vvERA,
              Jr-.tM States
              Environmental
              Agencv
              Regulations ana 5'anaarcs
              Catena ano Standards Divi
              Wasmngton 3C 20460
"980
Ambient
Water Quality
Criteria  for
Nitrophenols

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      AMBIENT WATER QUALITY CRITERIA FOR

               NITROPHENOLS
                 Prepared 8y
    U.S. ENVIRONMENTAL PROTECTION AGENCY

  Office of Water Regulations and Standards
       Criteria and Standards Division
              Washington, D.C.

    Office of Research and Development
Environmental Criteria and Assessment Office
              Cincinnati, Ohio

        Carcinogen Assessment Group
             Washington, O.C.

    Environmental Research Laboratories
             Corvalis, Oregon
             Duluth, Minnesota
           Gulf Breeze, Florida
        Narragansett, Rhode Is'and

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                              DISCLAIMER
      This  report  has  been reviewed by  the  Environmental  Criteria and
Assessment Office,  U.S.  Environmental  Protection  Agency,  and approved
for publication.   Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
                          AVAILABILITY  NOTICE
      This  document  is available  to  the public  through  the National
Technical Information Service, (NTIS), Springfield, Virginia  22161.
                                   11

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                               FOREWORD

    Section 304  (a)(l)  of the Clean Water Act  of  1977 (P.L. 95-217),
requires the Administrator  of the Environmental Protection  Agency to
publish  criteria for water  quality accurately reflecting  the latest
scientific knowledge on the  kind  and extent of all identifiable effects
on  health  and  welfare which  may  be  expected from  the presence of
pollutants in  any body of water, including ground water.  Proposed water
quality criteria  for the  65  toxic pollutants  listed under section 307
(a)(l) of  the  Clean Water  Act were developed  and  a notice  of  their
availability was published for public comment on March 15, 1979 (44 FR
15926), July 25,  1979 (44  FR  43660), and October 1, 1979 (44 FR 56628).
This document  is a revision  of  those  proposed criteria  based upon a
consideration of  comments received from  other  Federal  Agencies,  State
agencies,  special  interest  groups,  and  individual  scientists.    The
criteria contained in this document replace any previously published EPA
criteria  for  the  65  pollutants.    This criterion  document  is  also
published in satisifaction of paragraph 11 of the Settlement Agreement
in  Natural  Resources  Defense  Counci 1, et.  al.  ys. Train,  8 ERC 2120
(D.O.C. 1976), modified, 12 ERC 1833 (D.O.C.  1979).

    The term "water  quality criteria"  is used  in  two  sections of the
Clean Water Act, section 304  (a)(l) and section 303 (c)(2).  The term has
a different program impact  in  each  section.   In section 304, the term
represents a non-regulatory,  scientific   assessment of  ecological  ef-
fects. The criteria presented  in  this  publication  are  such  scientific
assessments.   Such water  quality criteria  associated with specific
stream uses when  adopted as  State  water quality  standards under section
303 become  enforceable maximum  acceptable  levels  of  a  pollutant in
ambient waters.  The water quality criteria adopted in the State water
quality standards could have the same numerical limits as the criteria
developed under section 304.  However, in many situations States may want
to adjust water quality criteria developed under section 304 to reflect
local   environmental  conditions   and  human exposure  patterns  before
incorporation  into  water  quality  standards.    It  is  not until  their
adoption as part  of the State water quality standards that the criteria
become regulatory.

    Guidelines to  assist  the  States  in  the modification of criteria
presented  in  this  document,  in  the  development  of  water  quality
standards, and  in other water-related programs of this Agency, are being
developed by EPA.
                                    STEVEN SCHATZOW
                                    Deputy Assistant Administrator
                                    Office of Water Regulations and Standards
                                   111

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                                 ACKNOWLEDGEMENTS
Aquatic Life Toxicology:

    William A.  Brungs, ERL-Narragansett
    U.S.  Environmental Protection Agency
John H.  Gentile,  ERL-Narragansett
U.S. Environmental  Protection  Agency
Mammalian Toxicology and Human Health Effects:
    Van Kozak (author)
    University of Wisconsin

    Steven 0. Lutkenhoff (doc. mgr.)
    ECAO-Cin
    U.S. Environmental Protection Agency

    Donna Sivulka (doc. mgr.) ECAO-Cin
    U.S. Environmental Protection Agency

    Kirby I. Campbell, HERL
    U.S. Environmental Protection Agency

    Ted Ericksen, HERL
    U.S. Environmental Protection Agency

    Sherwin Kevy
    Children's Hospital Medical Center

    V.M.S. Ramanujam
    University of Texas Medical Branch

    Alan B. Rubin, CSD
    U.S. Environmental Protection Agency
John Autian
University of Tennessee

C. Stuart Baxter
University of Cincinnati
Karen Blackburn, HERL
U.S. Environmental Protection Agency

Patrick Durkin
Syracuse Research Corporation

Karl Gabriel
Medical College of Pennsylvania

Jeanne Manson
University of Cincinnati

Joseph P. Santodonato
Syracuse Research Corporation
Technical Support Services Staff:  D.J. Reisman, M.A. Garlough, B.L. Zwayer
P.A. Daunt, K.S. Edwards, T.A. Scandura, A.T. Pressley, C.A. Cooper,
M.M. Denessen.

Clerical Staff:  C.A. Haynes, S.J. Faehr, L.A. Wade, D. Jones, B.J. Bordicks,
B.J. Quesnell, C. Russom, B. Gardiner.
                                     1v

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                                TABLE OF CONTENTS

                                                                    Page

 Criteria  Summary

 Introduction                                                         A-l

 Aquatic Life Toxicity                                                B-l
      Introduction                                                    B-l
      Effects                                                         B-2
          Acute Toxicity                                             B-2
          Chronic  Toxicity                                           3-3
          Plant Effects                                              3-3
          Residues                                                   B-6
          Miscellaneous                                              B-6
          Summary                                                    B-3
      Criteria                                                        B-8
      References                                                      B-19

                                Mononitrophenols                     C-l

 Mamalian  Toxicology and Human Health  Effects                         C-l
      Introduction                                                    C-l
      Exposure                                                        C-2
          Ingestion from Water                                       C-2
          Ingestion from Food                                        C-6
          Inhalation                                                 C-12
          Dermal                                                     C-13
      Pharmacokinetics                                                C-13
          Absorption and Distribution                                C-13
          Metabolism                                                 C-14
          Excretion                                                  C-16
      Effects                                                         C-19
          Acute, Subacute, and Chronic  Toxicity                      C-19
          Synergism and/or Antagonism                                C-24
          Teratogenicity                                             C-25
          Mutagenicity                                               C-25
          Carcinogenicity                                            C-26

                                 Dinitrophenols                      C-27

Mammalian Toxicology and Human Health Effects                        C-27
      Introduction                                                    C-27
      Exposure                                                        C-30
          Ingestion from Water                                       C-30
          Ingestion from Food                                        C-31
          Inhalation                                                 C-32
          Dermal                                                     C-32
      Pharmacokinetics                                                C-33
          Absorption                                                 C-33
          Distribution                                               C-33
          Metabolism                                                 C-34
          Excretion                                                  C-35

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     -I'-ects                                                         C-26
          Ac-Jte, Suoacjte, and  Chronic  Toxic'ty                      ;-36
          Synergism  and/or Antagcn'S~                                ~.i;
          Teratogenicity                                             2-~3
          Mutagenicity                                               C-4g
          Carcinogenicity                                            C-51

                                 Trinitropnenols                     C-52

Mammalian Toxicology and Human  Health Effects                        C-52
     Introduction                                                    C-52
     Exposure                                                        C-52
          Ingestion  from Water                                       C-52
          Ingestion  from Food                                        C-56
          Inhalation                                                 C-56
          Dermal                                                     C-56
     Pharmacokinetics                                                C-57
          Absorption                                                 C-57
          Distribution                                               C-57
          Metabolism                                                 C-58
          Excretion                                                  C-53
     Effects                                                         C-53
          Acute, Subacute, and  Chronic  Toxicity                      C-53
          Synergism  and/or Antagonism                                C-62
          Teratogenicity                                             C-62
          Mutagenicity                                               C-62
          Carcinogenicity                                            C-63

                                 Oinitrocresols                      C-64

Mammalian Toxicology and Human  Health Effects                        C-54
     Introduction                                                    C-6£
     Exposure                                                        C-67
          Ingestion from Water                                       C-57
          Ingestion from Food                                        C-67
          Inhalation                                                 C-68
          Dermal                                                     C-63
     Pharmacokinetics                                                C-68
          Absorption                                                 C-68
          Distribution                                               C-69
          Metabolism                                                 C-73
          Excretion                                                  C-75
     Effects                                                         C-77
          Acute, Subacute, and  Chronic  Toxicity                      C-77
          Synergism and/or Antagonism                                C-86
          Teratogenicity                                             C-86
          Mutagenicity                                               C-36
          Carcinogenicity                                            C-37

                              Criteria  Formulation                   C-88

     Existing Guidelines and Standards                               C-8S
     Current Levels of Exposure                                      C-3S
     Special Groups at Risk                                          C-39
     Basis and Derivation of Criterion                               C-90

                                   References                        C-95

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                               CRITERIA :oc'jy-N
                                 Aquatic Life
    The  available  data  for  m'troonenols   indicate  that  acute  toxicity  to
 freshwater  aouatic  life  occurs  at  concentrations  ds   low  as  230  ug/l  and
 would  occur at  lower  concentrations among  species  that  are more sensitive
 than  those  tested.   No  data are available  concerning the chronic toxicity of
 nitroohenols  to  sensitive freshwate- aoautic  life  but   toxicity to  one soe-
 cies of alqae occurs at concentrations as low as 150 ug/1.
    The  available  data  for  nitroohenols   indicate  that  acute  toxicity  to
 saltwater  aouatic  Tife  occurs  at  concentrations  as low as 4,350  ug/1  and
 would  occur at  lower  concentrations among  soecies  that  are more sensitive
 than those  tested.   No  data  are available  concerning the chronic toxicity of
 rvtroohenols to sensitive saltwater aouatic life.

                                 Human Health
    Due to  the  insufficiency in  the available  data  for mono-  and  trintro-
 ohenols,  satisfactory  criteria  cannot  be  derived  at  this  time,  using  the
present guidelines.
    For the protection of human health  from the toxic properties of dinitro-
ohenols  and  2,4-dinitro-o-cresol  ingested  through   water and  contaminated
aiuatic organisms,  the  ambient water criteria  are  determined to  be  70 ug/1
and 13.4 ug/1, respectively.
    For the protection of human health  from the toxic properties of dinitro-
ohenols and  2,4-dinitro-o-cresol  ingested  throuch  contaminated aouatic  or-
ganisms alone, the  ambient  water  criteria  are  determined to  be  H.3 mg.M  anc1
 755 uo/1,  respectively.
                                     vi i

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     wononitrophenol  has  three isomeric forms, distinguished by  the  position
 of  t^e  m'tro  grouo  on  the phenolic ring.   Three  isomeric forms are possible,
 namely  2-nitrophenol,  3-nitrophenol,   and  4-nitrophenol.   The compounds  are
 also  commonly  referred  to   as  o-nitrophenol,  m-nitrophenol,  and  p-nitro-
 phenol, respectively.
     Commercial  synthesis  of  2-nitrophenol and 4-nitrophenol  is  accomplished
 through  the hydrolysis  of the  appropriate  chloronitrobenzene  isomers  with
 aoueous  sodium  hydroxide  at  elevated temperatures  (Howard,  et  al.  1976).
 Production  of  3-nitrophenol   is  achieved  through  the  diazotization  and  hy-
 drolysis  of m-nitroani1ine  (Matsuguma,  1967).  The mononitrophenol  isomers
 are  used  in  the  United States primarily as  intermediates  for  the production
 of  dyes,  pigments,  Pharmaceuticals, rubber  chemicals,  lumber  preservatives,
 photograohic  chemicals,  and  pesticidal  and  fungicidal  agents  (U.S.  Inter-
 national  Trade Commission,  1976).   As  a result  of  this  use  pattern, the ma-
 jor  source for  environmental  release  of  mononitrophenols  is  likely  to  be
 from  production  plants and  chemical   firms  where  the  compounds   are used  as
 intermediates.   The mononitrophenols may  also be  inadvertently  produced  via
microbial  or  photodegradation  of  pesticides  which contain  mononitrophenol
moieties.  Approximately  10  to 15  million  pounds  of 2-nitrophenol are  pro-
duced annually  (Howard,  et al. 1976)   for uses including  synthesis of  o-ami-
nophenol,  o-nitroanisole,  and  other dye stuffs  (Matsuguma,  1967;  Howard,  et
al.  1976).  Although production figures for  3-nitrophenol  are  not available,
 Hoecker,  et  al.   (1977)  estimate  that  production  is less  than   one million
 pounds annually.  3-Nitrophenol is  used in the manufacture of  dye intermedi-
                                     A-l

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 ates  SUCH as  anisidine  and  Ti-aminophenol  (Kouris ana  Northcctt,  1963;  >v(at-
 suguma,  1967).  4-Nitrophenol  is probably the most  important  of  the mononi -
 trophenols  in  terms  of quantities used and potential environmental contamin-
 ation.   Demand for 4-nitrophenol was  35,000,000 pounds in  1976  and produc-
 tion  is  projected  to  increase  to 41,000,000 pounds by 1980  (Chemical Market-
 ing  Reporter,   1976).   Most  of  the 4-nitrophenol  produced  (87  percent)  is
 used  in  the  manufacture  of ethyl  and methyl  parathions.  Other uses (13 per-
 cent)  include the  manufacture  of  dye-stuffs  and  n-acetyl-p-aminophenol
 (APAP)  and  leather  treatments.   A  possible  source  of  human  exposure  to
 4-nitrophenol  is  as  a  result of  microbial  or photodegradation of  the  para-
 thions.   _In_ vivo  production of  4-nitrophenol  following absorption  of  para-
 thion or  other pesticides by  humans is another  possible source of human ex-
 posure.
    Physical and  chemical  properties of the mononitrophenols  are summarized
 in Table  1.
 Qini trpphenols
    Six  isomeric  forms  of  dinitrophenol  are  possible, distinguised by the
 position  of  the  nitro-groups  on the  phenolic  ring.   Of  the six  possible
 dinitrophenol  isomers,  2,4-dinitrophenol  is  by  far  the most important.   The
most  recent  production  figure  for 2,4-dinitrophenol is 863,000  lb.  reported
by the U.S.  International  Trade  Commission  (1968).   Approximate  consumption
per year  is estimated at 1,000,000  Ibs.  (Howard, et  al. 1976).  2,4-Oinitro-
phenol is used primarily as  a  chemical  intermediate for the  production  of
 sulfur dyes, azo  dyes,  photochemicals,  pest  control agents,  wood preserva-
tives,  and  explosives   (Matsuguma,   1967;  Perkins,  1919;  Springer,  et  al.
 1977a,b).
                                      A-2

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                                   TABLE 1



                       Properties of Mononitrophenols*

connula
Molecular Weight
Melting Doint (*C)
Boiling Point (°C)
Density
Water Solubility
fg/i)
Vapor Pressure
Ka
2-Nitrophenol
C6H5N03
139.11
44 -45
214-216
1.485
0.32 at 38*C
1.08 at 100'C
1 mm Hg at 49.3°C
7.5x10-8
3 -Mi trophenol
C6H5N03
139.11
97
279
1.485
1.35 at 25°C
13.3 at 90*C

5.3x10-9
4-Ni tropheno
CsH5N03
139.11
113-114
279
1.479
0.804 at 15"
1.6 at 25'C

7x10-8
1





C


*Sources: Windholz, 1976; Weast, 1975; Matsuguma, 1967.
                                      A-3

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            ion  *igures  and usage  data  for tne  remaining  f;ve  dinit
         are not available.  It  is  reasonable  to assume that  production  and
usage of these  compounds are extremely limited in the United States.
    Commercial  synthesis of 2,4-dinitrnnhenol  is  accomplished  by  the  hydrol-
ysis  of 2,4-
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                                    TABLE 2



                     Properties of  Dinitrophenol  Isomers*
Isomer
2,3-Dinitrophenol
2,4 -Cini trophenol
2,5-Dinitrophenol
2,6-Oinitrophenol
3, 4 -Oini trophenol
3,5-Dinitrophenol
m.p.
( C)
144
114-115
(sublimes)
104
63.5
134
122-123
K
(at 25'C)
1.3 x 10-5
1.0 x 10-4
7 x 10-5
2.7 x 10-4
4.3 x 10~5
2.1 x 10-4
Water
Solubility
(9/1)
2.2
0.79
0.68
0.42
2.3
1.6
Density
1.681
1.683


1.672
1.702
*Source: Harvey, 1959; Wirdholz, 1976; Ueast, 1975.
                                      A-5

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                                    "ABLE  3

                        Properties of Trinitrophenols*
    Molecular Weight
229.11
2,3.5-Trim'trophenol

    Molecular Weight
    Melting Point
229.11
119-120°C
2,3,6-Trinitrophenol

    Molecular Weight
    Melting Point
    Water Solubility
      Room Temperature
      Hot Water
229.11
119'C

Slightly Soluble
Very Soluble
2,4,5-Trini trpphenol

    Molecular Weight
    Melting Point
    Water Solubility
      Room Temperature
      Hot Water
229.11
96'C

Slightly Soluble
Soluble
2,4,5-Trinitrophenol

    Molecular Weight
    Melting Point
    Boiling Point

    Vapor Pressure
    Density
    Water Solubility
      Room Temperature
      100'C
229.11
122-123*C
Sublimates: Explodes at
  300'C
1 mm Hg at 195°C
1.763 g/cm3

1.28 g/1
6.7 g/1
*Source: Windholz, 1976; Weast, 1975; Matsuguma, 1967.
                                      A-6

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     According  to  Vatsuguma (1967) picric acid has  found  usage  as:  a  dye in-
 termediate,  explosive,  analytical  reagent,  germicide,   fungicide,  staining
 agent  and  tissue  fixative, tanning agent, photochemical,  pharmaceutical, and
 a  orocess  material for  the  oxidation  and eching  of  iron,  steel  and  copper
 surfaces.   The  extent to  which  picric  acid finds usage  in any  of  these ap-
 plications at the  present  time is unknown.
 Dinitrocresols
     Dinitro-ortho  cresol  is  a yellow  crystalline solid  derived  from  o-cre-
 sol.   There  are six possible isomers but the  4,6-dinitro-o-cresol  isomer  is
 the  only one of any commercial  importance.   In  fact,  a  comprehensive  search
 of  the literature  failed to  reveal  information  on  any of  the other  five
 dim'trocresol isomers.
     4,6-Oinitro-o-cresol  (hereafter  referred  to   as DNOC) is  produced  either
 by  sulfonation  of  o-cresol  followed  by  treatment with  nitric  acid  or  by
 treatment of o-cresol  in  glacial  acetic  acid  with nitric  acid at low  temper-
 ature.  Some important chemical  and physical  properties of  DNOC  are shown  in
 Table 4.
     The U.S.  Environmental  Protection  Agency (U.S.  EPA)  has  no  record  of
 DNOC being currently  manufactured in  the United  States  for use  as an  agri-
 cultural chemical.   Imports  of  DNOC have  also   decreased  in recent  years;
 from 217,899 Ibs.  in 1972 to 146,621 Ibs. in 1973  and  then  to 30,442  Ibs.  in
 1976  (National  Institute for Occupational Safety  and  Health  (NIOSH),  1978).
 Since DNOC  is not manufactured in  the  U.S., pesticide  formulators and  spray-
ers are the major  groups with potential  occupational exposure  to  DNOC.
     DNOC is used primarily as a  blossom-thinning  agent on fruit  trees  and  as
a  fungicide,  insecticide, and  miticide  on  fruit  trees  during   the  dormant
                                     A-7

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                                TABLE  4
                  Properties  of  4,6-Dinitro-o-cresol*
      Molecular Weight                   198.13
      Appearance                         Yellow Solid
      Melting Point                       85.8'C
      Vapor Pressure                       0.000052 mm Hg at 20*C
      Water Solubility                   100 mg/1 at 20*C
      pKa                                  4.46

*Source: Windholz, 1976; Weast, 1975; Matsuguma, 1967.

