any effect of 2,4-DNP 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. (1935). In a
later publication, Horner (1942) reviewed the acute and
chronic toxicity of 2,4-DNP (including cataract formation)
C-52
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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 aberations of taste and multiple
regional involvement affecting, particularly, the feet and
legs were recorded. Symptoms appeared after an average of
ten weeks, followed ordinary therapeutic doses and persisted
for weeks or months. Electrocardiographic evidence of
functional heart damage was offered by several investigators
and fragmentation of the heart muscle at autopsy in one fatal
case was reported. It was generaly agreed that 2,4-DNP was
rarely injurious to the liver and kidneys when administered
in therapeutic doses.
Over 100 cases of cataract formation following dinitro-
phenol therapy were reviewed by Horner (1942). Horner de-
scribed the following characteristic 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 or after
periods of dinitrophenol treatment. 3. An interval of
nonths or years might elapse between the time the last dose
was taken and the onset of blurred vision. 4. Lenticular
C-53
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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 gradual onset, the len-
ticular changes progressed with startling rapidity until the
vision was obscured. 6. Treatment was without effect in
staying their progress. 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 among 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 bearing on
the occurrence of cataracts. Individual susceptibility ap-
peared to be a more important factor. Horner (1942) estimat-
ed that the incidence of cataracts in patients who had taken
dinitrophenol exceeded one percent.
Formation of cataracts by acute exposure to DNP 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;
Bettman, 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 dis-
appear spontaneously in animals within 25 hours (Howard, et
al. 1976). Hence, the usefulness of data on the formation
C-54
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of cataracts in experimental animals following DNP
administration to 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 activity 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 DNP administration, differs
significantly from the situation seen in experimental 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 100
ug/egg of 2,4-dinitrophenol was nontoxic and nonteratogenic
after 96 hours of incubation. However, the combined adminis-
tration of insulin (a known teratogen) with 100 micrograms 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.
Both thyroid hormones and 2,4-dinitrophenol decrease the
efficiency of mitochondrial oxidative phosphorylation in vivo
and in vitro. The in vivo administration of both L-thyroxine
and 2,4-DNP results in larger changes in metabolic rate and
C-55
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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-dinitro-
phenol on the fertility, gestation, and fetal life of rats.
They administered 20 mg of 2,4-DNP/kg to female rats eight
days prior to the introduction of males. Dinitrophenol was
administered intragastrically twice daily until the respec-
tive litters were weaned. The average number born in each
litter was not affected by the use of dinitrophenol. Neither
did the treatment appreciably affect the body weight gains of
mothers during pregnancy. Neonatal 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 2,4-DNP mothers was 30.9 percent as
compared with 13.4 percent for young of control mothers. Two
possible explanations for this latter phenomenon were offered;
Dinitrophenol mothers neglect their young while in a febrile
state, and only the more vigorous of the offspring manage to
reach the mother for nursing; or, a toxic agent is passed to
the young through the milk. Data to distinguish between the
two possibilities are not available.
C-56
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Intraperitoneal (7.7 or 13.6 mg/kg) or oral (25.5 or
38.3 mg/kg) administration of 2,4-DNP to mice during early
organogenesis does not produce morphological defects in the
young, but embryotoxicity occurs at the higher dose levels
(Gibson, 1973). The higher doses also produced overt toxic
signs (hyperexcitability and hyperthermia) in the dams, but
were not lethal.
Bowman (1967) has studied the effect of 2,4-DNP on the
developing chick embryo in vitro. At 2,4-DNP concentrations
of 18 mg/1 or 370 mg/1 a syndrome of abnormalities resulted,
consisting of degeneration and sometimes complete absence of
neural tissue accompanied by a reduction in the number of
somites. The 2,4-DNP concentrations used in this study are
extremely high and the relevance of the experimental findings
to the in 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 incuba-
tion (Miyamoto, et al. 1975). Based on examination of puri-
fied myelin in the malformed embryos the investigators sug-
gested that 2,4-DNP administration resulted in deficient em-
bryonic myelination.
Based on the available data it appears unlikely that the
dinitrophenols pose a teratogenic hazard to humans. Further
investigations on this question are warranted.
C-57
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Mutagenicity
Friedman and Staub (1976) have developed an approach to
mutagenic testing which utilizes the measurement of induction
of unscheduled DNA synthesis in testes. These investigators
found a good correlation between a reduction in the residual
level of cell cycle associated DNA synthesis and the presence
of known mutagenic compounds. Testicular DNA synthesis in
mice was unaffected by administration of 2,4-DNP suggesting a
lack of mutagenic activity.
Bacterial mutagenesis of 2,4-DNP has been tested by
Demerec, et al. (1951), based on the production of back muta-
tions from streptomycin dependence to independence in E.
coli. Mutations were increased several-fold over control
values.
A recent study has been conducted on the effect of vari-
ous phenolic compounds including 2,4-DNP on chromosomes of
bone marrow cells from mice (Mitra and Manna, 1971). Mice
were injected intraperitoneally with 2,4-DNP and bone marrow
tissue was collected 24 hours after treatment. 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 administered to the mice by these investigators. The
water solubility of 2,4-DNP at 75.8°F is 3.01 mg/ml (Wind-
holz, 1976). If this value approximates the saturated solu-
tion used by Mitra and Manna (1971) and a three-to-four month
C-58
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old mouse weighs approximately 40 g, the following calcula-
tions result in three 2,4-DNP dose levels expressed as mg/kg
body weight.
- 18'8
(0'5 ml) ° - 37-6
75'3
The ability of 2,4-DNP to induce chromosomal damage
using an in vitro alkaline elution assay employing Chinese
hamster V79 cells (with or without a liver microsomal activa-
tion DNP system) was examined by Swenberg, et al. 1976). 2,
4-DNP failed to induce DNA damage in this system.
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-DNP to
promote tumor formation following a single initiating dose of
dimethylbenzanthracine. Although phenol itself has a promot-
ing 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.
C-59
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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 that 2,4-DNP does not possess
carcinogenic properties. Information on the other isomeric
dinitrophenols is not available.