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 season.   NIOSH  (1973)  estimates  that' 3,000  workers  in the  U.S.  are  poten-
 tially  exposed to ONOC.   In  view of the  small  amount of  DNOC used  in  the
 U.S., exposure of  the general public is expected to be minimal.
     In  general,  few  data are  available  regarding  the breakdown of  nitro-
 phenols  by natural communities  of microorganisms.  A  number  of  researchers
 have  isolated  microorganisms  capable of using nitrophenols as  a  sole  source
 of  carbon  in pure culture  (Simpson  and Evans,  1953;  Raymond  and Alexander,
 1971; Chambers,  et al.  1963;  Guillaume, et  al.  1963).   However, the  signifi-
 cance of such  studies as  related  to  the stability of nitrophenols in the en-
 vironment  is not known.
    Several  investigators  have  shown that  individual  species  of  aerobic  and
 anaerobic  bacteria, including Azotabacter chroococcum  and  Clostriduim  butyr-
 ium,  and  the fungus  Fusarium,  are capable  of reducing 2,4-dinitrophenol  in
 culture  (Radler,  1955;  Lehmber,  1956;  Madhosingh, 1961).   However,  the  pre-
 cise  pathway for metabolic  degradation  is  not  known.  Jensen and  Lautrup-
 Larson  (1967)  found that  Arthrobacter simplex,  Pseudomonas,  and Arthrobacter
were  able  to metabolize 2,4-dinitrophenol  and  2,4,6-trinitrophenol,  forming
nitrite.
    The actual degradation pathway of dinitro-o-cresol  has  been investigated
by Tewfik  and  Evans  (1966) in pure  cultures  of microorganisms.   It was  re-
ported that  in Pseudomonas  sp.  degradation  proceeded  by way  of formation  of
an aminocresol.   In Arthrobacter  simplex,  a hydroxylated  catechol is  formed
prior to ring cleavage.
    The significance of such studies as  related to the stability of  nitro-
phenols in  the environment  is not known.   Certain  investigators  have  postu-
 lated that  ambient  nitrophenol  concentrations may be  too  low  to  induce  the
appropriate microbial enzymes necessary to  facilitate  population  growth  and
metabolism of the compounds (U.S.  EPA,  1976).
                                     A-9

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         -nat i 3n  regadding the  TObilitv an
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                                   REFERENCES

 Chambers,  C.W.,  et al.  1963.   Degradation of  aromatic compounds by phenol-
 adapted  bacteria.   Jour. Water  Pollut. Control Fed.  35: 1517.

 Chemical Marketing  Reporter.  1976.  Chemical profile:  p-nitro-phenol.

 Gomaa,  H.W.  and  S.D. Faust.   1972.   Chemical  hydrolysis  and  oxidation  of
 parathion  and paraoxon in  aquatic  environments.  Adv. Chem. Ser.  Vol.  III.
 Iss.  Fate  Org. Pestic. in the Aquatic Environ.

 Guillaume,  J,  et  al.   1963.  Oxidation of  p-nitrophenol  by certain Mycobac-
 teria.   Comp. Rend.   256: 1634.

 Harvey,  O.G.   1959.  On the  metabolism  of some  aromatic  nitro  compounds  by
 different  species of  animal.  Part  III.   The  toxicity of the dinitrophenols,
 with  a note on the  effects  of high environmental temperatures.   Jour.  Pharm.
 Phamiacol.  11: 462.

 Hoecker,  J.E., et al.   1977.   Information profiles on potential  occupational
 hazards.   Nat. Inst. Occup. Safety Health, Cincinnati, Ohio.

 Howard,  H., et al.  1976.   Investigation  of selected potential  environmental
 contamination:  Nitroaromatics.   Off.  Tox.  Subst.    U.S.   Environ.   Prot.
Agency, Washington, O.C.
                                     A-ll

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 Jensen,  'J.L.  and  G.  Lautrup-larson.   1967.   Microorganisms  that  decorroose
 nitro-aromatic  compounds,  with  special reference  to  dinitro-o-creso 1.   Acta
 Agric. Scand.   17: 115.

 KouHs,  C.S.  and  J.  Northcott.   1963.   Aniline  and  its  derivatives.   _In_:
 Kirk-Othmer Encyclopedia of Chemical Technology.  2nd ed.  2: 411.

 lehmber, C.   1956.  Untersuchungen  uber  die Winbung von  Ascorbunsaure,  Stof-
 fwechselgifren  and Anderen  Faktoren  auf  den  Staffwechsel  von  Clostridium
 butyrium.  Beif. Arch. Mikrobiol.  24: 323.

 Madhosinggh,  C.  1961.  The  metabolic detoxification of  2,4,-dinitrophenol
 by Fusarium oxysponim.  Can. Jour. Microbiol.  7: 553.

Matsuguma,  H.J.  1967.  Nitrophenols.   lr±:  Kirk-Othmer Encyclopedia  of  Chem-
 ical Technology.  2nd. ed.  13:  888.

Mosinska, K.  and A. Kotarski.   1972.  Determination of 2-isopropyl-4,6-dini-
trophenol and 2,4-ONP  in  herbicides and in  technical  2-isopropyl-4,6-ONP  by
TCL.  Chemia Analltyezma.   17: 327.

Perkins, R.G.   1919.  A study of  the munitions intoxications in France. Pub.
Health Rep.  34: 2335.

Padler,  F.   1955.   Untersuchunger uber den  verlaug der  stoffwech Selvorgan-
gebei Azotobacter chroococcum.  Beig. Arch. Microbiol.  22: 335.
                                     A-12

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 Raymond,  C.  ,v.  and  vi.  Alexancer.   1971.  Vicrobial ~etabc'"S.T  anc  :o~e:a-
 bolism of nitrophenols.  Pestic. Siochem. Physio!.  1: 123.

 Saltzman, S.  and  S.  variv.  1975.   Infrared  study  of the  sorbtion  of pneno.
 and p-nitrophenol by  montmori1lonite.  Soil Sci. Soc. Am. ?roc.  39: 4/4.

 Simpson, J.S.  and  W.C. Evans.   1953.  The metabolism of  nitropheols  by cer-
 tain bacteria.  Siochem. Jour.  55: 24.

 Springer,  E.L.,  et  al.   1977a.   Chemical  treatment of  chips  for  outdoor
 storage.  Evaluation  of sodium n-methyldithiocarbomate and  sodium  2,4-dini-
 trophenol treatment.  Tapi.  60: 88.

 Springer, E.L., et  al.   1977b.  Evaluation of chemical  treatments  to  prevent
deterioration of wood chips during storage.  Tapi.  60:  93.

Tewfik,  M.S.  and  W.C. Evans.   1966.   The  metabolism  of 3,5^initro-o-crescl
 (DNOC) by soil microorganisms.   Biochem.  Jour.  99:  31.

U.S. EPA.  1976.   Investigation of  selected  potential environmental contami-
nants:  nitroaromatics.  Final Sep. Off.  Tox. Subst.  Washington,  D.C.

U.S. International  Trade Commission.   1967-73.   Synthetic  organic  chemicals:
U.S. production and sales.   Washington, D.C.

U.S. International  Trade  Commission.  1976.    Imports of  benzenoid  chemica's
and products, 1974.  Publ.  No.  762.   Washington, O.C.
                                     A-13

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Weast,  3.C.,  ed.   1975.   Handbook  of Chemistry  and  Physics.  57th ed.  C3C
Press.
        ,  i*., ed.   1976.   The Merck Index.  9th  ed.   Merck  and  Co.,  Rahway,
Mew Jersey.
    v, S.,  et  al.   1966.   Infrared  study of the absorotion  of  benzoic  add
and nitrobenzene in montomori llonite.  Isr. Jour. Chem.  4: 201.
                                     A-1

-------
 Aquatic  Life  Toxicology*
                                  INTRODUCTION
      Although fish and  invertebrate acute toxicity  data  and  plant toxicity
 data  are  available for  the groups of organic compounds which contain various
 numbers  of nitro  groups  substituted into  the  aromatic ring of  a  phenol  or
 cresol,  collectively referred  to  as  nitrophenols,  there  are  only limited
 data  available  for any individual  nitrophenol.   There  are  no  data available
 dealing with  chronic  effects  of any nitrophenol on freshwater aauatic organ-
 isms,  and  no  suitable substitute chronic value  can be determined from avail-
 able  toxicity information.  The  limited data  available preclude  deriving a
 criterion  for any  of the individual nitrophenol  compounds.   The derivation
 of  a  single criterion which would  protect  freshwater aauatic  organisms from
 all nitrophenols  is also  impractical because of  the  wide  difference in tox-
 icity of individual nitrophenols.
     The saltwater  data base  available  for the  various  nitrophenols is also
 limited.   For 4-nitrophenol  there  are  acute  test  results  for one  fish  and
 one invertebrate  species,  and one fish  chronic  test.   In addition, there are
 two invertebrate  lethal   threshold  values.   The  data base  for  2,4-dinitro-
 phenol consists of  acute  tests,  one algal  test, and miscellaneous effects on
one invertebrate  species.   Information  on 2,4,6-trinitrophenol  is  even more
 limited, consisting of acute results  for one  fish,  one algal,  and  one  in-
vertebrate species.
*The reader  is referred  to  the  Guidelines  for Deriving Water  Quality Cri-
teria for the Protection of Aquatic Life and  Its Uses  in  order to better un-
derstand the  following  discussion and recommendation.  The  following tables
contain the appropriate  data  that were found  in  the literature, and  at the
bottom of each  table  are calculations for deriving  various  measures  of tox-
icity as described in the Guidelines.
                                      3-1

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                                    ~~TECT$
 Acute  Toxicity
     The  data base  for freshwater  invertebrate species  (Table  1)  contains
 seven  data  points for four nitrophenol  compounds  with  two invertebrate spe-
 cies.   An  unspecified dinitromethylphenol  (reported by  Sanders and  Cope,
 1968  as dinitrocresol)  is the  most toxic  compound with  an  ICcg  value  of
 320  ug/l for  a  stonefly,  Pteronarcys californica.   This  compound is  followed
 in  order  of decreasing toxicity by 2,4-dinitro-6-methylpheno1,  2,4-dinitro-
 phenol,  4_nitrophenol, and 2,4,6-trinitrophenol.   The  2,4-dinitro-6-methy1-
 phenol  LCgQ for  Daphnia  magna  is  3,120 ug/l  (U.S.  EPA,  1978).   It appears
 that stoneflies  may  be more sensitive  to  dinitromethylphenol  than  are daph-
 nids,  although  this  is impossible  to verify since  the  compound tested with
 stoneflies  is  inadeauately identified  and  may  be a  slightly  different com-
 pound  than  that tested with  daphnids.   For 2,4-dinitrophenol,  the  two LC,-n
 values  for  daphnids  are Quite  close and are 4,710  ug/1  (Kopperman,  et  al.
 1974)  and  4,090  ug/l  (U.S.  EPA,  1978).   The  toxicity of  4-nitrophenol  to
 daphnids  shows  greater variation  between  investigators  with reported LC50
 values  of  8,396  ug/1  (Kopperman,  et  al.  1974) and  21,900 ug/l  (U.S. EPA,
 1978).  The least  toxic nitrophenol  to daphnids is 2,4,6-trinitrophenol with
 a reported LC5Q value of 84,700  ug/l  (U.S. EPA, 1978).
     The  freshwater  fish  acute  toxicity  data  base (Table  1)   consists  of
 seven  1C    values  for four  nitrophenols  and   two  fish  species.   Although
 differences in test methods make comparisons difficult,  it appears tluegills
 are  more  sensitive  than   fathead  minnows  with all  three  nitrophenols  for
which  data  are  available  for   both  species.    Comparisions  of   LCgQ   values
 for fishes  indicate that 2,4-dinitro-6-methylphenol  is  the most  toxic   nitro-
 phenol  with  LC5Q  va1ues   of  230   ug/1  (ILS<  EPA^  1978)  and  2,030 ug/l
                                      8-2

-------
 (Phipps,  et al.  Manuscript)  for the bluegill and fathead minnow, respective-
 ly;  2,4-dinitro-6-methylphenol  is  followed  in  order  of  decreasing toxicity
 by  2,4-dinitrophenol,  4-nitrophenol,  and  2,4,6-trinitrophenol.   The largest
 variation   in  species  sensitivity occurred  with  2,4-dinitrophenol  where
 LC5Q  values are  620 ug/1  for bluegills  (U.S.  EPA,  1978)  and  16,700 ug/l
 for  fathead minnows (Phipps,  et  al. Manuscript).  The  LCcn  values reported
 for  4-nitrophenol   for  bluegills  and  fathead minnows  are  8,280  ug/1  (U.S.
 EPA,  1978)  and 60,500  ug/1  (Phipps, et al.  Manuscript),  respectively.  The
 high  2,4,6-trinitrophenol  LC5Q of 167,000  ug/1  for  bluegills  (U.S.  EPA,
 1978)  indicates  the toxicity of nitrophenols does not increase directly with
 increasing  nitro-group substitution.
     The  order of  toxicity of  the four  nitrophenol  compounds  tested with
 both freshwater  fish and  invertebrate  species  is the same,  and  it appears
 from the  limited  data  available that  there  are  no large sensitivity differ-
 ences among these  species  with  any  of  these nitrophenols.  Invertebrate tox-
 icity values for 4-nitrophenol  and  2,4-dinitrophenol  fall between those cal-
 culated for bluegills  and fathead  minnows.   Daphnia magna  appeared slightly
 more  sensitive  to 2,4,6-trinitrophenol  than were  bluegills,   and  slightly
 less sensitive to 2,4-dinitro-6-methylphenol than were fathead minnows.
     As seen in Table  1 for  the saltwater acute data, 4-nitrophenol resulted
 in a  96-hour   1C    of  7,170 ug/1 with mysid shrimp,  whereas  the sheepshead
minnow  showed  greater  resistance   with  a  96-hour   LC5Q  value  of  27,100
 ug/1.  Both the mysid  shrimp and the  herring embryos  exhibited  greater sen-
 sitivity  to  2,4-dinitrophenol  (96-hour  LC5Q  values of  4,850  and  5,500
 ug/1, respectively) than  did the sheepshead  minnow  at 29,400 ug/1.  Of  the
 three  nitrophenols tested,  2,4,6-trinitrophenol   appeared  to  be  the  least
 toxic,  producing  96-hour  LCcg  values   of  19,700  ug/1  for  the  mysid  shrimp
                                      B-3

-------
 and  134,000 ug/1 for  the  sheesshead Tiinnow.  This  nitroohenol  was  also t^e
 least  toxic  of  the  tested  compounds  to freshwater fish and invertebrate spe-
 cies.   Although the invertebrate species was  consistently more sensitive- to
 all these compounds  than was  the sheepshead minnow,  the herring embryos also
 were very sensitive  to the toxic effects of  2,4-dinitrophenol.
 Chronic Toxicity
     There  are  no data available on the chronic effects  of any of  the var-
 ious nitrophenol compounds on freshwater aouatic  life.
     The chronic  effects  of  4-nitrophenol  and 2,4-dinitrophenol on  hatching
 and  survival  in an  early  life  stage  test  with  the sheepshead  minnow have
 been determined (U.S.  EPA, 1978).   Chronic values of  12,650  and 7,900 ug/1
were obtained  for  4-nitrophenol  and 2,4-dinitrophenol,  respectively   (Table
 2).  Although 4-nitrophenol may be   somewhat less  toxic  in the chronic test,
 the compounds were  of  similar acute  toxicity to  the  sheepshead minnow  (Table
 1).
     Acute-chronic  ratios  for   these  nitrophenols  may be  derived  for  the
sheepshead minnow.   For 4-nitrophenol  the   acute-chronic  ratio  is  2.1,  and
for 2,4-dinitrophenol the ratio  is 3.7.
     There are  no  nitrophenol chronic effects data  for  any saltwater   inver-
tebrate species.
Plant Effects
     Freshwater  plant  toxicity   values  (Table  3)  are  lower,  in  certain  in-
stances, than  acute LC--  values for  fish  and  invertebrate  species.   Expo-
sure to 4-nitrophenol  produced  toxic effects  in  ChloreT •- vulgaris  at 6,950
ug/l (Dedonder  and  Van  Sumere,  1971)  and  in Selenastrum  capricornutum  at
4,190  ug/1  (U.S. EPA,  1978).  These  plant effect  levels  for 4-nitrophenol
are  both  below  the lowest fish  or  invertebrate  LC5Q  values   for this com-
pound.   A 50 percent reduction  in chlorophyll  a_ also occurred  in Selenastrum
                                      B-4

-------
 cap^icoroutjn  in  95  hours  at  a  2,4,6-trini trooheno'  concentrat ; on  ,'ai,7CC
 ug/1)  t^at  is  below  the  lowest  LC-0  value  for  fish or  invertebrate  spe-
 cies.  As observed with  fish and  invertebrate species, 2,4,5-trinitropheno '
 is  less  toxic  than d_nitrophenol and 2,4-dini trophenol to Selenastrum  capri-
          (U.S. EPA, 1973).
      Dedonder  and  Van Sumere (1971) determined  that  9,200 ug/1  of 2,4-dini-
 trophenol  caused  a  70 percent growth  inhibition  in  Chlorella vulgaris in 80
 hours,  although  this concentration  caused  only a 25  percent growth   inhibi-
 tion  in  160 hours.
      Results of  tests which  examined the relative, toxicity of the three  iso-
 meric  foms  of mononi trophenol s  to  an  alga  (Huang  and Gloyna,  1967) indi-
 cated  that chlorophyll  synthesis  in  Chlorella pyrenoidosa  was inhibited  to a
 point  below  initial   control  levels  at  concentrations  of  25,000  ug/1  by 4-
 nitrophenol,  35,000  ug/1   by 2-nitrophenol ,  and  50,000  ug/l   by  3-nitro-
 phenol.   Studies  with three species of  algae  indicate that 4-nitropheno 1 is
 slightly  more  toxic  to  plants than  is  2,4-dinitrophenol  (Table  3).   The one
 exception to this toxicity trend  was observed by  Simon and Blackman   (1953),
who found  that  50 percent growth reduction  in  duckweed,  Lemna minor,  occur-
 red at  2,4-dinitrophenol  and 4-nitrophenol  concentrations  of  1,472  ug/1  and
9,452  ug/1,  respectively.    However,  these  observed effects  on  duckweed  oc-
curred under conditions  of low pH (5.2  and  5.4) and effects  at  a more  neu-
tral pH were not measured.
     All of the available  freshwater plant  effects data for nitrophenols are
based on  unmeasured  concentrations and  therefore there is  no Freshwater  Fin-
al Plant Value.
     Only one saltwater algal species  was examined for its response  to three
nitrophenols (U.S. EPA, 1978).  The  effects  of  these compounds on Skeletone-
ma_ costatum  are  summarized  in Table 3.  The 96-hour  LCrQ concentrations of
                                      B-5

-------
4-nitrophenol  (about  7,000  ug/1)  and 2,4-dinitroonenol  (about  95,000 yg/1)
were very  similar  for effects  on  both chlorophyll  a_ production and reduction
in cell  numbers.   However,  2,4,6-trinitrophenol  seemed  to have a greater ef-
fect on  chlorophyll a_,  with  a 96-hour  EC5Q of  62,7000 ug/1,  than  on cell
numbers  where  the  effective  concentration  was  141,000  ug/1.   Skeletonema
costatum was most sensitive  to 4-nitrophenol.
Residues
     No  measured,   steady-state  bioconcentration  factors are  available for
saltwater or freshwater organisms and any nitrophenol.
Miscellaneous
     No  miscellaneous  data  were  found  that would be  a  suitable substitute
for a  Final  Chronic Value for any  nitrophenol  compound  (Table  4),  although
some data are the  lowest  acute values or the only acute values available for
several  nitrophenol compounds.  Bringmann and Kuhn  (19_78) calculated  the 8-
day toxicity thresholds  (the  pollutant  concentration  causing  the  onset of
cell  multiplication inhibition)  for  nine  nitrophenol  compounds  with  two
algal   species,   Microcystis  aeruginosa  and  Scenedesmus  ouadricauda.   Data
from the most sensitive  species  tested  in  this  study  are the  lowest plant
values  available for 2-nitrophenol  (4,300 ug/1), 3-nitrophenol  (7,600 ug/1),
2,4,6-trinitrophenol  (40,000   ug/1),   and  2,4-dinitro-6-niethylphenol   (150
ug/1)   and  are  the only  plant data available  for  2-nitro-£-cresol  (3,800
ug/1),  4-nitro-m-cresol  (7,000 ug/1), and 6-nitro-m-cresol  (7,000 ug/1).  In
another  study,  Bringmann  and  Kuhn  (1977)  determined  the  24-hour  LC5Q
values  for  the same nine nitrophenols  with the  cladoceran,  Oaphnia magna.
This  study  provides the  only  invertebrate or fish  acute data available for
2-nitro-£-cresol (130,000 ug/1),  4-nitro-m-cresol  (33,000 ug/1),  and 6-ni-
tro-m-cresol  (43,000 ug/1).  and  also provides  a  toxicity comparison  of the
                                      3-5

-------
 three  isomeric  forms  of mononitrophenols  with daphnids.   They found 4-nitro-
 phenol  to  be the most  toxic compound  to  daphnids,  followed in order  of de-
 creasing toxicity  by  3-nitrophenol  and 2-nitrophenol.   Gersdorff  (1939) also
 found  the  same  relative order  of  toxicity  for  the  three mononitrophenols
 with goldfish  (8,000  ug/1 for 4-nitrophenol,  24,000  ug/1  for 3-nitrophenol,
 and  33,300 ug/l for  2-nitrophenol).   While  these are not  LC5Q  values, they
 represent  the lowest  acute effects  available for  these three mononitrophenol
 compounds.   Although  the  relative  order of  toxicity is  the  same for  the
 three mononitrophenols  with both  fish  and invertebrate species,  the toxicity
 order  appeared  to  be slightly different  in  the  alga,  Chlorella  pyrenoidosa
 (Huang and Gloyna, 1967, Table 3).
     Juvenile Atlantic  salmon were  much more sensitive to 2,4-dinitrophenol,
with a  lethal   threshold  value  of  700 ug/1   (Zitko,  et  al.  1976).   (Lethal
 threshold  is the geometric  mean  of the highest concentration  with no  deaths
and  the next higher  concentration  at  which  all   animals died).   The authors
tested  a  variety of  compounds  with both freshwater  and  saltwater species.
They did  not specify  whether  the  Atlantic salmon  was  tested  in salt  or
freshwater, but  the size  of the  tested organism  leads us to believe that the
test was probably conducted in freshwater.
     The only saltwater datum  for 2-nitrophenol   is  listed  in  Table 4.  This
result  is  very   similar to  that  for  4-nitrophenol  and  the  shrimp, Crangon
septemspinosa.    The soft  shell  clam responded to the  toxic effects of 4-ni-
trophenol  at about the  same concentration (McLeese,  et al. 1979).  2,4-Oini-
trophenol  also  adversely  affected sperm and  embryo  of two sea urchin species
at concentrations as  low as 46,000 ug/1.
                                      8-7

-------
Summary
     For  the  four  nitrophenols  for which  freshwater  acute data  are  avail-
able,  the order of most  to least  toxic  is 2,4-dinitro-6-methylpheno1,  2,4-
dinitrophenol,  4-nitrophenol,  and  2,4,6-trinitrophenol.   Acute  LC,-0  values
ranged  from  a   low  of  230 ug/1   for  bluegills  exposed   to  2,4-dinitro-6-
nethylphenol to  a  high  of 167,000 ug/1  for bluegills exposed to 2,4,6-trini-
trophenol.   There  are  no freshwater chronic  data available for  any  nitro-
phenol.   Plant   values  and miscellaneous  data for the  various  nitrophenols
showed that  effects occurred at  exposure concentrations ranging  from  a low
of  150  »g/l  for   2,4-dinitro-6-methy1phenol,  based  on  an 8-day  toxicity
threshold with  algae,  to  a high  of 210,000  ug/1  for  2-nitrophenol,  based on
a 24-hour LCg. with daphnids.
     For  the three  nitrophenols  having  a  saltwater acute toxicity data base,
the mysid shrimp was  consistently more  sensitive than the  sheepshead  min-
now.   In  comparing the  acute  toxicity of these  three  compounds, the least
toxic  for both  organisms was  2,4,6-trinitrophenol.   Acute  and  early  life
stage  tests  conducted on  the sheepshead  minnow yielded acute-chronic  ratios
of 2.1 for 4-nitrophenol  and 3.7  for 2,4-dinitrophenol.   There are no chron-
ic data for 2,4,6-trinitrophenol.
     Plant values  and miscellaneous data  for the various nitrophenols showed
a concentration  range of  effects  from a  low  of 7,370  ug/1  for 4-nitrophenol
on chlorophyll  a^ production in an  alga to a high of  141,000 ug/1 for  an ef-
fect of 2,4,6-trinitrophenol on cell numbers  of the same species.
                                    CRITERIA
     The  available data  for  nitrophenols  indicate that  acute  toxicity  to
freshwater  aauatic life  occurs  at concentrations  as  low  as  230  yg/1  and
would  occur  at   lower  concentrations among  species  that are  more sensitive
                                      3-8

-------
than fiose  tested.   Mo  data  are  available  concerning  the cnronic toxicity of
nitropnenols  to  sensitive freshwater aquatic  life  out  tcxicity  to  one spe-
cies of algae occurs at concentrations as low as 150 ug/1.
     The  available  data  for nitrophenols  indicate that  acute  toxicity  to
saltwater  aouatic  life  occurs  at  concentrations  as  low  as 4,350  ug/1  and
would  occur at  lower  concentrations among  species  that are more  sensitive
than those  tested.   No  data  are  available  concerning  tne chronic toxicity of
nitroohenois to sensitive saltwater aquatic  life.
                                      3-9

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Table t.  Acute values  for nltrophenols


Species

Cladoceran,
Daphnla magna
Cladoceran,
Daphnla Miagna
Cladoceran,
Daphnla magna
Cladoceran,
Daphnla magna
C 1 adoceran ,
Daphnla magna
Cladoceran,
Daphnla magna
Stonefly (naiad),
Pteronarcys call for n lea
Fathead minnow
( juvenl le).
Plmepnales promelas
Fdttiead minnow
(juvenl le).
Plroephales promelas
Fathead minnow
(juvenl le).
Plmephales promelas
Bluegill,
Lepomls macrochlrus
Bluegj 1 1,
Lepomis macrochlrus
Bluegill,
Ltipomls macrochlrus


Method*

S, U

S, U

S, U

S, U

S, U

S, U

S, U

FT, M


FT, M


FT, M


S, U

S, U

S. U



Chemical
FRtSHWAftR
4-nl trophenol

4-nl 1r ophenol

2,4-dlnltro-
phenol
2,4-dlnltro-
pnenol
2,4,6-trlnltro-
phenol
2,4-dlnltro-6-
methy (phenol
Olnltromethyl-
phenol**
4-nl trophenol


2,4-dlnltro-
phanol

2,4-dinitro-6-
methy (phenol **

4-nl trophenol

2,4-dlni tro-
phonol
2,4,6-trlnl tro-
phonol

LC50/EC50
(M9/D
SPECItS
8,396

21,900

4,710

4,090

84.700

3,120

320

60,500


16,700


2,030


8,280

620

167,000

Species Mean
Acute Value
(ug/l)

_

13,560

-

4,389

84 , 700

3,120

320

60.500


16,700


2,030


8,280

620

167.000



Reference

Koppurman, et al.
1974
U.S. EPA, 1978

Kupperman, ot al.
1974
U.S. EPA, 1978

U.S. EPA, 19/8

U.S. EPA, 1978

Sanders & Cope, 1968

Phlpps, et al.
Manuscript

Phlpps, et al.
Manuscript

Phlpps, et al.
Manuscr Ipt

U.S. EPA, 1978

U.S. EPA. 1978

U.S. EPA, 1978

                  B-10

-------
Table 1.  (Continued)
Species
Liluegl 1 1,
tupomts macrochlrus
Mysid shrlnp,
Mysldopsls bah ia
Mysid shrimp,
Mysldopsis bah la
Mysid shrimp.
Mysldopbis Uih i a
Herring (embryo),
Clupea harengus
Sheepbhoad minnow,
C/pr inodon varieyatus
ShHupbliuad in if mow,
Cypr inodon varieyatus
Slieupbhfcjd minnow,
Cypr inodon varieyatus

Method"
S, U
S, U
S, U
S, U
S, U
S, U
S, U
S, U

Chemical
2,4-dinitr 0-6-
mulhy t ptiunol
4-ni 1r ophonol
2,4-diul tro-
phbllol
2,4,6-1i Inl tro-
phonol
2,4-dlnitro-
phuliol
4-di tr'ophenol
2,4-dinltro-
phenol
2,4,6-tr Inl Iro-
pllaflol

LC50/LC50
(Mi/'J
230
UK SHEClES
7,170
4.050
19.700
5,500
27,100
29,400
134.000

Species Mean
Acute Value
(ug/D
230
7,170
4,050
19,700
5,'j()0
27,100
29,400
134,000

Reference
U.S. tt'A. 1970
U.S. EPA, 19/0
U.S. EHA, 1970
U.S. EHA, 19/0
Kosorilliol i Slc-l^or
1970
U.S. EHA, 1976
U.S. Ef'A, 19/0
U.S. EHA, 19/0

* S = italic, FT = flow-through, (I = unmeasured, M - measured



""Authors fuported results as 4,6-dlni tro-o-cresol or as only d lui 1ro<_rov>ol  (Sanders and Cope,  1960).
                                                     3-a.l

-------
Table 2.  Chronic values tor nltrophenols (U.S. tPA,  1970)
Species
Sheepshead minnow,
Cyprlnodon varleqatus
Sheepshead minnow,
Cyprlnodon varlegatus
* ELS = early life staye
Spec 1 os
Sheepshead minnow,
Cyprlnodon varlegatus
Sheepshead minnow,
Cypr Inodon varlegatus
Method"
SAL TW AUK
ELS
hLS
Chemical
SPEC Its
4-nl trophenol
2,4-dlnltro-
phenol
Limits
(My/I)
10.000-
16.000
5,200-
12,000
Chronic Value
(M9/I)
12.650
7,900
Acute-Chronic Ratio
Chemical
4-nl Trophenol
2,4-dlnltro-
phenol
Acute
Value
(ug/i)
27,100
29,400
Chronic
Value
tut}/ I )
12,650
7,900
Ratio
2.1
3.7
                          3-L2

-------
                               Table 3.  Plant values for nltrophenols
Species
AI yd.
Chloral la pyrenoldosa
Alga,
Cn I or e 11 a pyrenoldosa
Alga.
Chloral la pyrenoldosa
Alga.
ChIore I la pyrenoldosa
Alga,
Chior el la pyrenoldosa
Alga,
Chi or t, I la vulgar is

Alga.
Ch I or el la vulgar Is

Alga,
ChIoreIIa vuIgar is

Alija,
Selenastrum  caprI cornuturn

Alga.
Sfelenastrum  caprI cor nutum

Chemical
H.|
2-nltr ophenol



3-iiMrophbnol



4-nl trophenol



2,4-dinitro-
phenol


2,4-dlnitro-6-
methy I phenol *


4-ni trophenol

2,4-dlni tro-
phenol
2,4-dlnitro-
phenol
4-nl trophenol

2,4-dlnltro-
phenol

Effect
SMWAjifL^tc.!!
I nh Ibi t ion of
chlorophy 1 1
synthesis after
3 days
Inhibit Ion of
chlorophy 1 1
synthesis after
3 days
Inhibit Ion of
ch lot ophy I I
synthesis after
3 days
Inhibition of
ch lot ophy I I
synthesis after
3 days
Intrlbi t Ion of
chlorophy I I
synthesis after
3 days
50* growth Inhi-
bition lit bO hrs
70* growth Inhi-
bition In «0 hrs
25* growth Inhi-
bition In I60 hrs
96-hr EC50,
chlorophy 1 1 a
96-hr EC50,
ch lor ophy 1 1 a
Result
(M9/I)

35,000



50,000



25,000



50,000



50,000



6,950

9,200

9,200

4,190

9,200


Reference

Huang & G loyna.



Huang & G loyna.



Huang 4 G loyna.



Huang & G loyna.



Huang & G loyna.