060
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TRINITROPHENOLS
Mammalian Toxicology and Human Health Effects
EXPOSURE
Introduction
Six isomeric forms of trinitrophenol are possible, dis-
tinguished by the position of the nitro groups relative to
the hydroxy group on the six carbon benzene ring. The five
isomers are: 2,3f4-, 2,3,5-, 2,3,6-, 2,4,5-, 2,4,6- and 3,4,
5-trinitrophenol. Production volumes for the trinitrophenols
are not available. Usage of the trinitrophenol isomers is
apparently limited to 2,4,6-trinitrophenol, otherwise known
as picric acid. In fact, a comprehensive search of the
literature failed to detect a single citation dealing with
any of the trinitrophenol isomers except picric acid. Con-
sequently, the only information on these isomers presented in
this document are the chemical and physical properties found
in Table 8.
According to Matsuguma (1967) picric acid has found
usage as: a dye intermediate, 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, steel and
copper surfaces. The extent to which picric acid finds usage
in any of these applications at the present time is unknown.
061
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TABLE 8
Properties of Trinitrophenols
2,3,4-Trinitrophenol
Molecular Weight
229.11
2,3,5-Trinitrophenol
Molecular Weight
Melting Point
229.11
119-120°C
2,3,6-Trin itrophenol
Molecular Weight
Melting Point
Water Solubility
Room Temperature
Hot Water
229.11
119°C
Slightly Soluble
Very Soluble
2,4,5-Trinitrophenol
Molecular Weight
Melting Point
Water Solubility
Room Temperature
Hot Water
229.11
96°C
Slightly Soluble
Soluble
2,4,6-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
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Tr i n i tro^he no 1g
2,3,4-trinitrophenol 2, 3 , 5-trinif.rophenol 2 , 3 ,6- trinitrophenol
2,4,5-trinitrophenol
2,4,6-tr.in.r trophenol 3,4, 5-trini t-.roplvr-.ol
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Ingestion from Water
Monitoring data on the presence or absence of 2,4,
6-trinitrophenol (2,4,6-TNP) in water were not found. A
single report of 2,4,6-TNP contamination of ground water was
found, however (Cole, 1974). In 1955, 2,4,6-TNP (0.7 mg/1)
was detected in a well approximatley one mile from the former
site of an explosives manufacturing plant in England. The
plant was engaged in the manufacture of explosives from 1914
to 1918. The brief report by Cole (1974) failed to describe
either the types of explosives manufactured by the plant or
the disposition of the waste water during the period of ex-
plosives manufacture.
Harris,.et al. (1946) described an outbreak of hematuria
involving U.S. Navy personnel aboard ships anchored at Waka-
yama, Japan which resulted from ingestion of 2,4,6-TNP in the
drinking water. Approximatley three weeks prior to the out-
break of hematuria, more than 100 tons of confiscated Japan-
ese ammunition, (including 2,4,6-TNP) had been dumped in the
immediate vicinity of the anchorage. 2,4,6-TNP was apparent-
ly pumped into the ships' drinking water stills and carried
over with the vapor phase and into the freshwater supply, in-
ducing hematuria among those drinking the water. The inves-
tigators failed to detect 2,4,6-TNP in the sea water; how-
ever, analysis of the distilled drinking water yielded 2,
4,6-TNP levels of 2 to 20 mg/1.
Although it is not possible to precisely estimate either
the TNP water levels or duration of exposure necessary to in-
C-64
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duce hematuria, Harris, et al. (1946) detected levels of 10
ir.g/1 and 20 mg/1 in drinking water aboard two ships at the
time of the hematuria outbreak.
Hoffsonuner and Rosen (1973) have shown that the high ex-
plosive tetryl (N-methyl-N-nitro-2,4,6-trinitroaniline) dis-
solved in sea water at pH 8.1 and 25°C is largely converted
to 2,4,6-TNP in a few months. Although tetryl is no longer
manufactured in the U.S. (Howard, et al. 1976), these experi-
ments indicate that 2,4,6-TNP may be produced in water as a
result of degradation of other organic compounds. The nature
of other compounds which may give rise to 2,4, 6-TNP follow-
ing degradation is speculative.
The persistence of 2,4,6-TNP following release to the
environment is not 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 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 mg/1) by acclimated cultures of microorganisms
derived from garden soils, compost, and river mud in three to
six days. The extent to which microbial 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.
Ingestion from Foods
No information on human ingestion of trinitrophenols
from food was found.
No measured steady-state bioconcentration factor (BCF) is
available for any nitrophenol; however, an estimated value can
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be derived by using a log equation (Veith, et al., Manuscript)
based upon the octanol-water partition coefficient. Thus,
the weighted average BCF for 2,4,6-trinitrophenol and the
edible portion of all aquatic organisms consumed by Americans
is 6.0 (Table 2A).
Inhalation
No information on the presence or absence of trinitro-
phenols in air was found.
Dermal
Information on the dermal absorption of 2,4,6-TNP is
scant in the literature. During the 1920's and 30's, 2,4,
6-TNP was used both alone and in combination with butesin
(dinormalbutyl-p-aminobenzoate trinitrophenol) as an antisep-
tic 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 oint-
ments were properly used, there was no danger of toxic symp-
toms developing in humans.
A serious case of central nervous system dysfunction
following the topical application of 2,4,6-TNP was reported
by Dennie, et al. (1929). The patient recovered rapidly fol-
lowing removal from the 2,4,6-TNP treatment. No other infor-
mation 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 experimental animals is not available.
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Neurological complications following the topical admin-
istration of 2,4,6-TNP (Dennie, 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 is questionable.
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.
Distribution
Autopsy 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, and the blood vessels.
The results indicate that 2,4,6-TNP is distributed to many
tissues in the body. These investigators 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 the
distribution of 2,4,6-TNP would occur via the blood. No
other data on the tissue distribution of 2,4,6-TNP following
absorption were found.
Metabolism
In a review of the early literature. Burrows and Daere
(1975) indicated that elimination of 2,4,6-TNP from humans
occurs in both the free form and as picramic acid. In per-
fusion experiments with liver, kidney and spleen, the liver
exhibited the strongest capacity for reduction of 2,4,6-TNP.