Uedonder & Van
Sumero, 1971
Oedonder & Van
Sumere, 1971
Oedonder & Van
Suniere, 19/1
U.S. EPA, I97b

U.S. EHA, 197tt




1967



1967



1967



1967



1967













                                               3-13

-------
Table 3.   (Continued)
Species
Alga,
Sulunastrum capr Icornutuin
Duckweed,
lomna minor
Duckweed,
Lbmna minor
Duckweed,
Lemna mlnoi
Alga,
Skeletonema costatum
Alga,
Skeletonema costatum
Alga,
Skeletonema costatum
Alga,
Skeletonema costatum
Alga,
Skelfetonema costatum
Alga,
Skeletonemd costatum

Chemical
2.4,6-tr Inl Iro-
ptvenol
Effect
%-hour EC 50
ch lorophy 1 1 a
2-nl trophenol 50J growth
ruducl Ion
4-nl trophunol 501 growth
reduct ion
2,4-dinitro- 501 growth
phenol reduction
SALTWATER SPECIES
4-nl trophenol
4-nl trophenol
2,4-dlnl h-o-
phdnol
2,4-dlnl tro-
phenol
2,4,6-trlnltro-
pheiiol
2,4,6-trlnltro-
phenol
96-hr EC50,
ch 1 orophy 1 1 a
96-hr EC50,
eel 1 number
96- hr EC50,
ch lorophy 1 1 a
96-hr EC50,
ce 1 1 number
96- hr EC50,
ch lorophy 1 1 a
9<}-hr EC50,
eel 1 number
Result
(ug/l) Reference
41,700 U.S. EPA, 19/8
62,550 Simon & Blackmail,
1953
9,452 Simon & bldckman,
1953
1,472 Simon & blackmail.
1953
7,370 U.S. EPA, 1978
7,5/0 U.S. EPA, 1978
93,200 U.S. EPA, 1978
98,700 U.S. EPA, 19/8
62,700 U.S. EPA, 1978
141,000 U.S. EPA, 1978
* Authors  reported results as 4,6-dlnltro-o-cresol.
                                               B-14

-------
Table 4.  Other data  for nltrophenols
Species
Chemical
Duration
Effect
Result
(ug/l>
Reference

FKtSHWATEK SPtCltS
Cli lomydomonas sp.
Alya,
Microcyst Is
Alga,
Scenedesmus
Alga,
Microcyst Is
Alga,
Scenedesmus
Alga,
Microcyst Is
Alga,
Scenedesmus
Alga,
Microcyst Is
Alga,
Scenedesmus
Alga,
Microcyst is
Alga,
Scenedesmus
Alga,
Microcyst is
Alga,
Scenedesmus
aeruglnosa
quadr Icauda
aeruglnosa
quadr Icauda
aeruglnosa
quadr Icauda
aeruglnosa
quadr icauda
aeruglnosa
(juadr icauda
aeruginosa
quadr Icauda
D Inl trophenol
2-nl trophoni!
2-nl trophenol
3- nl trophenol
3-nl trophenol
4-nl trophenol
4-ni trophenol
2,4-dlnitro-
phenol
2,4-dlnltro-
phenol
2,4,6-tr I ni tro-
phenol
2, 4, 6-trlnl tro-
phenol
2-nl tro-p-
cresol
2-nltro-p-
cresol
30
8
8
8
8
8
8
8
8
8
8
8
8
sec
days
days
days
days
days
days
days
days
days
days
days
days
50* Inhibition
of f lagel lar
motl 1 1 1/
Toxlclty
threshold*
Toxlclty
tires hold*
Toxiclty
threshold*
Toxlclty
threshold*
Toxlclty
threshold*
Toxlclty
threshold*
Toxlcily
threshold*
Toxlcity
threshold*
Toxlcity
threshold*
Toxlclty
threshold"
Toxiclty
threshold*
Toxlclty
1 tires hold*
18,400
27
4
17
7
56
7
33
16
40
61
32
3
,000
,300
,000
,600
,000
,400
,000
,000
,000
,000
,000
,800
Marcus & Mayor, 1963
Br Inymdim
Brlngmann
1978
Brlngmann
1978
Br Ingmann
1978
Brlngmann
1978
Brlnc^nann
1978
Brlngmann
1978
Br Inymann
1978
Brlngmann
1978
Br Ingmann
1978
Br Inymann
1978
Brlnqmanri
19 10~
& Kulm,
& Kuhn ,
& Kuhn,
& Kuhn,
& Kul«i.
& Kuhn,
& Kuhn,
& Kulm,
& Kuhn,
& Kuhn,
& Kuhn,
& Kuhn,
               B-15

-------
Table 4.  (Continued)

Species
Alga,
Mlcrocystls aeruyioosa
Al.ja,
Scenedesmus quadrlcauda
Alga,
Hlcrccystls aerutjlnosa
Alga,
Scenadesmus quadrlcauda
Alga,
Mlcrocystls aerujjlncsa
Alga,
Scenedesmus quadrlcauda
A/ooo ba,
Amoeba proteus
Amoeba,
Amoeba proteus
Cladoceran,
Daphnla mayna
Cladoceran,
Daphnla magna
Cladoceran,
Daphnla macjna
Cladoceran,
Oaphnla magna
Cladoceran,
Daphnla magna
Cladoceran,
Udphnia nuitina

Chemical
4-ni tro-m-
cr osol
4-nl tro-m-
crosol
6-nltro-m-
cresol
6-nltro-m-
crt»sol
2,4-dinltro-6-
rnethyl phenol **
2,4-dinltro-6-
methyl phenol**
01 ni trophenol
Dial trophenol
2-nl trouhenol
3-nltrophenol
4-nl trophenol
2,4-dinltro-
phenol
2,4,6-trinltro-
phenof
2-ni tro-g-
cresol

Duration
6 d'tys
ti days
6 days
8 days
8 days
8 days
24 hrs
48 hrs
24 hrs
24 hrs
24 hrs
24 hrs
24 hrs
24 hrs

Effect
Toxlcl ty
threshold*
foxlcl ty
thrushold*
Toxic Ity
threshold*
Toxic Ity
thrushold*
Toxic Ity
threshold*
Toxlcl ty
threshold*
A6f reduction in
amoeba containing
go 1 g 1 bod i as
18* mortality
IC50
LC50
LC50
IC50
LC50
L.50
Result
(M9/D
13,000
7,000
34,000
7.000
150
13,000
92,000
92,000
210,000
39,000
35,000
19,000
145,000
130,000

Reference
UrinijirtdiiM &
1976
brlnijinaiin &
19/8
Br Inijihdfiu &
1978"
Brlnymann &
1978
Brlnynidnn &
1976
brlntjmann &
1978
Ft lckiny«r.
K 1 Icktnger,
brlngmann &
1977
Brinymann &
1977
iirlngmann &
1977
Brlnymann A
1977
Orln
-------
Table 4.  (Continued)
Spec 1 es
Cladoceran,
Ddphnla mayna
Cladoceran,
Daphnla magna
Cladoceran,
Daphnla magna
Southern bullfrog
(tadpole),
Kana gry 1 lo
Atlantic salmon (juvenile).
Sal mo salar
Goldfish,
Carasslus auratus
Goldfish,
Carasslus auratus
Goldfish,
Carasslus auratus
Bluegl 1 1 ( juvenl le),
Lepomls macrochlrus
blueglll (juvenile),
LepcMils macrochlrus
Shrimp,
C ran yon septemsplnosa
Shrimp,
Crangon septemsplnosa
Clam (soft-she! 1),
Mya arenarla
Chemical
4-n i tro-ni-
cresol
6-nl tro-m-
cresol
2,4-dlnl1ro-6-
methy (phenol *"
2,4-dlnltro-
phenol
2,4-dlnltro-
phenol
2-nl trophenol
3-nltrophenol
4- n I trophenol
2-nl trophenol
2-nl trophenol
2-nl Irophenol
4-nl trophenol
4-nl trophenol
Duration
24 hrs
24 hrs
24 hrs
7 hrs
96 hrs
8 hrs
8 hrs
8 hrs
24 hrs
48 hrs
SA11WAUR
96 hrs
96 hrs
96 hrs
Effect
LC50
LC50
LCt>0
Increased
resplrat Ion
Lethal threshold
value
38} mortality
53> mortality
42* mortality
LC50
LC50
SPECIES
Lethal threshold
value
Lethal threshold
value
Lethal threshold
value
Result
(U9/D
33,000
43,000
6,600
5,520
700
33, 300
24,000
8,000
66,900
46,300-
51,600
32,900
26,400
29,400
Reference
br Ifiijrtiann &
1977
Brlnymanri &
I9//
Urintjmann &
19/7
Kullll,
Kuhn ,
Kuhn,
Lewis 4 Frleden, 1959
Zltko, et al. 1976
Gersdorff, 1939
GersdorH, 1939
Gersdorff, 1939
Lammorlncj & burtwnk,
1960
Ldnmierlny & BurUanK,
I960
McLeese, et al. 1979
McLeese, tit
McLeese, et
al. 1979
al. 1979
                                                 B-17

-------
Table 4.  (Continued)
Species
Sud urchin (sporni),
Sfroiujy locentrotus
£urj)urdlus
bud urchin (ombryo),
Pseudocdiitrotus dupressus

Chemlcdl
2,4-dinltro-
plionol
2,4-Ulnltro-
phono 1

Ourdtlon tttect
|i Irs Inhibit cosplra-
t UMI, mul flit/
2 lirs AUiornial cluavdtjo

Result
w.ooo
4b,000

Refer enco
,»M*t.,«,


* Toxlclty  threshold = the pollutant  conctmti at ion  ciiusin^  the  onsot of col \ mul t Ip 11 ccit ion  Inhibition.



"Authors reported results as 4,6-dini tro-o-cresol.
                                                      B-I8

-------
                                  REFERENCES

Bernstein,  G.S.   1955.   Effect  of  2,4-dinitrophenol  on  sea  urchin  sperm.
Proc. Soc. Exp. Biol. Med.  90: 28.

Bringmann, G.  and R.  Kuhn.   1977.   Befunde der Schadwirkung wassergefahrden-
der Stoffe gegen Oaphnia magna.  Z.F. Waser-und Abwasser-Forschung.  5:  161.

Br-fngman, G.  and  R. Kuhn.   1978.   Testing of substances  for  their  toxicity
threshold: Model organisms Microcystis  (Diplocytis)  aeruginosa  and Scenedes-
mus quadricauda.  Mitt. Inter. Verein. Limnol.  21: 275.

Qedonde-r, A.  and  C.F.  Van Sumere.  1971.   The effects of  phenolics  and  re-
lated compounds  on   the  growth and  respiration of  Chiore 11 a  vulgaris.   Z.
Pflanzen. Physiol.  65: 70.

Flickfnger,   C.J.   1972.   Influence  of  inhibitions  of energy metabolism  on
the formation of Golgi bodies in Amebae.  Exp. Cell Res.   73:  154.

Gersdorff, W.A.   1939.   Effect of the  introduction  of the nitro  group  into
the phenol  molecule  on  toxicity  to  goldfish.  Jour.  Cell.  Comp.  Physiol.
14: 61.

Huang, J. and  E. Gloyna.   1967.   Effects of toxic  organics on  photosynthetic
reoxygenation.  Environ. Health Eng. Res. Lab.  PB 216-749.
                                     8-19

-------
•'7"''~3,  v.<.   I960.   ~^e er~fect  cf  DN? anc  \aN->  on '"erti • : zee eaas  of  tne
                                                 ^                  ~* "
sea  j*-cMn  with  sDecia* reference  to  the  induction  cf the  abncrma'  cleav-
age.  Embryologia.  5: 71.
Kcope'-re",  -.!_.,  et  a1.   197^.  Aoueous  chlorination  and  ozonation studies.
I. Structure -toxicity  correlations  of  phenolic  compounds  to  Daphnia  magna.
Oem. 3ioT. Interact.  9: 245.
      -ing, M.W.  and  N.C.  Surbank.  1960.  The  toxicity  of phenol,  o-chloro-
o^enol and o-nitrophenol to  bluegill  sunfish.   Eng.  Bull.  Purdue Univ., Eng.
Ext. Serv.  106: 5*1.

Lewis, E.J.C.  and  E. Frieden.  1959.   Biochemistry  of amphibian metamorpho-
sis: Effect of  triiodothyronine,  thyroxin,  and  dinitrophenol  on the respira-
tion of the tadoole.  Endocrinology.  65: 273.

Marcus, M. and  A.M.  Mayer.   1963.   Flagellar movement in Chlamydomonas snow-
iae and  its  inhibition  by ATP and  dinitrophenol.   Iri; Studies on Microalgae
and PHotosynthetic  Bacteria.  Jap. Soc.  Plant  Physio!.,  Univ.  Tokyo  Press,
Tckyo, Jaoan.
   eese, O.W., et  al.   1979.   Structure-lethality relationships for phenols,
an-j lines,  and  other aromatic  compounds in  shrimp and  clams.   Chemosphere.
q:
ph'oos, G.L.,  et  al.  The  acute  toxicity of  phenol  and substituted phenols
to the fathead minnow.  (Manuscript)
                                     B-20

-------
 Rosenthal,  H.  and  R.  Stelzer.   1970.   Wirkungen  von  2,4-  und 2,5-dinitro-
 phenol  auf  die  Embryonalentwicklung  des  Herings   Clupea  harengus.   Mar.
 B1ol.   5: 325.

 Sanders,  H.O.  and  O.B.  Cope.   1968.   The  relative  toxicities  of  several
 pesticides  to  naiads  of  three  species  of  stoneflies.   Limnol.  Oceanogr.
 13: 112.

 Simon,  E.W.  and  G.E. Blackman.   1953.   Studies in  the principles of phyto-
 toxicity.   IV.   The effects of  the degree of  nitration  on  the  toxicity of
 phenol  and other substituted benzenes.  Jour. Exp.  Bot.  4: 235.

 U.S.  EPA.   1978.   In-depth  studies on  health  and  environmental  impacts of
 selected water pollutants.   U.S.  Environ.  Prot. Agency.  Contract No. 68-01-
 4646.

 Zitko,  V.,  et  al.   1976.   Toxicity  of  alkyldinitrophenols  to  some  aauatic
organisms.  Bull. Environ. Contam. Toxicol.  16: 508.
                                     3-21

-------
                               MONONITROPHENOLS
      san Toxicology and uuman ^ealth Effects
                                 INTRODUCTION
                    has three  isomeric  forms,  distinguished by the  oosition
of  the  m'tro- group  on the  phenolic  rinq.   Three isomeric fcrms  are  oossi-
ble, namely  2-nitroohenol,  3-nitrophenol,  and 4-nitrophenol .   The  comoounds
are  also  commonly referred to as o-m'troohenol,  m-nitroohenol, and  o-m'tro-
ohenol, respectively.
    Commercial  synthesis  of  2-nitroohenol  and 4-nitroohenol  is accomplished
tHrouoh  the  hydrolysis of the  appropriate  chloronitrobenzene isomers  with
aqueous  sodium hydroxide  at  elevated  temperatures   (Howard,  et  al.  1976).
Production of  3-nitropheno 1  is  achieved  through the  diazotization and  hy-
drolysis  of  ii-ni troani 1 ine  (Matsuguma,  1967).   The  mononitroohenol  isomers
are used  in  the  United States  primarily as  intermediates  for  the  production
of  dyes,  pigments,  Pharmaceuticals, rubber  chemicals,  lumber  preservatives,
photoaraohic  chemicals,   and  pesticidal  and fungicidal   agents  {U.S.  Int.
Trade  7omi.  1976).   As a  result of this use  pattern,  the major  source for
environmental  release  of  rnononitroonenols  is  likely to  be from  production
plants  and  chemical  firms where the  comoounds  are  used  as  intermediates.
The  mononitrophenols  may  also be  inadvertently produced  via microbial  or
photodearadation  of   pesticides which  contain  mononitrophenol   moieties.
Approximately  10 to 15 million pounds  of  2-nitrophenol  are produced annually
(Howard,  et  al.  1976) for  uses  including synthesis  of  o-aminophenol,  o-
nitroanisole,  and  other  dye stuffs  (Matsuquma,  1967; Howard,   et  al.  1976).
Although  production figures  for  3-m'trophenol  are not available,  Hoecker,  et
al.  (1977) estimate that  production is less than one million  pounds annual-
ly.  3-Nitroohenol  is  used  in the  manufacture of dye  intermediates such  as
                                      C-l

-------
 anis^dine  and  Ti-a^inoohenol  >Kouris  and  Nortncott,   1953; ^atsusuma,  '.9571.
 4-Nitrophenol is Probably the most  important of  the  monoitroohencls  in  terms
 of quantities used  and  ootential  environmental  contanination.   Demand  for  
-------
                                   TABLE  1
                       Properties of Mononitrophenols*
Formula
Molecular Weight
Melting =>oint (°C)
Soiling Point
Density
Water Solubility
  (g/D
Vapor Pressure
Ka
2-Nitrophenol
C6H5N03
139.11
44-45
214-216
1.485
0.32 at 38*C
1.08 at 100'C
1 mm Hg at 49.3'C
7.5x10-8
3-Nitroohenol
C6H5N03
139.11
97
279
1.485
1.35 at 25'C
13.3 at 90"C

5.3x10-9
4-Nitrophenol
C6H5N03
139.11
113-114
279
1.479
0.804 at 15'C
1.6 at 25°C

7x10-3
*Sources: Windholz, 1976; Weast, 1975; Matsuguma, 1967.
                                     c-3

-------
CH

                            2-nitrophenol
OH

        MO,
                            3-nitrophenol
                            4-nitrophenol
            FIGURE 1



         MononitrophenoU
               C-4

-------
operation  around  1930.   2-Nitrophenol was  detected  at  unidentified  levels  in
2 river water  samples  and  in  4 samples  of chemical plant  effluent;  3-nitro-
phenol  was found  in  one  chemical  plant  effluent  sample  (U.S. EPA,  1976).
Systematic monitoring  for  mononitrophenols in  the  environment  has  not  been
done.   It  is  reasonable to assume that measureable  (although perhaps  trans-
ient) levels of the mononitrophenols may be present  in  localized  areas  where
organophosphate pesticides are in use.
    Little data are available  regarding  the breakdown  of mononitrophenols  by
natural communities of microorganisms.  Alexander and Lustigman  (1966)  stud-
ied the degradation of  mononitrophenols  by a  mixed  population of soil  micro-
organisms.  The inoculum was derived  from  a  suspension of Niagara  silt  loam
soil.   Their  results  indicated that  2-nitrophenol was more  resistant  to de-
oradation than either  3-nitrophenol or 4-nitrophenol.  Utilizing  the absorb-
ancy  of  small soil   inoculums  to  estimate   the  loss- of  mononitrophenol,
3-nitrophenol  was  found  to  degrade  completely  within   a  4-day  period.
4-Nitrophenol  degraded  fully within  a 16-day  period,  while 2-nitrophenol re-
sisted degradation over a 64-day period.
    Brebion, et al.  (1967) examined  the  ability of microoganisms  to  attack
4-nitrophenol.  The bacteria were  derived  from soil, water  or  mud  and  grown
on a  porous  mineral  bed and  were cultivated   in  a  mineral  nutrient solution
to which nitrophenols were added  as  the  sole   source of  carbon.   The experi-
mental findings revealed no  significant  removal of the  compound  under  these
conditions.
    In contrast to these reports, a number of investigators have found that
the mononitrophenols are readily and rapidly  degraded by  acclimated popula-
tions  of  microorqanisms.  Tabak,  et  al.  (1964)  studied the ability of ac-
climated cultures derived  from garden soil,  compost, and  river mud to de-
                                      C-5

-------
grade  the  morionitroonenols.    Phenol-adapted   bacteria  derived  from  these
sources  were  found  to  readily  degrade  all   three  rcionitroghenol   isomers.
Ninety-five  percent  degradation  (measured spectrophotometrically)  occurred
within three  to  six days.  Pitter  (1976) reported  greater  than 95  percent
degradation  of  the  three mononitrophenol  isomers  in  an acclimated  sludge
system.   The  nitrophenols served as  the sole  source  of organic carbon  and
degradation was complete within 120 hours.
    A  recent  study  (Haller,  1978) reports  on the  ability of  unacclimated
microorganisms to  degrade the  mononitrophenols.  Either  sludge obtained from
the primary  settling  tank of  the city  of Ithaca, N.Y.  wastewater  treatment
plant  or  a  Windsor loamy fine sand soil  were  used as  the  source of the ino-
culum.  2-Nitro-,  3-nitro-, and 4-nitrophenol   (16 mg/1) were  completely  de-
graded in three  to five  days  by the sludge system.  Soil inocula degraded 16
mg of 3-nitrophenol/1 in  three  to five  days while a  similar  concentration of
2-nitrophenol  and  4-nitrophenol  required 7 to  14  days  for complete degrada-
tion.
    Although  definitive  conclusions cannot be  derived  from this limited num-
ber of studies,  it appears that the mononitrophenols are readily and rapidly
degraded by microbial populations present  in the environment.
Ingestion from Food
    No  data  were  found  demonstrating  the presence of mononitrophenols' in
food.  One possible source of  mononitrophenol  exposure for humans is through
the food  chain as  a result of  the  ingestion  of food crops contaminated with
pesticides  containing the nitrophenol  moiety.   The production  of  4-nitro-
phenol by microbial metabolism  of parathion  is well  documented (Munnecke and
Hsieh, 1974,  1976; Siddaramappa,  et al. 1973;  Sethunathan and Yoshida, 1973;
Katan  and lichtenstein,  1977;  Sethunathan,  1973).  Microbial  metabolism of
                                      C-6