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Other studies dealing with the metabolism of 2,4,6-TNP
in humans or in experimental animals were not found.
Decompositon of 2,4,6-TNP by an atypical strain of
Corynebacterium simplex with the production of nitrites has
been reported by Gunderson and Jensen (1956). This alterna-
tive 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 administration of a lethal dose in dogs was re-
ported by Dennie, et al. (1929). The presence of 2,4,6-TNP
in the urine of humans following oral exposure was reported
by Harris, et al. (1946). These studies indicate that 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, Sub-acute, and Chronic Toxicity
According to Windholz (1976) ingestion or percutaneous
absorption of 2,4,6-TNP may cause nausea, vomiting, diarrhea,
abdominal pain, oliguria, anurea, yellow staining of skin,
pruritus, skin eruptions, stupor, convulsions, and death.
Although Dennie, et al. (1929) state: "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 reports of human
fatalities resulting from 2,4,6-TNP exposure were found in
the literature. Gleason, et al. (1968) reports 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
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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 result in lethal
effects in humans. The limited acute toxicity information
for experimental animals has been compiled and presented as
fable 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 non-lethal doses of trinitrophenol
(TNP) appears to be an allergic or irritative dermatitis
(Anon. 1937; Ehrenfried, 1911). According to Dennie, et al.
(1929) about four percent of people treated with TNP are sen-
sitive and develop a local dermatitis. Reactions may also
appear in unexposed areas. An intense itching and burning,
printus, skin eruptions, and irritability are common. Skin
eruptions are characterized by irregular-shaped macules,
popules, 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 charac-
teristic, even in distant lesions.
More severe reactions can lead to diffuse, often severe
erythema and desquamation of affected areas (Sulzburger and
Wise, 1933; Am. Conf. Gov. Ind, Hyg., 1971). The reaction
may last from several weeks to almost a year (Sulzburger and
Wise, 1933).
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TABLE 9
Acute Toxicity of Trinitrophenol Isomers3
o
I
Species
Dog
Dog
Dog
Rabbit
Frog
Frog
Cat
Human
Dose
(mgAg)
100-125
60
60
120
200
200-300
500b
5
Route of
Administration
2,4 ,6-Trinitrophenols
S.C.
S.C.
?
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
aAcute toxicity data for trinitrophenol isomers other than 2,4,6-TNP were not found.
dose in milligrams.
-------
Ingestion from Water
Monitoring data on the presence of DNOC in ambient water
are not available. An unspecified amount of DNOC was de-
tected in the wastewaters of Fison's Pest Control Limited in
Harston, Cambridge, England (Jenkins and Hawkes, 1961). Webb,
et al. (1973) detected 18 mg/1 DNOC in the wastewater of a
specialty chemical plant. The extent to which human exposure
to DNOC results from the ingestion of contaminated water is
unknown.
Ingestion from Foods
No data are available on the presence or absence of DNOC
residues in food for human consumption. Since the primary
usage of the compound involves treatment of fruit trees dur-
ing the dormant season, it appears unlikely that contamina-
tion of human food stuffs would occur to any large extent.
No measured steady-state bioconcentration factor (BCF) is
available for any nitrophenol; however, an estimated value can
be derived by using a log equation (Veith, et al., Manuscript)
based upon the octanol-water partition coefficient. Thus,
the weighted average BCP for DNOC and the edible portion of
all aquatic organisms consumed by Americans is 7.5 (Table 2A).
Inhalation
An evaluation of the literature (Natl. Inst. Occup.
Safety Health, 1978) indicates that occupational 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. Per-
sons at risk include those manufacturing, formulating, or ap-
plying the compound as an aerosol. Inhalation esposure to
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the general public is expected to be minimal although data
addressing this point are not available Dermal
As mentioned in the proceeding section, occupational in-
toxication by exposure to DNOC has occurred as a result of
inhalation and dermal exposure where the compound is manu-
factured, formulated or applied. Dermal exposure of the gen-
eral 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 gastrointestinal tract and respiratory tract in humans
(Natl. Inst. Occup. Safety Health, 1978). Although most
cases of occupational intoxication resulting from DNOC expo-
sure contain both a respiratory and a dermal component, human
intoxication has been reported as a result of dermal contact
to DNOC aloneo
In a report from the Russian literature (Buchinskii,
1974; reviewed by NIOSH, 1978) a four-year-old boy was fa-
tally intoxicated after a rash had been treated with 50 g of
an ointment to which 25 percent DNOC was added by mistake.
Stott (1956) reported 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 so-
lutions of DNOC. Since neither man worked near the actual
operation, and both denied blowing into the spray jet to
clean them, Stott (1956) concluded that the major route of
exposure was skin contact,.
Work by Harvey, et al0 (1951) indicates that DNOC is
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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 maxi-
mal from two to four hours after ingestion. Van Noort, et
al. (1960; reviewed by NIOSH, 1968) investigated the effec-
tiveness of personal protective equipment used by 24 sprayers
in Holland. Serum DNOC levels and the quantity of DNOC used
were determined in a three-week spraying period. Protective
equipment ranged from usage of full body covering and masks
to individuals who failed to use any type of protective
equipment. Their findings indicated that both inhalation of,
and dermal contact with, DNOC can lead to an appreciable
absorption into the blood stream.
Experimental animal studies, reviewed by NIOSH (1978),
also have confirmed the toxicity of DNOC in humans exposed by
the oral, inhalation, and dermal routes.
Distribution
Whether absorption of DNOC occurs through the skin, gas-
trointestinal tract, or respiratory tract, the compound is
transported in and distributed by the blood (Natl. Inst.
Occup. Safety Health, 1978). Harvey, et al. (1951) described
the effect of DNOC taken orally by five male volunteers.
Capsules containing 75 mg of pure DNOC were administered
daily for five consecutive days amounting to a dose of 0.95
to 1.27 mg/kg/day. The concentration of DNOC in the blood
increased in the first three to four days and reached concen-
trations of 15 to 20 mg/kg. After concentrations of 15 to 20
had been obtained, additional doses appeared to cause
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temporary high blood concentrations which were associated
with toxic symptoms.