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 *luoridifen  (o-nitroonenyl,  a,a,a-trifljoro-2-oitro-p-tolyl  ether-)  results
 in  the  intermediate  formation  of  4-nitroohenol  (Tewfik  and  Hamdi,  1975).
 The  major  degradation  product  of  fluoridifen  following  uptake by  peanut
 seedling  roots was  4_nitroohenol   (Eastin,  1971).    4-,Nitropheno 1  was  also
 detected  in  soyoean  roots  following  absorption  of  fluoridifen  (Rogers,
 1971).   °hotodecomDO"sition  of the herbicide  nitrogen (2,4-dichloroohenyl-o-
 nitrophenyl  ether)  in anueous  susoensions  under sunlight or  simulated  sun-
 light  is  characterized  by rapid cleavage of  the  ether  linkage to  form  2,4-
 dichloroohenol  and  4-nitrophenol  (Nakagawa and  Crosby,  1974).   El-Refai  and
 Hopkins  (1966)  have investigated the  metabolic  fate  of  parathion  following
 foliar  applicatin  or  root  absorption  by  bean  plants,  Phaseolus  vulgaris.
 Detectable amounts  of 4-nitroohenol  were  found  in chloroform  rinses  of para-
 thion-treated  leaves after four days.
    In  another experiment,  analysis  of nutrient solutions containing para-
 thion  in  which  plants  were  grown  for  root   absorption  studies  revealed
 4-nitroohenol,  oaraoxon,  and traces of  degradation  products.   Since these
 compounds  were also  detected  in   control  solution  which did  not  contain
 olants, the  authors  concluded that  possible photochemical oxidation process-
 es  had occurred  in  the   aaueous  medium.   The  authors   believed  that  the
 4-nitrophenol  detected  following  foliar application  of parathion was  due to
 ohotochemical  degradation.   4-Nitrophenol  was  not detected  in  bean  plants
 followinq injection of parathion directly into  the  stems  of bean plants (El-
Refai and Hook ins, 1966).
    4-Nitrophenol  has also  been detected  as  a  photoalteration product  of
 parathion following application to cotton plants (Joiner and Baetcke,  1973).
    Archer (1974) has examined  the  dissipation  of  parathion and its metabol-
 ites  from  field spinach.   Field plots  were sprayed  with  either 0.5  or  one
                                      C-7

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oound  of  active oarathion per  acre.   Application r-econneidations for
thion  are: not  less  than 14 days  before harvest at  the  -ate  of 0.5 pounds
active  ingredient  oe"  acre.   Spinach samples  were  analyzed daily for  para-
thion  residues  and a  number  of known  metabolites  including  4-nitrooheno i.
Levels of 
-------
                                    TABLE 2
         Levels  of 4-Nitrophenol  Following Application  of  Parathion  to
               Field Spinach at Two Different Application  Rates*
                                     4-Nitrophenol Residue (wg/kg)a»b
Sample Day

1
2
3
4
5
6
7
8
9
10
11
12
13
14
0.5 Ib. Parathion/Acre
172
88
73
76
73
72
35
40
34
34
31
38
28
33
1.0 Ib. Parathion/Acre
453
305
240
188
136
216
117
18
19
18
18
22
16
19
*Source: Archer, 1974.

^Calculated on a fresh weight basis.  Percent moisture from 86.4 to 89.2.

bllnsprayed spinach control samples taken prior to any spray treatments con-
  tained 95 ug/kg.
                                     C-9

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that  the  average  urine void is 1.4  I/day  per  70  kg  person,  initial  exposure
levels can  be  estimated from residual  levels  found  in urine.  For  example,
the  exposure level  leading to the  1.0  yg/1  residue can  be calculated  as
fo"lows:
    Exposure =  (10.0 ug nitrpphenol/1)  (1.41  of urine/day) = Q.Q2 ug/kg/day
                                   70 kg man
A  similar  calculation  using  the  maximum  urine  residue  level  observed  by
Kutz, et al. (1978) (113 ug/1) gives an exposure of 2.26  pg/kg/day.
    Knowles, et al.  (1975)  have demonstrated the  production  of a  wide number
of  mononitroohenols  including  2-nitrophenol  in  a  model  system  simulating
gastric  digestion  of  smoked  bacon.   These  studies,  utilizing  nitrosated
liquid smoke, were conducted under conditions favorable to  nitrosation,  and
since the  temperature,  pH,  and  duration emoloyed  approximated those encount-
ered  during gastric digestion, their  results  indicated  that  nitrosation  of
phenols  in  smoked bacon may  occur in the  stomach with  resultant  production
of 2-nitrophenol.
    Mononitrophenols may also  be  formed in vivo  via  metabolic degradation of
pesticides  such as  parathion by humans.   Excretion of 4-n-;trophenol, a meta-
bolite  of  the  organophosphorous  pesticides,  parathion,  methylparathion,
0-ethyl  0-(p-nitrophenyl) phenylphosphonothioic acid  (EPN),  and dicapthon is
a good indicator  of human  exposure  to  these pesticides  (Wolfe, et  al. 1970;
Broadway and Shafik,  1973;  Elliott,  et al.  1960; Roan, et al.  1969).   4-Ni-
trophenol has also been detected   as  a  urinary metabolite  of nitrobenzene in
humans (Myslak, et al. 1971).
    A bioconcentration  factor (BCF) relates the  concentration  of  a chemical
in aauatic  animals to  the  concentration  in the water  in  which  they live.
The steady-state  3CFs  for a 1ioid-soluble compound  in the  tissues of various
                                     C-10

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 snuatic  animals  seem  to be  proportional  to the  percent  Hpid in  the  tis-
 sue.   Thus the  oer  capita  inqestion  of  a  1ioid-soluble  chemical  can be esti-
 mated  from the  oer capita consumption of  fish and  shellfish,  the weighted
 average  percent lipids  of  consumed  fish and  shellfish,  and  a  steady-state
 3CF for  the chemical.
    Data  from  a recent  survey  on  fish  and   shellfish  consumption  in  the
 United  States  were  analyzed  by SR!  International  (U.S.  EPA,  1980).   These
 data were  used  to estimate  that the  per capita consumption of freshwater and
 estuarine  fish  and  shellfish  in the United  States  is  6.5  g/day  (Stephan,
 1930).   In addition, these data were used with data on  the  fat  content  of
 the edible portion  of the same  species  to estimate that the weighted average
 percent  lipids  for  consumed  freshwater and  estuarine fish  and  shellfish  is
 3.0 percent.
    No measured steady-state  bioconcentration  factor tBCF)  is available for
any nitrophenol,  but the eiuation "Log  BCF  =  (0.85  Log  P)  - 0.70"  can  be
 used (Veith,  et al. 1979)  to estimate  the steady-state BCF  for anuatic or-
ganisms  that  contain about  7.6 percent  lipids (Veith,  1980)  from the  oc-
tanol/water partition coefficient  (P).   The  log P  values were obtained  from
Hansch and Leo  (1979)  or were  calculated by  the  method described therein.
The adjustment  factor of 3.0/7.6 = 0.395  is  used to adjust the estimated BCF
from the 7.6  percent lipids on  which the eouation is based to  the  3.0  per-
cent lipids that is the weighted average for  consumed  fish and shellfish in
order to obtain the weighted  average bioconcentration factor  for  the  edible
portion of all  freshwater and estuarine  anuatic organisms  consumed by Ameri-
cans.
       Chemical
     2-nitrophenol
     4-nitrophenol
                                     C-ll
loa P
1.73
1.91
Estimated
Steady-State
BCF
5.89
8.38
Weighted
Averaae
BCF
2.33
3.31

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Inhalation
    Quantitative  data  were  not  found regarding  the  presence of  rnononitro-
phenols in air.  Lao, et al. (1973) discussed the application of a gas chro-
matograph  quadrapole Tiass soectrometer-data  processor  combination for  rou-
tine  analysis  of  air  pollutants.   During  a  sample  run  of  urban  ambient
particulate matter  (location not designated) these  investigators  identified
the presence of 4-nitrophenol as well  as  a large number of other  air  pollu-
tants.  Quantitative data were not  provided, however.   Ambient  air levels  of
4-nitrophenol  in  a  Boeing plant  where the compound  was used  for the  preser-
vation of  the  cork  surfaces of  the Minuteman  Missile were equal  to  or  less
than 0.05 mg/m  of  air (Butler and Bodner, 1973).
    4-Nitrophenol may be produced  in  the  atmosphere  through the  photochemic-
al  reaction  between benzene  and nitrogen monoxide.   Nojima, et  al.  (1975)
irradiated a combination of  benzene vapor  and nitrogen  monoxide  gas  for  five
hours with a  xenon lamp and characterized the  resulting  photochemical  prod-
ucts.  The production  of nitrobenzene,  2-nitrophenol, 4-nitrophenol,  2,4-
dinitrophenol  and 2,6-dinHrophenol  was described by  the  authors.  Identity
of the compounds  was confirmed using  thin layer chromatography,  gas  chromat-
ography,   gas  chromatography-mass   spectrometry,  and  infrared  spectrometry.
The  authors  suggested that  these  nitro-compounds may be the  cause of  the
characteristic symptoms  of seriously  stricken  victims  of  photochemical  smog
in Japan  including:  headache, breathing  difficulties,  vomiting,  rise  in  body
temperature, and  numbness in the estremeties.
    In a  second  paper  (Nojima,   et  al. 1976), the photochemical reaction  of
toluene with nitrogen monoxide was  investigated.  It was  felt that the prod-
ucts  of  photochemical  reaction  of toluene  with nitrogen monoxide might  be
                                     C-12

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more  important  >n  the production of photochemical smog since  the  concentra-
tion of toluene in urban air  is  higher  than  that  of  benzene.   Compounds  pro-
duced as  a  result  of this reaction  included  o-cresol,  m-nitrotoluene,  4-ni-
troohenol,  2-methyl-5-nitrophenol,   3-methyl-4-nitrophenol ,   2-methyl-4-ni-
troohenol,  and  2-methy1-4-nitrooheno1.   These  compounds  were  identified  by
gas chromatography-mass spectrometry.   In another  experiment,  the  investiga-
tors  examined  the  organic compounds  present  in rain.  An  analysis  of  rain-
water  yielded  4-nitrophenol, 2-methyl-6-nitrophenol  and  2-methyl-4-nitro-
phenol.   The  authors  suggested  that the nitrophenols produced  by  the photo-
chemical  reactions, described above, dissolve  in  rain.   It  seems  likely that
in  areas  where severe  photochemical  smog exists,  humans  may  be  exposed  to
substantial levels of mononitrophenols.   However, it is impossible  to  esti-
mate  the  levels at which  humans are exposed to  these  compounds  via inhala-
tion, based on available data.
Dermal
    Roberts, et  al.   (1977) used  human  autopsy epidermal membranes  in  an  _m
vitro system  to determine  the  permeability  of human  skin  to various  com-
pounds.   Both   3-nitrophenol  and  4-nitrophenol  were shown  to permeate  the
skin  and  to produce  damage at threshold concentrations  of  0.8 and  0.9  per-
cent  (w/v),  repsectively.   According to  Patty (1963)i  2-nitrophenol may  be
absorbed  through the  intact  skin.   No  information on possible human dermal
exposure to the mononitrophenols was found.
                               PHARMACOKINETICS
Absorption and Distribution
    Data  specific  to  the absorption  and tissue  distribution of the monoitro-
phenols were not available.   It  is reasonable to  assume, based on the  rapid
urinary elimination  of  the mononitrophenols,  that the  compounds  may be re-
stricted primarily to the blood and urine following absorption by humans.
                                     C-13

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Metabol ^sm
    Metabolism  of  the  rnononitrophenols  prcoably occurs  via  one  of  three
•nechanisms  in  humans.   The  major route of mononitrophenol metabolism  is  un-
Jouotealy  conjugation  and  the  resultant  formation of either  glucuronide  or
sulfate conjugates.  Other  possible routes  of metabolism  include  reduction
of amino-compounds or oxidation to dihydric-nitrophenols.
    Sulfate  and  glucuronide conjugative processes are  two  of the  major  de-
toxification mechanisms  in  many  species,  including  mammals  (Quebbemann  and
Anders, 1973).   In  recent years,  4-nitrophenoi has been used  as  a  preferred
substrate for  biochemical analysis of  the  giucuronidation reaction  in  a wide
number  of  species  (Aitio,  1973; Sanchez  and  Tephly,  1974;   Ranklin,  1974;
Heenan  and  Smith,  1974;  Yang and Wilkinson,  1971).   This  use  reflects  the
simple  techniques  available for  quantitating  the disappearance  of 4-nitro-
phenol  and  the  synthesis  of  the glucuronide  conjugate.   The  relevance  of
many of these  in vitro studies towards an assessment of the  metabolic fate
of tne  mononitropnenols  in  humans  is  questionable;  thus only those  _j_n vivo
studies with direct relevance to the  metabolic fate of  mononitrophenols  in
humans or experimental  animals are discussed here.
    It has  been  known  for some time that  levels  of  the mixed function oxi-
dases and the  enzymes responsible for  conjugation of  many compounds are gen-
erally  highest  in  the mammalian liver.    Litterst,  et  al.   (1975)  assayed
liver,  lung,  and  kidney  tissue  from  the  rat,  mouse,  rabbit,  hamster,  and
guinea pig  for  standard  microsomal  and soluble fraction  enzymes  involved in
drug  biotransformation.   These studies  included  an  analysis  of glucuronide
conjugation of  4-nitrophenol  by  these  tissues-.   For  all species,  liver  was
the most  active  organ.   Kidney  and  lung  activities  were  usually  15  to  40
                                     C-14

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 percent  of that found in  liver, with  kidney  slightly Tiore active than lung.



 UDP-glucuronyl-transferase  activity  toward the acceotor,  4-nitrophenol,  was



 higher  in  hamsters and rabbits than  in other species.



    Conjugation  activity  need  not  be constant even  within  the  same  species.



 Pulkkinen  (1966b)  noted  that  sulfate  conjugation  of 4-nitrophenol  is  de-



 creased  during  pregnancy  in   rabbits.    The  author  suggested  that  large



 amounts  of estrogens  may  cause  more  protein binding, thus inhibiting the re-



 action.   In another  study  (Pulkkinen,  1966a)  it  was  noted  that  conjugation



 capacity  increases  with  age in the rat,  guinea pig,  and  man.   The  human



 fetus does  not have a very  high  capacity  to form  sulfate or glucuronide con-



 jugates  of mononitrophenols or  other  compounds.   In addition,  Moldeus,  et



 al. (1976)  noted that the  relative rate of glucuronide versus sulfate conju-



 gation  of  4-nitrophenol  may depend  on  the levels of  substrate present.   In



 in  vitro  tests utilizing  isolated rat  liver cells,  ttre  investigators  noted



 that at  4-nitrophenol  concentrations of 25 uM, the  rate of glucuronide con-



 jugation was  low and  over  75 percent of the conjugation  products were found



 to  be  sulfates.  The glucuronidation  increased  more  rapidly  than  did  the



 sulfate conjugation with  increasing  substrate conjugation.  At 250  uM,  con-



 jugation of 4-nitrophenol   with  sulfate  was inhibited  almost  completely and



more than 95 percent of the conjugates formed were found to be glucuronides.



    Robinson,  et al.  (1951)  studied  the metabolic  detoxification of  the



mononitrophenol  isomers in  rabbits.   They showed  that, with  doses of 0.2 to



 0.3 g/kg,  conjugation _in_ vitro  with  glucuronic  and sulfuric acids was almost



 complete.   Only small  amounts  (less  than one  percent)  of  the  unchanged



 nitrophenol were excreted.  With  all  three of the  mononitrophenol  isomers,



 the major  conjugation product  was  nitrophenyl-glucuronide,  which  accounted



 for about  70  percent  of  the dose.   The corresponding sulfate conjugates were
                                     C-15

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also  excreted.   Reduction of  the  nitroohenols occurre:  to  a small  extent,
the reduction  of the 4-  isomer  being  slightly greater than  that  of the 2-
and 3-  isomers.   The mononitrophenols were  also  shown to undergo  oxidation
to a very small  extent  (less than one  percent).   2-NitroDhenol yields traces
of  nitroouinol;  3-nitroohenol  yields  nitroouinol  and 4-nitrocatechol;  and
4-nitrophenol yields 4-nitrocatechol.
    A summary  of the metabolism of the  mononitrophenols is shown  in  Table
3.  Data  directly  addressing the metabolic  fate  of the  mononitrophenols  in
humans  are  not available.  However, it  is  expected that  following  exposure
to  the  mononitrophenols  humans  will   rapidly  excrete both  glucuronide  and
sulfate conjugates in the urine.
Excretion
    Data directly addressing the excretion  of  the mononitrophenols following
exposure  of humans  were  not  found  in the  literature.    However,  excretion
patterns  for 4-nitrophenol  following  human exposure  to  parathion  may  shed
some liaht on  their  elimination  kinetics.   Arterberry, et al.  (1961) studied
the pharmacodynamics  of 4-nitrophenol  excretion  following exposure  to para-
thion.   They noted  that  the  excretion  of  4-nitrophenol  in  the  urine  was
nuite rapid  "as  might be expected in  the  case of a water-soluble  metabolite
of  a  substance  which  is luickly  broken  down  by th-e  animal   organism."
4-Nitrophenol  usually had disappeared from  the urine within about  48 hours
after cessation  of  exposure.   In  a similar study  of orchard  spraymen  in-
volved  in  the  application  of parathion,  Wolfe, et  al.  (1970)  noted  that
urinary  levels  of  4-nitrophenol rose  promptly in response  to parathion  and
returned  to the nondetectable  level  after  several  days.   Myslak,  et  al.
(1971)  reported  on  the  excretion of 4-nitrophenol  from  a 19-year-old female
subject  following a  suicidal oral  dose of nitrobenzene.   Large quantities of
                                     C-16

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                                            TABLE 3

                      Urinary Metabolites of Mononitrophenols in Rabbits*
Percentage of Dose Excreted as
Nitrophenol



2-Nitrophenol
3-Nitrophenol
4-Nitrophenol

Nitro
Compounds
(N)
82
74
87

Amino
Compounds
(A) (N + A)l
3 85
10 84
14 101


Glucuronides
(G)
71
78
65

Ethereal
Sulfates
(E)
11
19
16



(G + E)
82
98
81
*Source: Robinson, et al. 1951.