Blood analysis of humans displaying symptoms of DNOC
toxicity has invariably revealed concentrations exceeding 10
mg/kg (Natl. Inst. Occup. Safety Health, 1978).
In studies conducted to determine the kinetics of ab-
sorption and distribution, DNOC has not been shown to accumu-
late in the blood of various 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 de-
creased despite the administration of two additional doses.
DNOC is more rapidly eliminated from the blood of ani-
mals 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 DNOC,
elimination from the serum of rabbits was nearly complete.
Four days were necessary for serum clearance in rats and
cats, while six days were required 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 administration by stomach tube (King and Harvey, 1953a),
The accumulation of DNOC in the blood of humans follow-
ing DNOC exposure has been well documented (Harvey, et al.
1951; Bidstrup, et al. 1952). The accumulative effect may
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reflect the binding of DNOC with albumin in the blood and a
subsequent slow rate of excretion in humans (Harvey, et al.
1951).
DNOC is slowly eliminated from humans. The investiga-
tions 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 volun-
teers. Another study (Van Noort, et al. 1960; reviewed by
NIOSH, 1978) showed that it took two to eight weeks for DNOC
to be cleared from the serum.
Parker, et al. (1951) studied the tissue distribution of
DNOC 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 contained
the high levels of DNOC but the investigators postulated that
these levels were the highest due to the high 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 DNOC could be ac-
counted for, of which 72 percent was in the blood.
DNOC content of a number of tissues was determined in
rats receiving a single subcutaneous injection of the com-
pound (Parker, et al. 1951). The results, presented as Table
11, clearly indicate the DNOC failed to accumulate in the
tissues.
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TABLE 11
DNOC Content of Blood and Tissues" of Rats Killed at Intervals After Subcutaneous Injection
of One Dose of 1.5 mg DNOCa
Time
After Injection
n
i
00
30
1
2
3
4
5
6
mins.
hr.
hrs.
hrs.
hrs.
hrs.
hrs.
Serum
(mg/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.
7.
11.
11.
4.
4.
7.
5
5
0
0
5
5
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
8.0
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
aSource: Parker, et al. 1951.
content of tissue mg/kg net weight.
-------
In another experiment Parker, et al. (1951) failed to
detect significant DNOC accumulation in liver or kidney tis-
sue of rats after 40 successive daily injections of 20 mg/kg
DNOC.
In a single study reviewed by NIOSH (1978) Sovljanski,
et al. (1971) discussed tissue distribution of DNOC in
humans. Autopsy results of two suicide victims, 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 quantita-
tive 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 detoxi-
fication and excretion are very slow or there is some storage
of DNOC in body tissues. Based on their calculation of ex-
cretion kinetics in man, the investigators suggested that
detoxification and excretion of DNOC are inefficient and slow
in humans.
None of the available data suggest significant accumula-
tion of DNOC in specific tissues of humans or experimental
animals (Natl. Inst. Occup. Safety Health, 1978).
Metabolism
The metabolism of DNOC in humans has not been studied.
Several investigators have conducted experiments to determine
the fate of DNOC after its administration to animals, however,
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Truhaut and De Lavaur (1967; Reviewed by NIOSH, 1978)
reported on the metabolism of DNOC in rabbits. Following the
administration of DNOC by gastric intubation into rabbits,
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 animals. 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, very little 6-amino-4-nitro-o-cresol
was detected in either the urine or tissues. The authors
considered the metabolism of DNOC 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 6-amino-4-nitro-o-cresol
to DNOC might be a useful indicator in evaluation of the
severity of exposure to DNOC.
The metabolic fate of DNOC in rabbits was also investi-
gated by Smith, et al. (1953). Following administration of
20 to 30 mg/kg DNOC 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 per-
cent was detected as free DNOC, and 0.7 percent as DNOC con-
jugates. The conjugates were not characterized by the -in-
vestigators. Most of the urinary metabolites (about 12 per-
cent of the dose) were derivatives of 6-amino-4-nitro-o-
cresol. About 1.5 percent of the dose was excreted as
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6-acetamido-4-nitro-o-cresol, and 9 to 10.5 percent as the
hydroxyl group conjugate. Traces of 6-amino-4-nitro-o- cre-
sol, 4-amino-6-nitro-o-cresol, and 3-amino-5- nitro- salicyl-
ic acid were also detected.
Since the detoxification and excretion of DNOC in man
are very slow compared with rats or rabbits (King and Harvey,
1953b), the applicability of the experimental animal detoxif-
ication mechanism to the human situation is questionable.
The elucidation of DNOC detoxification mechanism in humans
awaits further investigation.
Excretion
Available data indicate that DNOC is rapidly excreted
following administration to experimental animals. Parker, et
al. (1951) found that DNOC injected subcutaneously disap-
peared from the blood at various rates in different species.
Single 10 mg/kg doses of DNOC were administered subcutane-
ously 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 DNOC was approximately three
hours in the rabbit, 15 hours in the rat, 20 hours in the
cat, and 36 hours in the dog.
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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 excre-
tion of DNOC from a seriously poisoned man in Great Britain.
The man was admitted to the hospital and full biochemical in-
vestigations were carried out 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; Reviewed by NIOSH, 1978) mea-
sured the serum DNOC levels in ten sprayers on a weekly basis
for two months after the spraying period ended. They found
the DNOC was eliminated from the serum slowly and that the
rate varied from individual to individual. Two to eight
weeks elapsed before DNOC was cleared completely from the
serum of these workers. The amount of time needed for DNOC
to be totally eliminated was directly related to the quantity
of DNOC in the serum on the last day of exposure.
In experiments where DNOC was orally administered to
five human volunteers, Harvey, et al. (1951) demonstrated
C-86
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that DNOC, absorbed by ingestion at intervals of 24 hours,
accumulates in the human body and is excreted slowly. Forty
days after the. last dose of DNOC was administered by mouth, 1
to 1.5 mg/1 DNOC was still present in the blood.