1(N + A)  should be roughly equal  to  (G + E) since the  amounts  of free phenols excreted were
 very small.  Both glucuronides  and ethereal  sulphates  include  nitro  and  amino conjugates.
                                            C-17

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4-nitrooherol and  4-aminoohenol  were detected in the urine.   Elimination  of
d-nitroonenol   in   the   urine  was  exoressed   by   the  aquation   V  /y
                                                                      i.' 0
 —0 '"'OP*1
e~ ""  "  where  V   and  7   denote  the  excretion  rate  at  the  interval
time  0 and t measured in hours.   THe half-life for  excretion  corresoonded  to
about 34 hours.
    Shafik,  et  al.  (1973)  studied  the  urinary  excretion of  4-nitroohenol
following  administration of  the  pesticide  EPN.   Following oral  administra-
tion  of the  pesticide for three  days,  animals were  maintained and urine sam-
oles  collected  at  24-hour  intervals.   Three days were  required  'or  complete
excretion  of 4-nitropnenol  under these  conditions.   The foregoing  studies
indicate that 4-nitroohenol is  rapidly excreted  following its  production  _i_n_
vivo  *rom other organic compounds.
    Only one study was found that  examined excretion  of  4-nitrophenol  fol-
lowing direct administration  of  the compound.  Lawford, et al.  (1954)  stud-
ied the elimination of  various  nitrophenolic  compounds  from  the blood of ex-
oeriTiental  animals.   Elimination of  4-nitrophenol   by  the monkey following
oral  and  intraoeritoneal  doses  of  20  mg/kg body weight  was  complete within
five  hours.   Elimination  by mice,  rats,  rabbits,  and  guinea pigs  was  also
rapid.   Vost doses  were  eliminated  completely  from  the  blood  within  two
hours of administration.  Experimental  animals eliminated  4-nitrophenol  from
the blood  in the  following  descending order  of efficiency:  mouse,  rabbit,
guinea pig,  rat, and monkey.
    In  summary,  the  available  data indicate  that   the  mononitrophenols  are
excreted rapidly  via  the  urinary route and  that  total elimination  time  is
likely not  to  exceed  one  week.    The mononitrophenols  are highly water solu-
ble and  accumulation  or bioconcentration in various  tissues  is  not  expected
to occur to  a  larae  extent.   However,  much  more  data are needed to precisely
define  the  transport  distribution   and  elimination  of  these  compounds  in
humans.
                                     C-13

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                                    EFFEC~S
    Threshold  concentrations  for  odor,  taste,  and  color  for  2-nitro-,
3-m'tro-, and  4-nitrophenols  in  reservoir water  have  been  reported  in an ab-
stract  of  a oaoer  from the  Russian  literature  (Makhinya,  1964).   Reported
threshold concentrations for  2-nitroohenol were  3.83  mg/1  for  odor,  3.6 mg/1
for taste,  and 0.6  mg/1  for color.   Concentrations  for  4-nitroohenol  were
53.3,  43.4,  and  0.24 mg/1  for  odor,  taste,  and  color,  respectively.   The
values for 3-nitrophenol were given  as 339,  164.5, and  26.3 mg/1.   Accepta-
bility thresholds from  the  standpoint  of  human consumption were not  reported
by these investigators.
Acute, Subacute, and Chronic Toxicity
    Known effects  of  4_nitrophenol  demonstrated  in  animal experiments  are
methemoglobinemia, shortness  of  breath,  and  initial  stimulation  followed  by
proaressive deoression (von Oattinqen, 1949).
    Acute toxicity information for the  mononitrophenol  isomers  has  been corn-
oiled  and  presented  as  Table 4.   4-Nitrophenol  is  the  most  toxic  of  the
mononitrophenols  followed   by 3-nitroohenol   and  2-nitroohenol  in  relative
toxicity.  Toxicologic  symptoms  of  mononitrophenol  poisoning  have  not been
well described in the  literautre.   Sax  (1968)  noted that 2-nitrophenol expo-
sure produced  kidney and  liver  injury in experimental animals.   Methemoglob-
in formation as a  result  of mononitrophenol  exposure has  also  been  reported
(Patty,  1963).   Grant  (1959),  however, was  unable  to  detect  methemoglobin
formation after oral  administration  of 3-nitro- and  4-nitroohenols  to rats.
Small  inconstant  amounts  of methemoglobin  were formed after  3-nitrophenol
administration.  Smith, et  al.  (1967) were  able to  show  that  the  reduction
products  of  mononitrophenols, 2-  and  4_aminophenol,  would  produce  methemo-
globin  in  female mice.   Methemoglobin formation,   therefore,  may depend  on
                                     C-19

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                                             TABLE A

                            Acute Toxic ity of Mononitrophenol  Isomers
Species
Dose
(mg/kg)
Route of
Administration Effects
References
Frog
Mouse
Rabbit
Cat
Doq
Rat
Mouse
Guinea Pig
Doq
Rat
Mouse
Frog
Rabbit
Cat
Dog
Rat
Mouse
Rat
 300
 600
1700
 600
 100
2830
1300
 900
  83
 930
1410
  50
 600
 197
  10
 620
 470
 350
2-Nitrophenol

     s.c
     i.m.
     s.c.
     s.c.
     i .v.
     Oral
     Oral
     s.c.

3-Nitrophenol

     i.v.
     Oral
     Oral

4-Nitrophenol

     s.c.
     s.c.
     s.c.
     i.v.
     Oral
     Oral
     Oral
Lethal Dose
Lethal Dose
Lethal Dose
Lethal Dose
Lethal Dose
LD50
L050
Lethal Dose
Minimum Lethal Dose
L050
LD50
Minimum Lethal Dose
Minimum Lethal Dose
Minimum Lethal Dose
Lethal Dose
LD50
LH50
Spector, 1956
Spector, 1956
Spector, 1956
Spector, 1956
Spector, 1956
Vernot, et al. 1977
Vernot, et al. 1977
Spector, 1956
Spector, 1956
Vernot, et al. 1977
Vernot, et al. 1977
Spector, 1956
Spector, 1956
Spector, 1956
Spector, 1956
Vernot, et al. 1977
Vernot, et al. 1977
Fairchild, 1977
                                            :-20

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the capacity  of  the organism  to  reduce  the morionitroohenoIs.  As  mentioned
in  the  metabolism section  of  this document,  reduction  of the nitroohenols
does not normally occur to any large extent.
    Oqino and Yasukura (1957)  reoorted the  development of  cataracts  in  vita-
min  C-deficient  guinea  Digs  following  administration  of   4-m'troohenol.
Cataracts developed  in two  of three guinea Digs on  days  7 and 11  following
daily intraperitoneal  adminstration  of  8.3 to 12.5  mg 4-nitroohenol/kg  body
weight.    Subchronic  administration  of  4-nitrophenol  over  a  20-day   test
period produced  cataracts  while  2- and  3-nitroohenols did not.   The  authors
concluded that  the  oara-oositioning  of the  hydroxyl- and nitro- grouos  is
necessary for cataract induction.
    Several   deficiencies  in  this  study preclude  definitive  conclusions  on
the cataractogenic properties  of 4-nitrophenol.  The  investigators  failed  to
report results on  control  animals, either  totally untneated  or treated  with
the nitrophenols and a vitamin C supplement.   Thus,  it  is  oossible,  based  on
the results  reported,  to  conclude that vitamin  C  deficiency  itself  caused
cataracts in some of the animals tested.   The  small  size  of the experimental
grouos (three animals/test  compound)  also  make definitive conclusions diffi-
cult.    The  reported  conclusions   must  be  taken with  considerable  caution
based on the above considerations.
    In  contrast,  Dietrich  and Beutner  (1946)  found 2-nitro- and  4-nitro-
phenol s  to  be devoid of cataract-forming  activity   in seven-day-old  chicks.
Animals  were  fed on  a  commercial  brand  of  chick  food containing 0.25 percent
nitrophenol.  Although  cataracts  developed rapidly  (within 24 to  48  hours)
when  the animals were  fed  2,4-dinitrophenol,  no  cataracts developed within a
three-week  period  when animals were fed  the  mononitrophenol  isomers.   The
capacity for  cataract  formation  in humans  following mononitrophenol exposure
is unclear.
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    Both  2-nifo-  and  4-nitroohenol  have  been  shown  to  inhibit  oorcine
malate  denydrogenase  in  vit^o (Wedding,  et  al.  1967).   The  compounds  acted
as comoetitive  inhibitors  for NAD in the forward  direction  of  the enzynatic
reaction.  The clinical significance of these findings is unknown.
    The  ventilatory effects  of  the mononitrophenols  have been examined  in
anesthetized rats  (Grant,  1959).   Test comcounds were administered  by  stom-
ach tube:  2-nitroohenol,  60 to 120 mg; 3-nitrophenol,  20  to  45 mg; 4-nitro-
phenol,  7 to  12  mg.  Significant  increases  in  respiratory volume  ranging
from 15 to 30 percent were reported in these experiments.
    Neither  carbon dioxide output  nor  oxygen uptake were affected by  •sub-
lethal  doses of  2-nitrophenol in rats  (Cameron,  1953).   In  contrast,  oxygen
uptake was decreased  in  3-nitrophenol-treated  rats while carbon dioxide  out-
put was  increased  following  4-nitroPhenol  administration.   Rectal  tempera-
ture was  depressed in rats  receivina  any of  three  isomers.   These  results
suggest  that mononitrophenol  isomers are not  potent  uncouplers of oxidative
ohosohorylation,  in contrast  to the  chemically  similar  compound 2,4-dini-
trophenol.
    Although the  mechanism of  toxic action of  the  mononitrophenols  is  not
well  understood,  the  following  studies suggest  that  an action directly  on
cell  membranes  may  occur.    3-Nitroohenol  binds  readily  to  red  blood  cell
(R8C)   membranes.    Expansion   of RBC   ghosts  occurs  following  nitrophenoi
treatments, as measured  by  the resistance of such ghosts to hemolysis (Macn-
leidt,  et  al.   1972).   2-Nitrophenol  and  4-nitroohenol  inhibit  chloride
transport  in red  blood cells  (a metabolism-independent  process) suggestion a
direct action on  the  cell  membrane (Motais, et al.  1979).   Further informa-
tion on  the  acute or  chronic  toxicity  of the  mononitrophenols  to  humans w
not found.
                                     C-22

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     The  National  Institute  for  Occupational   Safety  and  Health  (NIOSH),
 recently  undertook  a health  hazard  evaluation  determination at  the  request
 of  an  erioloyee  of  the Boeina Company who had routinely handled 4-nitrophenol
 (Sutler  and  Sodner,   1973).   A  15  percent  solution  of  4-nitrophenol  and
 methylphenol  was  painted  on the exposed cork surfaces of  the Minuteman Mis-
 sile before arrival  at the  assembly plant.  If  the  surface was  damaged  in
 transit  it  was  necessary  to  apply small amounts  of  the 4-nitrophenol  solu-
 tion to  the repaired areas of cork.   The worker in auestion was  engaged  in
 such touch-up operations.   Workers routinely wore an  organic vapor cartridge
 respirator,  a face shield,  cotton  gloves, rubber gloves,  and were completely
 covered  with protective clothing.   The employee complained of fatigue,  joint
 pain,  abdominal  cramos and  diarrhea,  and  attributed  these symptoms to  his
 exposure  to both the  treating  solution  and the  dried cork  impregnated with
 4-nitrophenol durina  his  work as a mechanic.   Medical~examination failed  to
 detect 4-nitrophenol  in the  urine  but  revealed  a complete absence of the im-
munoalobins  IqA and  IqQ in  the employee.  Based on medical judgement  and the
 existing  literature,  the  study concluded that  the employee's condition  stem-
med from the  lack of  IgA  and IgD and that this  deficiency was  not caused  by
 exposure to 4-nitrophenol.
    Gabor,  et al.  (1960)  reported a uniaue  effect  of 2-nitrophenol  on  blood
platelet levels.  When 31  rats were  administered 2-nitrophenol  by intraperi-
 toneal  injection  at  1 mg/kg  body weight,  the  platelet  count  increased sig-
nificantly.   Even at  doses  of 0.1 mg/kg a similar  effect  was  produced.  Ad-
ministration  of  3-nitro or 4-nitrophenol did not produce  a  rise in platelet
 levels.   Additional  data  are not  available to  explain   this  unioue  pheno-
menon,  nor  is the clinical significance of these findings  known.
                                     C-23

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    A  reoort  from  the  Russian  literature  (Makhinya,  1969)  reports  that
2-nitro-,  3-m'tro-,  and  4-nitroohenols possess  distinct  cumulative  proper-
ties.    Chronic  administration  of  any of  the  mononitroohenols  to  mammals
caused alterations  of  neurohumoral  regulation  and  patholoaical changes  in-
cluding colitis, enteritis,  hepatitis,  gastritis, hyperplasia  of  the spleen,
and neuritis.   Limiting  doses for  the  disruption  of conditioned reflex  ac-
tivity were  establised as  0.003  mg/kg (equivalent  to  0.006 mg/1  of water)
for 2-nitroohenol  and  3-nitroohenol  and 0.00125 mg/kg (eouivalent  to 0.0025
•ng/1 of water)  for 4-nitrophenol.  Unfortunately a report of  this  study was
available in abstract  form  only.  Details of  the experiment  including animal
species, mode  of administration,  duration  of  the  treatment, and a complete
description of  the observed biological  effects, were not reported.   The  re-
sults  must  be  considered  Questionable  until  evaluation of  the experimental
protocol is possible.
Synergism and/or Antagonism
    Only one  report was found  dealing  with possible synergistic effects of
the mononitroohenols.  Cairns,  et al.  (1976)  studied  the  effects  of  a  sub-
lethal exposure  to zinc  and subseouent toxicity of  4-nitrophenol  to snails,
Goniobasis  livescens.   Snails  were exposed  for 96 hours  to  two  sublethal
concentrations  of  zinc (1.54 mg/1 and  3.08 mg/1 corresponding  to 0.2 and 04
of  the  48-hour LC50 dose,  respectively)  followed  by an acutely  lethal  dose
of  4-nitrophenol  (1,000  mq/1).   A  significantly reduced  survival  time  fol-
lowing  exposure to 4-nitrophenol  was  reported.    In  a  second  experiment,
snails  were  exposed  to  sublethal  levels  of  4-nitrophenol  (13.2  mg/1)  and
subjected  to  a lethal temperature  shock  96 hours  later.   A significant de-
crease  in  the  median survival time  of  the   snails  during the  temperature
shock  was  noted.   The applicability of these data to humans  or  mammals is
unknown.   Data  regarding  synergistic  or   antagonistic  effects  of  the  the
•nononitrophenols in mammals were  not  found.
                                      C-24

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Teratocenici -•/
    Pertinent  information  could not  be  located in the  available  literature
regarding teratogenic properties of the mononitrophenols.
Mutaoem'c'ity
    Szybolski   (1953)  tested   the  three  mononitrophenol  isomers  for  their
ability  to  induce  streptomycin-independence  in streptomycin-independent  E_.
co 1 i.  All three isomers gave negative results.
    Buselmaier, et al.  (1976)  tested  4-nitrophenol  for mutagenic  activity  in
mice with the  host mediated  assay  and the dominant lethal  method,  using Sal-
monella  typhimurium  G46  His",  Serratia  marcescens  a21 leu~, and  Serratia
marcescens a31  His", as indicator organisms.   Spot tests j_n  vitro  were  al-
so performed.   Mutagenic activity was not demonstrated.
    4-Nitropnenol  also  failed  to  induce mutations  in   Salmonel1 a both  with
and without microsomal activation (McCann, et al. 19751^
    Fahrig  (1974)  demonstrated a weak mutagenic activity  when 4-nitroonenol
was  tested  for mitotic  gene  conversion  in  Saccharomyces cerevisiae.   This
test  system  allows  the detection of a  genetic alteration whose  molecular
mechanism is  presumably based  on  the formation of single-strand breaks  of
DNA.
    Adler, et  al.  (1976)  used the  difference  in  growth  inhibition  of wild
type  Proteus  mirabilis  and the  corresponding  repair-deficient strain  as  an
indication of  DNA  damage.   4-Nitrophenol  showed some evidence of  DNA damage
in this system.
    Effects on mitosis  and  chromosome fragmentation  have been  reported  in
plants.  Sharma and  Ghosh  (1965) examined the  mitotic effects  of  the mononi-
troohenol  isomers  in  root tips  of  A11i urn  cepa.   Inhibition  of  mitosis  in
root  tips  was  reported   for  all  three  mononitrophenol   isomers  but  only
4-nitrophenol   induced  detectable  chromosome  fragmentations.   Amer  and  Ali
                                     C-25

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^Ia69j  studied  the  ejects  of  2- and  i-ni trooheno 1 s  on  the  "ate-'a1  '•oot
mitoses  of  V i c • a  f aba  seedlirgs.   The  -nitotic  Index  was  reduced  at  con-
centrations  of  these compounds  ranging  from 0.025  oercent to O.I  percent.
Induction of anaohase  bridges by  both isomers was  noted but  (in  agreement
with the work of  Levin  and  Tjio  (19*3) with A11ium ceoa)  chromosome fragmen-
tation was not  detected.  The relationship  of these  changes  in  plants  to al-
terations in mammalian  cells  has not   been  established.   Based  on  the  avai"-
able data, the mononitrophenols  do  not appear  to pose a  mutagenic  hazard  to
humans.
Carcinogenicity
    Data on  the possible carcinogencity  of  the  mononitrophenols are scant  in
the  literature.   Soutwell   and Bosch   (1959) have  studied  the  ability  of  a
number of  phenolic compounds to promote  tumor formation  on mouse  skin  fol-
lowing a  single  initiating  dose  of dimethylbenzanthracene.  Although  phenol
itself  has  demonstrated a  promoting   capacity  in  this  system, both  2- a^c
4-nitroohenols  failed  to  promote tumor  development  in mice.   No  othe»-  data
on possible carcinogenic potential  of   the mononitrooherols were found.
    4-Nitrophenol  has  been  selected by  the National Cancer  Instritue (NCI)
for testing under the Carcinogenesis Bioassay Program.
                                     C-26