The experimental evidence suggests, therefore, that a
substantial difference in the excretion patterns of humans
vs. experimental animals exists. 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 (Natl. Inst. Occup. Safety Health,
1978) have long utilized serum levels of DNOC in order to as-
sess when humans are exposed to dangerous amounts of the com-
pound. A review of the literature (Natl. Inst. Occup. Safety
Health, 1978) indicates that workers with DNOC concentrations
of 40 mg/kg of whole blood (approximatley 80 mg/1 of serum)
or greater will most likely develop toxic effects. In addi-
tion, in the concentration range between 20 and 40 mg/kg of
whole blood (probably because of variation in individual sus-
ceptibility) some workers are affected and others show no ad-
verse effects. Most workers with blood DNOC levels below 20
mg/kg are not affected, although, again because of individual
susceptibility, some exhibited mild effects. The blood level
of 20 mg/kg has been used as a maximum permitted level for
industrial or agricultural workers utilizing the compound
during their employment.
Bidstrup, et al. (1952) recommended that a person should
be removed from further contact with DNOC for at least six
C-87
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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, Sub-acute, 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 DNOC in humans,
it has been estimated (Fairchild, 1977) that 5 mg/kg may
prove lethal to humans.
A large number of occupational and nonoccupational poi-
sonings of humans by DNOC have been reviewed by NIOSH (1978).
The available literature concerning humans indicates that
DNOC may be absorbed in acutely toxic amounts through 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
metabolism. Symptoms of poisoning include profuse sweating,
malaise, thirst, lassitude, loss of weight, headache, a sen-
sation of heat, and yellow staining to the skin, hair,
sclera, and conjunctiva.
In addition to the effects associated with increased me-
tabolism, other effects occasionally reported in humans poi-
soned by DNOC included kidney damage, diarrhea, unspecified
C-88
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TABLE 12
Acute Toxicity of 4,6-Dinitro-o-creosol
o
1
00
VO
t ..
•'f--
Species
Mouse
Rabbit
Guinea Pig
Rat
Rat
Rat
Rat
Mouse
Mouse
Hare
Cat
Pheasant
Partridge
Rat
Rat
Mouse
Rat
Goat
Dog
Dog
Dog
Pigeon
Dose
(mg/kg)
187
1000
500
85
30
40
30
47
16.4
24.8
50
8.4
8.3
26-39
20
24.2
24.6
50
15
5
10
5
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
LD 50
LD 50
100% Lethal
LD 50
MLD
100% Lethal
LD 50
LD 50
LD 50
LD 50
LD 50
LD 50
LD 50
LD 50
MLD
LD 50
LD 50
LD 50
LD
LD
LD
LD
References
Arustamyan, 1972; Reviewed by NIOSH,
Burkatskaya, 1965; Reviewed by NIOSH
Spencer, et al. 1948
Burkatskaya, 1965; Reviewed by NIOSH
Ambrose, 1942
Ambrose, 1942
Spencer, et al. 1948
Burkatskaya, 1965; Reviewed by NIOSH
Arustamyan, 1972; Reviewed by NIOSH,
Janda, 1970; Reviewed by NIOSH, 1978
Burkatskaya, 1965; Reviewed by NIOSH
Janda, 1970; Reviewed by NIOSH, 1978
Janda, 1970; Reviewed by NIOSH, 1978
Harvey, 1952
Ambrose, 1942
Parker, et al. 1951
Spector, 1956
Ambrose, 1942
Spector, 1956
Spector, 1956
Spector, 1956
Spector, 1956
1978
, 1978
» 1978
, 1978
1978
-------
changes in the gastrointestinal tract, in the cardiovascular
system, and in the peripheral blood and central nervous
systems.
It is generally believed that the toxic effects of DNOC
result from its ability to uncouple the oxidative phosphory-
lation process. DNOC is an extremely potent uncoupler of
oxidative phosphorylation. At a biochemical level, this
effect results in the decreased formation of adenosine tri-
phosphate (ATP) and a resulting inhibitory effect of enzyme
reactions requiring ATP. Such a toxicant is expected to have
extreme and profound effects on all tissues where the concen-
tration of the chemical is high enough to severely affect
oxidative phosphorylation. 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 investigations have correlated blood DNOC levels
with the severity 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 animals, DNOC accu-
mulates in the blood of humans. Accumulation is believed to
occur as a result of DNOC binding to albumin in the blood
(Harvey, et al. 1951). 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
C-90
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effects are compiled and presented as Table 13. The data
show 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 the concentration
range between 20 and 40 mg/kg of whole blood, some workers
are affected and others show no adverse effect (probably
because of differences in individual susceptibility). Most
individuals with blood levels of DNOC below 20 mg/kg were not
affected, although some exhibited mild effects. As the data
in Table 4 suggest, most investigators have concluded that
blood DNOC levels are associated with the severity of intoxi-
cation in humans (Natl. Inst. Occup. Safety Health, 1978).
In comparing studies on blood DNOC levels, certain pre-
cautions must be taken when correlating the results. It has
been reported that over 90 percent 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 ex-
pressed as weight/weight of whole blood can therefore only be
done by approximate conversions. Any given DNOC serum level
will have a lower value when expressed a^ the amount of whole
blood.
It is impossible to develop a dose response relationship
for occupational DNOC poisoning in humans since air concen-
trations 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 estimated.
C-91
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TABLE 13
Relationship of Blood DNOC Levels and Effects in Humans3
Route
of Exposure
No. of Individuals
and Occupation
Blood DNOC
Level (mg/kg)
Effects
to
to
Inhalation, Dermal
Inhalation, Dermal
Inhalation, Dermal
Inhalation, Dermal
Inhalation, Dermal
Inhalation,
Inhalation,
Oral
Inhalation,
Inhalation,
Inhalation,
Inhalation,
Inhalation,
Inhalation,
Inhalation,
Inhalation,
Inhalation,
Inhalation,
Inhalation,
Inhalation,
Inhalation,
Oral
Dermal
Dermal
Dermal
Dermal
Dermal
Dermal
Dermal
Dermal
Dermal
Dermal
Dermal
Dermal
Dermal
Dermal
Dermal
1 Agricultrual Worker
1 Agricultural Worker
1 Agricultural Worker
1 Agricultural Worker
1 Agricultural Worker
1000b'c
200b»c
75
60
60b'c
1 Agricultural Worker 55
2 Agricultural Workers 44-55
5 Experimental Subjects 40-48
4 Agricultural Workers 20-40b
5 Agricultural Workers 30-40
6 Agricultural Workers 21-40b
32 Agricultural Workers 7-37b
1 Agricultural Worker 30b
16 Agricultural Workers 20-30
1 Agricultural Worker 25b
21 Agricultural Workers 10-20
149 Agricultural Workers <10
4 Agricultural Workers 4-9b»c
23 Agricultural Workers l-8b'c
1 Agricultural Worker <5b»c
2 Manufacturing Workers 10-20
5 Experimental Subjects 20
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
aSource: Modified from NIOSH, 1978
bReported as mg/1
cSerum or Plasma DNOC level
-------
This lack of data makes assessment of a minimum toxic dose
for humans extremely difficult. Several studies however,
where the oral toxicity of DNOC has been assessed in humans,
shed some light on this question.
Harvey, et al. (1951) orally administered DNOC to five
male volunteers and discussed both the resulting blood levels
and toxic effects seen in the volunteers. Each man was given
capsules containing 75 mg of pure DNOC daily for five consec-
utive days, amounting to a dose of 0.92 to 1.27 mg/kg/day.
The men experienced 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 tox-
icity. 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.
DNOC was introduced in 1933 as an alternative to dini-
trophenol for the treatment of obesity (Natl. Inst. Occup.
Safety Health, 1978). Many poisonings, and some deaths, re-
sulting from overdoses were reported, as well as the develop-
ment of cataracts in some patients, months after they had
stopped taking DNOC. 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
metabolic rates, appeared in three persons who had taken as
C-93
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little as 0.35 to 1.5 mg/kg/ day of DNOC for up to nine weeks
(Plotz, 1936). Hunter (1950; reviewed by NIOSH, 1978) noted
that, although, less than one percent of those individuals
treated with DNOC 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 discon-
tinued.
Although DNOC is considered a cumulative poison in hu-
mans, probably as a result of slow metabolism and inefficient
excretion, true chronic or subacute effects (with the pos-
sible exception of cataract formation) have never been re-
ported 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 lev-
els (and by inference, total body burden). It is generally
agreed that the toxic manifestations of DNOC result from its
potent effects on metabolism (Natl. Inst. Occup. Safety
Health, 1978).
Several long-term studies designed to determine dietary
levels of DNOC needed to cause toxic symptoms in experimental
animals have been conducted. Spencer, et al. (1948) main-
tained rats on a diet containing DNOC for six months. Growth
curves, periodic blood counts, analyses of urea-N, organ
weights, and histopathological examinations were carried out
on all animals. No adverse effects on these parameters were
detected among rats fed on diets containing 100 mg DNOC/kg
food. Higher concentrations in the diet produced effects
C-94
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that may be attributed to the action of DNOC as a metabolic
stimulant. Such effects included: weight loss or poor weight
gain, marked emaciation, a hungry, thin, and unkempt appear-
ance, and minor histopathological effects on the liver, kid-
neys, and spleen at the highest dose level (1000 mg DNOC/kg
food). For water, a concentration of approximately one-half
the dietary intake will result in the equivalent dosage on a
body weight basis (assuming a fluid intake two times the dry
matter intake). Thus, the no observable effect level for
DNOC in rats if all DNOC were derived from drinking water
would be 200 mg/1.
In a similar study Ambrose (1942) reported no observable
effect on rats fed diets containing 63 mg DNOC/kg food for
105 days. At DNOC levels of 125 mgAg food, 60 percent of
the animals died. At necropsy and histopathological examina-
tion, the tissues of all rats receiving the drug for 30 days
or more failed to show any characteristic lesions that could
be ascribed to the drug. The calculated no effect level for
DNOC in drinking water is 126 mg/1.
When DNOC was administered in the diet of rats by
Parker, et al. (1951) poisoning was only observed when the
calculated daily intake of the drug greatly exceeded the
single lethal dose. At a level of 200 mg DNOC/kg food, rats
grew normally over an observed period of 18 weeks.
A Federal workplace environmental limit of 0.2 mg/m3
for DNOC has been recommended by NIOSH (1978). The limit was
based on the following considerations: A study from the Rus-
sian literature (Burkatskaya, 1965; reviewed by NIOSH, 1978)
C-95
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documented the lowest airborne DNOC levels found in the
•
literature associated with health effects in humans. Expo-
sure to airborne DNOC at concentrations that averaged 0.9
mg/m3 produced unspecified changes in the cardiovascular
system, the central and autonomic nervous systems, the gas-
trointestinal tract, and the cell pattern of the peripheral
blood of workers involved in manufacturing and applying DNOC.
In agricultural workers exposed to DNOC at an average concen-
tration of 0.7 mg/m3, slight unspecified changes in the
blood and autonomic nervous system were observed.
Another study (Batchelor, et al. 1956) revealed that
agricultural sprayers exposed to an airborne DNOC concentra-
tion of about 0.23 mg/m3 failed to demonstrate adverse ef-
fects of the compound. No symptoms of poisoning were ob-
served and blood DNOC levels were well below those associated
with toxic effects.
In the study by Burkatskaya (1965? reviewed by NIOSH,
1978) the effect of airborne DNOC on cats was examined. Cats
exposed at 0.2 mg/m3 for two or three months had slightly
increased body temperatures and leucocyte counts and de-
creased hemoglobin concentrations, erythrocyte counts, and
catalase and peroxidase activity. 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) concluded "since only slight
effects were seen in workers exposed to DNOC at an average
C-96
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concentration as low as 0.7 mg/m3 for an unspecified dura-
tion, and since short-term exposure at 0.2 mg/m3 had no
lasting effect on cats," NIOSH recommends that the current
Federal workplace environmental limit of 0.2 mg/m3 be
retained.
It is possible to calculate the anticipated daily expo-
sure of a 70 kg human male exposed to 0.2 mg/m3 DNOC for an
eight-hour period. If one assumes the average minute volume
was 28.6 liters of air/minute (average minute volume for a
man doing light work—NIOSH, 1978) the anticipated daily ex-
posure is 39 ug/kg/day.