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                                OINITSOPHENOLS
Mammalian Toxicology ana Human Health Effects
                                 INTRODUCTION
    Six  isomeric  forms of dinitrophenol  are possible,  distinguished  by  the
position  of the  nitro- groups  on  the  phenolic  ring.   Of the  six  possible
dinitrophenol  isomers,  2,4-ainitrophenol  is by far the most  important.   The
most  recent  production figure  for  2,4-dinitrophenol  is  363,000 Ibs.  reported
by  the  U.S.  International  Trade Commission  (1968).   Approximate  consumption
per year  is  estimated  at  1,000,000 Ibs.  (Howard,  et al. 1976).  2,4-Dinitro-
phenol  is  used primarily as  a chemical  intermediate  for the  production  of
sulfur  dyes, azo dyes,  photochemicals,  pest control agents,  wood  preserva-
tives,  and   explosives  (Matsuguma,  1967;   Perkins,  1919; Springer,  et  al .
1977a,b).
    Production  figures and usage  data  for  the remairnng  five dinitrophenol
isomers are  not available.   It is  reasonable  to  assume  that  production  ana
usage of these  compounds are extremely limited in the United  States.
    Commerical  synthesis of 2,4-dinitrophenol  is  accomplised  by the  hydroly-
sis  of 2,4-dinitro-l-chlorobenzene  with  sodium   hydroxide  at  95  to  100°C
(Matsuguma,   1967).   As  a result  of  the   use  pattern  of  2,4-dinitrophenol
(2,4-DNP) the major source for  environmental release  of 2,4-DNP is  likely to
be from production  plants  and chemical  firms where  the compound is  used  as
an  intermediate.    It   is  possible  that  2,4-DNP   may  also  be produced  via
microbial  or photodegradation  of  compounds which  contain  the  nitropnenol
moiety, such as parathion  (Gomaa  and Faust,  1972).    2,4-DNP  has also  been
identified  as   a  impurity in  technical  preparations  of  the  herbicide  DN?
(2-isooropyl-4,6-dinitrophenol) by Mosinska  and Kotarski (1972).
    The physical  and   chemical  properties  of  the dinitrophenol  isomers  are
summarized in Table 5  and Figure 2.
                                     C-27

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                                    TABLE  5
                     Properties of Dinitrophenol Isomers*
Isomer
2,3-Oinitrophenol
2,4-Dinitrophenol
2,5-Oinitrophenol
2,6-Oinitrophenol
3,4-Dinitrophenol
3,5-Oinitrophenol
m.p.
CO
144
114-115
(sublimes)
104
63.5
134
122-123
K
(at 25*
1.3 x
1.0 x
7 x
2.7 x
4.3 x
2.1 x
C)
10-5
10-4
10-6
10-4
10-5
10-4
Water
Solubility
(9/1)
2.2
0.79
0.68
0.42
2.3
1.6
Density
1.681
1.683


1.672
1.702
*Source: Harvey, 1959; Windholz, 1976; Weast, 1975.
                                       C-28

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               MO,
                                    OH
                                     NO,
2,3-dinitrophenol
2,4'-dinitrophenol
2,5-dinitrophenol
                                     OH
                                     OH
2,6-dinitrophenol
3,4-dinitrophenol
3,5-dinitrophenol
                                   FIGURE  2
                                Oinitrophenols
                                      C-29

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                                   EXPOSURE
Ingestion from Water
    No data  were  available regarding  human  exposure via ingestion  of  dini-
troohenols from water.
    The enhancement of  biological  waste water treatment by  2,4-ONP  has been
examined (Shah, 1975; Shah, et  al.  1975).   Addition of 0.92  mg/1  2,4-ONP to
waste water  systems  results  in an increase  of  85  percent  in waste degrading
rate and a decrease of  70  percent  in cell  growth.   Shah, et  al.  (1975) note
that the optimum concentration  for 2,4-DNP in this system  (0.92 mg/'l)  is un-
dersirably high from  the standpoint  of current  Federal  effluent  regulations
but  that  the compound  is  completely  eliminated  by adsorption on  activated
carbon which  Generally  follows biological  treatment of waste waters.   It is
not  known whether  this treatment  method  is currently used in  the  United
States.  Theoretically,  such  usage might result in  2,4-ONP  contamination of
surface waters.
    Games and Hites  (1977)  detected  dinitrophenol  (isomer  not identified) in
the effluent  waters  of  a dye  manufacturing  plant.  Dinitrophenol  at  300 to
400  ug/1  was  detected   in  raw waste  water, prior  to  biological  treatment.
The final plant effluent contained dinitrophenol at  42  to  270 ug/1.  Mud and
river water  samples  downstream from  the effluent  point were analyzed  by gas
chromatoqraphy/mass  spectrometry.   Dinitrophenol  was  not  detected  in  these
samples.
    The persistence  of  dinitroohenol  isomers in ambient waters  has not  been
well studied.  A number of  investigators have studied the bacterial degrada-
tion  of  the  dinitrophenols  utilizing  acclimated  populations of microorgan-
isms.  Phenol-adapted bacteria obtained from garden soil,  compost, and river
                                      C-30

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mud  degraded 2,--dinitropreno1  in  seven to  ten  days (Tabak, et  al.  196^;.
2,5-DinitroDhenol was  degraded very slowly in this system.   2,4-,  2,5-,  and
2,6-Dinitrophenols  were  tested for biological degradability  by  an activated
sludge  culture  obtained  from a sewage treatment plant  (Fitter,  1976).   2,5-
Dinitro- and 2,5-dinitrophenols were  not degraded  in  this system although 35
percent  removal  of  2,4-dinitrophenol  was  achieved  within 20  days.   Further
degradation  of  2,4-dinitrophenol  did  not occur   in  this system,  however.
Bacteria  isolated  from  parathion-treated flooded  soil   (Sudhakar-Barik,  et
al.   1976)   degraded   2,4-dinitrophenol   after  an   exceptionally  long  lag
period.  Nitrite was  produced  only  in  trace amounts  after  25   days.   Even
after 50 days, only eight percent nitrogen was accounted for as nitrite.
    The  available data indicate that  dinitrophenols  are  susceptible  to par-
tial  degradation by certain  microorganisms.   Of the dinitrophenol  isomers,
2,4-ONP appears  to be  most easily degraded.   It  may be speculated that  dini-
trophenols  will  be  subject  to microbial attack  in environmental  situations
where  acclimated mirobiological  populations   exist  (e.g., sewage  treatment
plantsK  The  persistence of dinitrophenols  in  the environment  where accli-
mated microbial populations do not exist is speculative.
Ingestion from Food
Pertinent data  could  not  be  located  in the   available  literature regarding
exposure to dinitrophenols via ingestion of food.
    No  measured  steady-state bioconcentration factor  (BCF)  is available for
any nitrophenols, but  the equation "Log BCF  »  (0.85  Log P)  - 0.70" can be
used  (Veith,  et al. 1979)  to estimate  the steady-state  BCF  for  aquatic or-
ganisms  that  contain  about  7.6 percent  lipids  (Veith,  1980)  from  the  oc-
tanol/water partition  coefficient  (P).   The  log P  values  were obtained from
Hansch  and  Leo   (1979)  or  were calculated  by the  method  described  therein.
                                     C-31

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The adjustment factor of  3.0/7.5  =  0.295  is  jsea to adjust tne est;~ateJ  3CF
from the  7.6  percent  lipids  on which the equation  is  basea to the  3.0 oer-
cent lipids that  is  the weighted average for  consumea  fish and  shellfisn  in
order to  obtain  the  weighted average bioconcentration  factor  for  the  edible
portion of all freshwater and  estuarine  aquatic  organisms  consumed oy  Ameri-
cans.
Inhalation
    Dinitrophenol isomers may  be  produced in  the atmosphere through a  photo-
chemical  reaction  between benzene  and   nitrogen monoxide.   Nojima,  et  al.
(1975)   irradiated  a  combination of  benzene  vapor and  nitrogen  monoxide  for
five hours  with  a  xenon lamp  and  characterized   the following  resulting
photochemical   products:  nitrobenzene,   2-nitrophenol,  4-nitrophenol,  2,4-
dinitrophenol,  and   2,6-dinitrophenol.   The   authors   suggested  that   these
nitro-  compounds may be the  cause of the  characteristic symptoms of serious-
ly stricken victims  of  photochemical smog in  Japan, which include headache,
breathing difficulties,  vomiting, rise  in  body temperature  and  numoness  in
the  extremities.   In the  absence  of  monitoring data it  is impossible  to
estimate  the extent of  human exposure to dinitrophenols as a result of their
photochemical  production in the atmosphere.
Dermal
    2,4-ONP is  rapidly absorbed  through the intact  skin (Gleason,  et  al.
1969).   Although no direct information on the other dinitrophenol  isomers is
available, it  is  reasonable to  suppose  that  dermal absorption  will readily
occur with these  compounds  as  well.  Since  2,4-ONP is used  primarily as  a
chemical  intermediate,  dermal  exposure  is expected  to  occur most often in an
inductrial setting.   2,4-ONP  is  also used  occasionally  as  a spray against
aphids   and mites,  as a fungicide for certain molds and  mildews,  as  a weea
                                     C-32

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killer,  and  as an  ingredient  in  some wood preservative  formulations  (Glea-
son, et  al.  1969).  Dermal  exposure  to humans  may  occur among  individuals
handling 2,4-DNP  in these  aoolications.   Data on the  importance  of  the  der-
•nal exposure route of dinitrophenols in humans are not available.
                               PHARMACOKINETICS
Absorption
    Absorption  of dinitroohenol   (isomer  unspecified) through  the skin  and
following inhalation occurs, readily (von Oettingen,  1949).
    Gehring and Buerge  (1969b) reported that  2,4-ONP  is  absorbed  very  rapid-
ly  by  ducklings  and rabbits  followina  intraperitoneal  adminstration.    In
fact,  imnature  rabbits  absorbed  the administered DNP  so  rapidly  that  an ab-
sorption constant  could  not  be calculated from  the data.  ONP  concentration
is serum peaked within five minutes of administration.
    Other Quantitative  information on  the  rate of absorption  of the  dinitro-
ohenol  isomers was not found.
Distribution
    Blood  levels   of the  dinitrophenols  rise  rapidly following  absorption
(Gehring and Buerge,  1969b;  Harvey,  1959) suggesting  that the  dinitrophenol
isomers  are  transported by  the blood regardless of  the  mode of  absorption.
2,4-ONP binds  to  serum  proteins  following intraoeritoneal administration  to
rabbits and ducklings.   Early after the administration of 2,4-ONP,  the  con-
centration of free  DNP  in  serum  is much  greater  than the bound form,  and  at
later times the reverse is true (Gehring and Buerge,  1969b).
    Based on the  available data,  the  dinitrophenol  isomers do  not  appear  to
be stored  to  any  significant extent  in the  tissues  of human  or experimental
animals  followino absorption.  Gisclard  and  Woodward  (1946)  unsuccessfully
attempted  to  extract 2,4-dinitrophenol or its metabolites from  the  tissues
of two human victims of fatal  intoxication.
                                     C-33

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    It  seems  likely,  based  on  the  short half-lives  of  these compounds  in
mammals, that  the  large majority of  any dose will  be rapidly excreted  via
the urine.  On  the other  hand,  von  Oettingen (1949)  reported  both  dinitro-
phenol  (unspecified  isomer)   and  aminonitrophenol  in  the  liver,   kidney,
brain,  blood,  and  spinal  fluid  of  dogs after fatal doses  of dinitrophenol.
Recent  work on  the tissue distribution  of the dinitrophenols  following  ab-
sorption in mammals was not found.
Metabolism
    In  a  study of  the munitions industry in  France  (Perkins, 1919)  it  was
reported  that  the  urine  of  men  fatally  poisoned  by  2,4-DNP  contained:
2-amino-4-nitrophenol,  4-amino-2-nitrophenol,  diamino-phenol,  and a  number
of nitrogen compounds  resulting  from  a combination  of two  molecules  of  ami-
nonitrophenol   or  of  diaminophenol.    It  has  frequently  been  reported  that
2-amino-4-nitrophenol  invariably exists  in  the  urine of  persons  suffering
from  serious  intoxication by  2,4-DNP.   Williams  (1959) stated  that  2,4-DNP
is excreted in mammals  in  the  following forms: partially  unchanged;  partial-
ly  conjugated  with   glucuronic  acid;  reduced   to  2-amino-4-nitrophenol,
2-nitro-4-aminophenol  and probably   2,4-diaminophenol.   Rats  orally  dosed
with  2,4-DNP  at 1.5  to 12 rag/kg excreted both free  dinitrophenol  (78  per-
cent)  and 2-amino-4-nitrophenol (17 percent)  (Senszuk,  et al. 1971).
    Although  the jji vitro metabolism  of 2,4-dinitrophenol  has not  been  ex-
tensively studied in mammalian systems,  Parker (1952)  examined the enzymatic
reduction  of  2,4-DNP  by  rat  liver  homogenates  and  found 4-amino-2-nitro-
phenol  to  be  the   major metabolite.    The metabolite 2-amino-4-nitrophenol
comprised less than  10  percent of  the total  metabolites formed; 2,4-diami no-
phenol  was  found  in  trace  amounts.   Presumably  the  latter  metabolite  was
formed  from  the reduction of  the remaining  nitro- group  of one  of  the  two
aoove compounds.
                                     C-34

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     In  contrast,  Eiseman,  et a!.  (1974)  reported  that  2-amino-i-m'troonenoi
was  the  major  metabolite (75 percent of  total  ami no).   In the  latter  study
4-amino-2-nitrophenol  was  formed  in  considerably smaller  amounts  (23  per-
cent) when  ?,4-QNP  was  enzymatically  reduced in vitro by  rat  liver  homoaen-
ates.   These  investigators  also detected only  traces of  diaminoohenol  indi-
catinq that  it  nay be a secondary  reduction  product  as suagested by  Barker
(1952).  A  precise  definition of  the  metabolic  fate  of  the dinitroohenoIs in
humans awaits further investigation.
Excret ion
    Data on the elimination  kinetics  of  the  dinitrophenols or  their  metabol-
ic products  in  humans were not  found.   Edsall  (1934) stated:  "Judging  from
the  metaoolic  response, DNP  appears  to  be  eliminated  entirely in  three or
four days; in the presence of liver or kidney damage  it  is possible  that the
drug will be retained  over  a  longer oeriod."  Information on  the elimination
kinetics of  the dinitrophenols  from  experimental  animals  is  also scant  in
the literature.
    Genring and  3uerge  (1969b)  have  developed  enuations which  describe the
elimination of 2,4-DN?  from  the serum of ducklings, mature rabbits,  and im-
mature  rabbits following  intraperitoneal  administration  of  the  compound.
Serum levels of 2,4-ONP  in  the  mature rabbit declined to  less than  one per-
cent of  their  original  high values  within  seven hours.   Twenty-four  hours
were renuired before the serum  levels  in  the immature rabbit  declined  to two
percent  of  their  orignial   values.   Ducklings  eliminated 2,4-DNP from the
serum over a similar time frame (96 percent elimination  in 24  hours).
    Lawford. et  al.  (1954)  also studied the elimination of  various  nitro-
phenolic  compounds  (including  2,4-dinitrophenol).   Elimination  from  the
blood of  mice,  rabbits, guinea  pigs,  rats,  and monkeys  was  complete  within
                                     C-35

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30 hours.   Harvey  (1959)  calculated the  elimination  rates of all six  dini-
trophenol isomers from  the  blood  of mice and  rats  following a single  large
dose given  intraoeritoneally.   Data are  presented in Table  6.  The  data  de-
velooed by  these  investigators  must be  taken  with  caution  since  the  actual
elimination of  the  dinitrophenols or  their  metabolits  in urine was not  di-
rectly measured.   In  view of the lack of  data suggesting  concentration  of
the  dinitrophenols  in  mammalian  tissues and  the high  water solubility  of
these compounds, their  elimination  via the  urine may  be  a  rapid  process  in
humans.
                                    EFFECTS
Acute, Subacute, and Chronic Toxicity
    All of  the  dinitrophenol  isomers are potent metabolic poisons.  Most of
the  literature  available deals  with  2,4-dinitrophenol  since this  compound
has been used extensively for more  than  70  years.   A number of excellent re-
views on the uses, chemistry, mode  of  action,  and mammalian  toxicity of 2,4-
dinitrophenol   are  available  (Edsall,  1934;  Metcalf,  1955;  Homer,   1942;
Simon, 1953; Slater,  1962;  Parascandola, 1974;  Howard,  et al. 1976)  and no
attempt will be made to duplicate the information found in these documents.
    2,4-Oinitrophenol  is  considered a classic  uncoupler of  oxidative  phos-
phorylation and  is  widely used by  biochemists to determine whether a given
biochemical  process is  energy dependent.  Hence,  an  enormous body of litera-
ture has been generated dealing with the biochemical  effects of 2,4-dinitro-
phenol on  cellular and biochemical  processes  both _in_ vivo  and j£  vitro.
Only those  studies  with direct  relevance to the  acute or chronic effects of
the dinitrophenols on humans are reviewed in this document.
    The toxic action  of the dinitrophenols  is  generally  attributed  to their
ability to  uncouple oxidative  phosphorylation.  These  compounds  prevent  the
                                     C-36

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                                TABLE  5

   Elimination  Rates  of  Dinitroohenol  Isomers  from the Slood of Mice
        and  Rats  Following  a  Single  Large  Intraperitoneal Dose*
Isomer

2,3-Dinitrophenol
2,4-Dinitrophenol
2,5-Dinitrophenol
2,6-Dinitrophenol
3,4-Dinitrophenol
3,5-Dinitrophenol

2,3-Dinitrophenol
2,4-Dini troohenol
2,5-Oinitrophenol
2,5-Dinitrophenol
3,4-Dinitrophenol
3,5-Dinitrophenol
Dose
(mg/kg)
MICE
90
20
ISO
30
60
30
RATS
90
20
90
25
90
30
Half-time for
El imination
( m i n . )

2.7
54.0
3.3
233.0
3.5
2.7

12.5
225.0
13.0
210.0
11.5
2.1
*Source: Harvey, 1959.
                                  C-37