If one assumes that absorption of DNOC across the res-
piratory tract is identical to gastrointestinal absorption,
and that a 70 kg human male consumes 2.0 liters of water
daily, the following calculation indicates the maximum allow-
able levels of DNOC in drinking water based on the NIOSH air
standard values.
39 ug/kg/day x 70 kg = 2.73 mg/day
2.75 mg/dav _ , .
2 I/day - -1-38 mg/1
Although NIOSH (1978) states "the standard was not de-
signed for the population-at-large, and any extrapolation
beyond the occupational environment is not warranted," devel-
opment of a base-line level for chronic human effects using
the same data used by NIOSH appears to be a reasonable way in
which to approach the development of a water criteria.
C-97
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In summary, daily human exposure to 0.35 mg/kg DNOC 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 this effect cannot be calculated.
Although true "chronic" effects of DNOC have never been
documented, 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 effect 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 (1978) has recommended a
Federal workplace limit of 0.2 DNOC/m^ air. Based on an
estimate of human exposure for an eight-hour work shift, it
was calculated that a drinking water level of 1.4 mg/1 would
result in a similar exposure to the general population.
Synergism and/or Antagonism
No information was found describing synergistic or an-
tagonistic effects associated with DNOC.
Teratogenicity
No information was found regarding the presence or ab-
sence of teratogenic properties of DNOC.
Mutagenicity
Andersen, et al. (1972) reported an evaluation of the
ability of 110 herbicides, including DNOC, to produce point
C-98
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mutations in histidine-dependent 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 DNOC were no greater
than the spontaneous rates.
Nagy, et al. (1975) tested DNOC for its ability to in-
duce back-mutations of her"1" and her" derivatives of E.
coli WP2 try-bacteria. DNOC failed to induce reverse muta-
tions in this system.
The difference in growth inhibitions of wild type Pro-
teus mirabilis and the corresponding repair-deficient strain
has been used by Adler, et al. (1976) as an indication of DNA
damage. Evidence of DNA damage in the presence of DNOC was
reported.
Information on the potential mutagenicity of DNOC for
mammals is not available.
Carcinogenicity
DNOC has not been tested for Carcinogenicity, although
Spencer, et al. (1948) failed to report tumor formation in
rats maintained on diets containing DNOC for six months.
Similarly, no tumors were reported in rats maintained on
diets containing DNOC for 105 days (Ambrose, 1942) or 126
3ays (Parker, et al. 1951).
No further information was found regarding the presence
or absence of carcinogenic properties of DNOC.
C-99
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CRITERION FORMULATION
Existing Guidelines and Standards
No U.S. standards for exposure to the nitrophenols or
dinitrocresols in drinking or ambient water have been set.
The following limits for toxic substances in drinking
water have been set in the U.S.S.R. (Stofen, 1973):
2-nitrophenol .06 mg/1
3-nitrophenol .06 mg/1
4-nitrophenol .02 mg/1
2, 4-dinitrophenol .03 mg/1
Based on organoleptic considerations a limit of 0.5 mg/1
for 2,4,6-trinitrophenol has been set by the U.S.S.R.
(Stofen, 1973).
The maximum air concentration established by the Ameri-
can Conference of Governmental Industrial Hygienists (1971)
is 0.1 mg/m3 for 2,4,6-trinitrophenol and 0.2 mg/m3 for
4,6-dinitro-o-cresol for an eight-hour exposure (TLV).
The Code of Federal Regulations (40 CFR Part 180) estab-
lishes a tolerance of 0.02 mg/kg for residues of 4, 6-di-
nitro-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 samples of urban ambient particulate
matter.
C-100
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The photochemical reaction between benzene vapor and
nitrogen monoxide results in the production of 2-nitrophenol ,
4-nitrophenol, 2 , 4-dinitrophenol, and 2 , 6-dinitrophenol under
laboratory conditions and 4-nitrophenol has been detected in
rainwater in Japan. Available data indicate that the general
public may be exposed to nitrophenols in the atmosphere when
severe photochemical fog conditions develop. Quantitative
estimates of such exposures are not possible at the present
time.
4-nitrophenol has been detected in the urine of 1.7 per-
cent of the general population at levels as high as .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 1.7 ug/1 residue can
be calculated as follows.
Exposure = U° . = 0.20
A similar calculation using the maximum urine residue
level observed (113 ug/1) gives an exposure of 2.26 ug/kg/
day.
These urine levels are not believed to result from di-
rect exposure to 4-nitrophenol, however. A number of widely
used pesticides, including parathion, are readily metabolized
to 4-nitrophenol in the human body and are believed to be the
source of 4-nitrophenol residues in human urine.
C-101
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Current levels of human exposure to the nitrophenols or
dinitrocresols (with the possible exception of 4-nitrophenol)
are either very low, non-existent, 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 ex-
posure to the nitrophenols are industrial workers involved in
the manufacture of compounds for which the nitrophenols are
intermediates. Since picric acid (2,4,6-trichlorophenol) may
find some usage as an explosive, germicide, tanning agent,
fungicide, tissue fixative, and industrial process material,
a higher risk of exposure exists among personnel engaged in
such operations.
Although 4,6-dinitro-o-cresol (DNOC) is no longer manu-
factured in the U.S., a limited quantity is imported and used
as a blossom-thinning agent on fruit trees and as a fungi-
cide, 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 com-
pound .
Basis and Derivation of Criterion
Mononitrophenols no criterion
Dinitrophenols 68.6 ug/1
Trinitrophenols 10 ug/1
Dinitrocresols 12.8 ug/1
Uncertainty factors used for criteria formulation have
been loosely adapted from Drinking Water and Health (Natl.
Acad. Sci., 1977) .
C-L02
V
-------
The organoleptic thresholds for mononitrophenols in
water range from 0.24 to 389 mg/1. These levels extracted
from the Russian literature are detection thresholds. Ac-
ceptability thresholds from the standpoint of human comsump-
tion are not available.
With the exception of a single study abstracted from the
Russian literature, data on chronic mammalian effects of the
mononitrophenols are absent from the literature.
The Russian investigation (Makhinya, 1969) was reported
in abstract form only. Attempts to obtain the full report
proved fruitless. The investigators reported distinct cumu-
lative properties of the mononitrophenol isomers in mammals.
Threshold levels for effects of mononitrophenols on condi-
tioned reflex activity were reported but details of the ex-
periment including animal species, mode of administration,
duration of the experiment, and the exact parameters measured
are not available. Hence, it does not seem prudent to
develop a criteria based on these results.
In the absence of data on chronic mammalian effects no
water criteria for human health can be established for the
mononitrophenol isomers at this time.
Information on the dinitrophenol isomers is limited to
2,4-dinitrophenol. Spencer, et al. (1948), in a six-month
feeding study with rats demonstrated the no-observable-effect
level (NOEL) for 2,4-dinitrophenol to be between 5.4 mg/kg
and 20 mg/kg. Taking the lower of the two figures and assum-
ing a 70 kg man consumes 2 liters of water daily and 18.7 grams
of contaminated fish having a BCF of 2.4, the NOEL for humans
0103
-------
based on the results obtained in rats may be calculated as
follows:
5.4 mgAg x 70 kg = 378 nig
378 mg
2 liters + (2.4 x 0.0187) x 1.0
Based on these calculations no biological effect would
«
» * •
be predic-ted in a man direct ly-cOK^-indirectly exposed to ambient
..'•••>'
water containing "135.3 mg/1 2, 4-DNP.
Experience with the use of 2,4-DNP as an anti-obesity
drug in the 1930 's indicates that adverse effects, including
cscs"Hct forms u i.on . ivisv occur i.n hmusris exposed? ^~o s'3 ^it^"^ —
as 2 rr.g/kg/day. The drug v?as • frequently used in an
uncontrolled manner and the available data do not allow the
calculation of a no-adverse-effect level in hurnans. It is
clear/ however, that ingestion of 2 mg/kg/day 2,4-DKP for a
protracted period may result in adverse effects, including
cataracts, in a small proportion of the population. Assuming
a 70 Jcg nan consumes 2 .liters of water daily and 18.7 grams
of contaminated fish having a BCF of 2.4 and assuming 100
percent gastrointestinal absorption of 2,4-DNP, a 2 mg/kg
dose of 2,4-DNP would result if ambient water contained 68.6
mg/1 of 2,4-DNP.
. _ 140 mg/day _ fi mg/-|
. 2 liters + (2.4 x 0.0187) x 1.0 D°
C-104
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These data taken together with the demonstrated bacte-
rial mutagenicity of 2,4-DNP (Demerec, et al. 1951) and the
suspected ability of the compound to induce chromosomal
breaks in mammals (Mitra and Manna, 1971) suggest that an un-
certainty factor of 1,000 should be used in criteria formu-
lation.
The suggested water criterion for 2,4-DNP is, there-
fore :
68.6 mg/1 _ ,fi , ..
1,000 ~ 68'6 ug/1
The available data are insufficient to enable calcula-
tion of water criterion levels for the remaining dinitro-
phenol isomers. For the present, it seems reasonable to as-
sume that the 2,4-dinitrophenol criterion would be appro-
priate for the other isomers.
Chronic mammalian toxicology data for the trinitro-
phenols 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. Although it is not possible to precisely
estimate either the 2,4,6-trinitrophenol water level or dura-
tion of exposure required for the development of hematuria
2,4, 6-trinitrophenol levels of 10 mg/1 and 20 mg/1 were de-
tected in drinking water aboard two ships at the time of the
outbreak.
C-105
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Two studies (Demerec/ et al. 1951; Yoshikawa, et al.
1976) have demonstrated mutagenic activity of 2,4, 6-trinitro-
phenol in bacterial systems. Auerbach and Robson (1947)
failed to detect mutagenic activity in Drosophila, however.
Based on the presumed development of hematuria in humans
at drinking water levels of 10 mg/1 and the evidence indicat-
ing nutagenic activity in bacteria, an uncertainty factor of
1,000 is suggested for formulation of the 2 , 4, 6-trinitro-
phenol water criteria:
Since available data are insufficient to enable calcula
tion of water criterion levels for the remaining trinitro-
phenol isomers, it seems reasonable to assume, for the pres-
ent, that the 2 , 4, 6-trinitrophenol criterion is appropriate
for the other isomers.
Although 4,6-dinitro-o-cresol (DNOC) is considered a
cumulative poison in humans, probably as a result of slow
metabolism and inefficient excretion, true chronic or sub-
acute 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-
terin animal studies is of questionable value.
The no-observable-effect level (NOEL) for DNOC respira-
tory exposure in humans has been reported as 0.2 mg/m^ air
(Natl. Inst. Occup. Safety Health, 1978). NIOSH (1978) has,
in fact, recommended that the current Federal workplace
environmental limit of 0.2 mg/m3 be retained, based on the
available data.
C-106
-------
It is possible to calculate the anticipated daily expo-
sure of a 70 kg human male exposed to 0.2 mg/m^ for an
eight-hour period. If one assumes the average minute volume
is 28.6 liters of air/minute (average minute volume for a man
doing light work — NIOSH, 1978) the anticipated daily exposure
is 39 ugAg/day. Since the NOEL's calculated from long-term
experimental animal studies are considerably higher than this
value, it will be used as a basis for the suggested water
criterion.
If one assumes that absorption of DNOC across the res-
piratory tract is identical to gastrointestinal absorption,
and that a 70 kg human male consumes 2 liters of water daily
and 18.7 g of contaminated fish having a BCF of 7.5, the fol-
lowing calculations indicates the maximum allowable levels of
DNOC in drinking water based on the NIOSH air standard
values:
39 ug/kg/day x 70 kg = 2.73 mg/day
_ 2.73 mq/dav __
(2/1 + (7.5 x 0.0187) x 1.0
,
mg/J-
In view of the lack of data indicating chronic effects
and the existence of a very recent Federal guideline for
human exposure, an uncertainty factor of 100 is chosen for
the protection of the general public. The suggested crite-
rion for 4 , 6-dinitro-o-cresol (and in the absence of adequate
data, the other dinitrocresol isomers) is
1.28 mg/1 n _ _
100 = 12'8
C-107
-------
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