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utilization of the energy orovided by cellular  respiration  and  glyco'vsis  b/
inhibiting the formation of  high energy phosphate bonds.  All  energy  depen-
dent biochemical  orocesses  are  therefore affected by the action  of  the  com-
oounds (Metcalf,  1955).  The  large  number of clinical effects  attributed  to
dinitrophenol  toxicity  result   essentially  from   the   shortcircuiting  of
metabolism in cells which absorb sufficient dinitrophenol.
    All  six  dinitroohenol  isomers are  ootent uncouplers of  oxidative  phos-
phorylation.  The relative potencies of  the six  dinitrophenols  in uncoupling
phosphorylation  in  rat  liver  mitochondria were found  to  be  (in  declining
order):  3,5-> 2,4->  2,6- =  3,4-> 2,3- =  2,5-dinitrophenol  (Burke and  White-
house,  1967).    3,5-Dinitrophenol  is approximately  five times  more  potent
than 2,5-dinitophenol as measured in this  system.  The relative _in_ vivo  tox-
icities  of the dinitrophenol  isomers have been  determined by a  number  of in-
vestigators  (von  Oettingen,  1949;  Harvey, 1959; Cameron, 1958; Grant,  1959;
Levine,  1977) and the order  of  relative potency of  the  isomers  determined  in
these  investigations  frequently differs  form the order developed  by  Burke
and  Whitehouse   (1967).   Several explanations   for  these  discrepancies  are
possible: (1) differential tissue absorption  of  the  isomers  or  (2) different
metabolic detoxification mechansims  for  the  isomers  or  (3)  the  presence  of
cellular  or  biochemical effects unrelated to   the  uncoupling  of  oxidative
phosphorylation.  Resolution of  this Question awaits further investigation.
    At  concentrations  higher  than  those  necessary to uncouple  oxidative
phosphorylation,  a number of  inhibitory effects of  the dinitrophenol isomers
on certain enzymatic reactions may occur.
    Both  2,4-dinitro- and  3,5-dinitroohenol   inhibit porcine  heart  malate
dehydrogenase in  vitro  (Wedding, et al. 1967).  Inhibition  of the reaction
                                     C-38

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occurred -at nitrophenoi concentrations  10  to  100  times  tnose  causing  jn
ling,  and  resulted from  a  competitive  inhibition  with NAD  in the  forward
direction of  the  malate dehydrogenase  reaction.   In  a similar  study  Stock-
dale and Selwyn  (1971)  reported in the  j_n_ vitro  inhibition of  both  lactate
dehydrogenase  and  hexokinase  by  2,4-dinitro-,   2,5-dintro-,   2,6-dinitro-
phenols.
    The  dinitrophenols  may  also  act  directly on  the cell  membrane,  thus
causing toxic effects oh ceils which do  not depend  on  oxidative phosphoryla-
tion  for their energy  requirements.    2,4-Dinitro-,  2,5-dinitro-,  and  2,5-
dinitroohenols inhibit oassive oermeabi1ity to chloride (a  metabo lically  in-
dependent process) in red blood cells  (Motais, et al.  1978).
    Acute toxicity  information  for the dinitroohenols  has been  compiled  and
oresented in Table 7.
    Numerous occasions  of  human poisoning  by 2,4-DNP  -have been  reported  in
the literature.   The  earliest  cases of  fatal   2,4-DN?  intoxication  relate  to
its usage as a component of  explosives during World War I.   Thirty-six cases
of  *atal  occupational  dinitrooh^nol  poisoning occurred  among   employees  of
the munitions  industry  in France  between  1916 and 1918  (Perkins,  1919).   A
literature  review by  von  Oettinqen  (1949)  revealed  27  reported  cases  of
fatal  occupational  dinitrophenol  poisoning  in   the  United  States for  the
years 1914 to 1916.
    Gisclard and  Woodward  (1946)  reported two fatal  cases  of  dinitrophenol
poisoning during manufacture of  picric  acid where 2,4-DNP was produced as  an
intermediate.   Swamy  (1953)  describes  a case  of   suicidal  poisonina  by
2,4-DNP.
    Early in the  1930s, 2,4-dinitrophenol  was widely  recommended as a treat-
ment for obesity.  Oinitrophenol  was  received with overwhelming  popularity
                                     C-39

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                TABLE 7



Acute Toxicity of Oinitrophenol  Isomers
Soecies
Oose
(mg/lcg)
Route of
Administration
Effects
References
2,4-Dinitrophenol
Rat
Rat
Rat
Rat
Rat
Mouse
Mouse
Guinea Pig
Rabbit
Rabbit
Rabbit
Ooq
Dog
Doa
Dog
Dog
°iqeon
Pi aeon
Human

Human
Human
25
35
30
23.5
31
36
26
700
30
200
100
30
20-30
22
20
30
7
15-20
40 mg/m^

1-3 a
d.3 '
s.c.
i.o.
Oral
i.p.
i.p.
i .p.
i.p.
Dermal
s.c.
Oral
i.p.
UNK
Oral
s.c.
i .m.
i.v
i.m.
i.v.
Inhalation

Oral
Oral
LD50
l°50
L05Q
L050
LDlOO
LD50
i-050
Lethal Oose
LD50
LOso
Lethal Oose
MLD
L050
LD50
L050
LD50
Lethal Dose
Lethal Dose
Lethal Cone.

Lethal Dose
Lethal Dose
von Oettingen, 1949
Harvey, 1959
Spector, 1956
Lawford, et al. 1954
Gatz and Jones, 1970
Harvey, 1959
Lawford, et al. 1954
Spencer, et al . 1948
von Oettingen, 1949
Spector, 1956
Spector, 1956
Harvey, 1959
Spector, 1956
Spector, 1956
Spector, 1956
Spector, 1956
Spector, 1956
Spector, 1956
MacBryde and Taussig,
1935
Sax, 1968
Geiger, 1933
2,3-Oinitrophenol
Rat
Mouse
Dog
190
200
1000
i.p.
i.o.
UNK
L050
LD50
MLD
Harvey, 1959
Harvey, 1959
Harvey, 1959
                  C-40

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                                  TABLE  7  (continued)

                        Acute  Toxicity of  Dinitrophenol  Isomers
Species
 Dose
(mg/kg)
   Route of
Administration
Effects
   References
Rat
Mouse
Dog
Rat
Mouse
Dog
150
273
100
38
45
50
                                  2,5-Dinitrophenol
i.D.
i.p.
UNK
LD50
MLD
                                  2,6-Dinitrophenol
i.p.
i.p.
UNK •
LD50
LD50
MLD
                                  3,4-Oim'trophenol
Harvey, 1959
Harvey, 1959
Harvey, 1959
Harvey, 1959
Harvey, 1959
Harvey, 1959
Rat
Mouse
Dog

Rat
Mouse
Dog
s.c.
i.p.
i .m.
i.v.
UNK
MLO
98
112
500

45
50
500
subcutaneous
intraperitoneal
intramuscular
intravenous
unknown
minimum lethal dose
i
i
U

i
i
U






.p.
• P.
NK
3?
.p.
• P.
NK






LDSO

MLO
5-Dinitroohenol
LDSO

MLO






Harvey,
Harvey,
Harvey,

Harvey,
Harvey,
Harvey,






1959
1959
1959

1959
1959
1959






                                         C-41

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(Homer,  1942)  as  a slimming agent in spite of warnings  of  harmful  sicie  ef-
fects  caused  by disruption  of  the metabolic  rate.   It  was estimated  that
during the  first  15 months follwoing its  introduction, 100,000  persons  took
the drug  for  weight reduction (Homer,  1942).   More  than  1,200,000  capsules
of  0.1  g each  were dispensed from a single clinic  in San  Francisco.   More
than 20  drug  houses offered to supply  both  dinitrophenol and mixtures  con-
taining  the drug.   Many  of  these  remedies  could be procured  without  pre-
scription  and  with no  further  directions than  to take  "one capsule  three
times daily after  meals."   In view  of this widespread and uncontrolled usage
of  the  compound,  it  is  not surprising  that  both  toxic  side  effects  and
fatalities  resulted.  Horner  (1942)  reported  a total  of  nine  deaths result-
ing from the use of dinitrophenol  as a slimming agent.
   . Parascandola  (1974)  reviewed  the  history  and public concern which  de-
veloped  over  dinitrophenol in  the  United States.  An  article  appearing  in
Newsweek  (1933) entitled  "Diet  and Die  with Excess Alpha Dinitrophenol"  was
typical of public  concern  generated by misuse  of  dinitrophenol.   In  the  wake
of  reports  that cataract  development  in  humans attributable to dinitrophenol
was occurring, the drug was finally withdrawn  from use in  1937.
    The  toxic manifestations  of dinitrophenol  exposure  as reviewed  by Horner
(1942),  included   subacute symptoms  such as  gastrointestinal  distrubances
(nausea,  vomiting, colic,  diarrhea,  anorexia),  profuse  sweating,  weakness,
dizziness,  headache,  and   loss  of  weight.  Acute  poisoning has resulted  in
the  sudden onset   of  pallor,  burning  thirst,  agitation,   dyspnea,  profuse
sweating,  and  hyperpyrexia.   Intense and  rapid  onset of  rigor  mortis  after
death has  also  been described.  A  physician who  ingested  a fatal overdose of
dinitrophenol   (estimated  at  2.5  to 5  g) was literally  "cooked  to  death"
(Geiger, 1933).  Rectal  temperature at death  exceeded  110'F.
                                     C-42

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     Perkins  (1919)  made  the interesting observation  that postmortem examina-
tion  of  dinitrophenol  victims  demonstrated  no  characteristic  lesions.   Acute
edema  of the  lungs  was  mentioned  but  was  believed  to  be secondary  to  the
toxic effects  on the  vasomotor  system.   Microscopic  lesions of  the liver  and
kidney cells were inconstant and typical changes were lacking  elsewhere.
    Spencer, et al.  (1948)  studied  the  chronic toxicity  of 2,4-dinitrophenol
in  rats.  Male rats  were  fed  diets  containing 0.01,  0.02,  0.05,  0.10,  or
0.20  g  of  2,4-dinitrophenol  per  100  g of food.    Rats  were maintained  on
diets  containing   2,4-dinitrophenol  for six   months  and  both  hematological
pathological investigations on  surviving animals were performed.   Hematolog-
ical  examination   included  erythrocyte counts,  hemoglobin  concentrations,
leukocyte counts,  differential  counts,  and bone marrow  counts  at  autopsy.
Both  gross   and microscopic  examination  of  liver,  kidney,  spleen,  lung,
heart,  adrenal,  pancreas,  and  stomach  tissues  were  also performed.   Rats
maintained on  diets containing  0.02 percent 2,4-DNP  (corresponding to 5.4 to
20 mq/kg body weight/day) grew  at a normal  rate  and  the  investigators failed
to detect discernible  ill  effects of pathological changes  at  autopsy.   Sim-
ilarly,  pathological changes were not  found upon microscopic examination  of
tissues  from  rats  receiving diets  containing  0.05  percent 2,4-DNP (corres-
ponding  to 13.5 to  50  mg/kg/day)  although growth of these rats fell  five to
ten percent below that of  the  controls  throughout  the six-month experimental
period.  At autopsy  the  only changes observed  in  these  animals  were  a very
slight  deoletion  of  body  fat  and  a  very  slight   increase  in  the  average
weight of the  kidneys.   At  higher doses of 2,4-dinitrophenol in  their diets
(54 to  200  mg/kg  body weight/day) rats  occasionally died  and  survivors lost
weight  rapidly.   Examination of  surviving  animals  revealed  marked  emacia-
tion, an empty gastrointestinal tract,  a slightly enlarged and dark spleen,
                                     C-43

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and undersized  testes.   Microscopic  examination  showed  slight  congestion  and
cloudy swelling of the  liver,  very slight  parenchymatous  degeneration  of  the
epithelium of  the  renal  tubules,  slight congestion and hemosiderosis  of  the
spleen and testicular atrophy.  No significant pathological  changes  were  ob-
served in  the  lung,  heart, adrenals, pancreas,  or  stomach  of  these  animals.
Based  on  the work of  Spencer, et  al.  (1948),  a  no-observable-effect-level
for 2,4-DNP  in rats lies between 5.4 and 20 mg/kg body weight/day.
    Information  on  the  subacute  or chronic  effects  of  the  other  dinitro-
phenol  isomers  in  experimental  animals   was  not  found.   Langerspectz  and
Tarkkonen  (1961)  failed to  detect  histological  changes  in the adrenals  or
the  liver during  2,4-dinitrophenol   treatment  of  Swiss  albino male  mice.
2,4-Oinitrophenol was  administered  via the subcutaneous  injection  of  10  mg
of 2,4-ONP/kg twice daily for 30 days.
    Arnold,  et  al.  (1976)  examined  the effects on  the  kidney of  a  single
large  dose of 2,4-DNP.  Although  a  dose   close  to  the  ID5Q was chosen,  (20
mg/kg) only  small areas  of  cortical  tubular necrosis  were  observed  in  a  few
of the '•ats treated.
    Tainter  and Cutting  (1933)  administered 2,4-DNP  to dogs at intervals  of
three or more days over  a period of  two to three months.   Abnormal  liver  and
kidney  pathology were   not  detected  but   an  effect  on   spleen tissue  was
noted.  Over  large areas of the material   containing  "numerous large faintly
staining cells with vesicular polyhedral nuclei."
    The widespread use  of  2,'4-dinitrophenol  as a  weight  reducing  agent  in
humans during  the  1930s  provides  some  information  regarding the chronic  ef-
fects of this compound  in  man.  Recommended theraputic doses  of 2,4-DNP  for
weight control  on  humans ranged from  2 to 5 mg/kg body  weight/day  (Dunlop,
1934;  Horner, 1942; Tainter, et  al.  1933).  Tainter,  et  al.  (1933)  adminis-
tered  2,4-DNP to  113  obese  patients  for as  long  as  four  months  without
                                     C-44

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 demonstrating  evidence  of cumulative  or  toxic effects.  The most  important
 side  effect  noted by the  investigator was a  skin  rash observed in about  7
 percent  of the  patients  treated.   The rash  was manifested usually after  a
 one-day  period  of mild  itching and consisted  of  a  maculopapular  or urticar-
 ial  type of  rash.  The itching was  intense and  in  some cases there was  con-
 siderable  swelling.   Symptoms subsided in  two to five days following with-
 drawal  from  the  drug.   The  next  most  important side effect  noted by  the
 authors  was  a  loss of taste  for salt  and  sweets observed in 5.3 percent  of
 the  patients.   This  effect also subsided  following  withdrawal  from 2,4-DNP.
 The  investigators  failed to detect  any effect of 2,4-ONP on liver  or  kidney
 function,  pulse,  blood  pressure,  or oxygen capacity of the  blood.   No cases
 of anemia, agranulocytosis, or malignant  neutropenia appeared.   Three cases
 of mild  gastrointestinal upset were reported, however.
    The  development  of  cataracts following  dinitrophenol therapy  was  first
 described  by Horner,  et al.  (1936).   In  a later publication,  Homer  (1942)
 reviewed the acute and  chronic toxicity of use  of  2,4-DNP  (including  catar-
 act formation)  resulting from therapeutic  use of the compound.   Gastrointes-
 tinal  symptoms  consisting of nausea,  vomiting,  and  loss  of   appetite  were
 common as  a  result  of  2,4-DNP  administration.   Cutaneous  lesions  were  the
most  frequent  side effect with  an  incidence  of  8  to   23 percent.   Although
 the majority of lesions were  mild, others were  severe.  Bone marrow effects
of dinitrophenol  have also been  reported.   Eight  cases of agranulocytosis
were  reported,  with  three fatalities.  Thirty  cases   of neuritis  including
 aberrations of  taste  and multiple regional  involvement, particularly affect-
 ing the  feet and legs were recorded.   Symptoms  appeared  after  an average  of
 ten weeks,  followed  ordinary  therapeutic  doses  and persisted  for  weeks  or
                                     C-45

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months.   Electrocardiograohic  evidence of  functional  heart  damage  was o*-
fered by several investigators and fragmentation of the heart muscle was re-
oorted  at  autopsy  in one  fatal  case.   It was generally agreed that  2,4-ONP
was  Barely  injurious  to the  liver  and kidneys when  administered in  thera-
oeutic doses.
    Over  100  cases  of  cateract  formation  following  dinitrophenol   therapy
were reviewed  by Homer  (1942).  Horner described the following characteris-
tic  features  of  2,4-DNP  induced  cataracts:  (1) they occurred in  young  women
who were physically  normal  save  for varying degrees of  obesity  and  were  in
an age group  in which  senile  cataracts  do not  occur;  (2)  they were bilateral
and appeared  either  during  of after periods of dinitrophenol treatment; (3)
an interval  of months or years might  elapse between  the time the last  dose
was  taken  and  the onset  of  blurred  vision;  (4)  lenticular  changes  were
strikingly  similar and  could  be demonstrated  with the  biomicroscope  at   a
time when vision for distance  and  reading was still normal;  (5)  after  arad-
ual  onset,  the lenticular  changes  progressed  with startling rapidity  until
the vision was obscured;  (6)  treatment was  without effect in staying  their
progress; and  (7)  surgical  removal of  the  lens was uniformly successful  in
restoring vision.
    The  length of  time  that  2,4-DNP was  taken  and the  amount  of the  drug
consumed varied widely amonq cataract victims.  In  29 cases,  the  duration  of
treatment varied from 3 months to  24 months with  an  average of 11  months.
Neither the length of treatment nor the  total  dose seemed to have any  bear-
ing  on  the occurrence  of  cataracts.   Individual  susceptibility  appeared  to
be a more  important  factor.  Horner  (1942)  estimated  that the incidence  of
cataracts in patients who had taken dinitrophenol  exceed  one percent.
                                     C-46

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     Formation  of cataracts by  acute  exposure  to ONP was  first demonstrated
 in  animals  almost ten years after the  problem  was  known to exist  in  humans
 (Gehring  and  Buerge,  1969a; Ogino and  Yasukura,  1957;  Feldman, et al.  1959,
 1960;  Settman,  1946).  Experimental   cataracts,  first  produced  in  ducks  and
 chickens, differ  from DNP-induced  human cataracts in that  they can be formed
 in  acute  exposures and may appear  in  less  than  one  hour.  Furthermore,  these
 lesions  will  disappear spontaneously in animals with  25  hours  (Howard,  et
 al.  1976).  Hence,  the usefulness of data  on  the formation of cataracts  in
 experimental  animals  following ONP administration  in  assessing human  hazard
 to  dinitrophenol  is questionable.
     The  available data  do not allow  the  calculation  of  a  minimum  effect
 level  for 2,4-DNP-induced  cataract formation in  man.   Cataractogenic  activ-
 ity in humans has been observed  in a small  proportion  of  patients receiving
 as  little as  2  mg/kg  body weight/day.  An  assessment of the no-effect-level
 for  cataract  formation awaits  further  investigation.   Such  an assessment  is
 further complicated by the fact that  cataract  formation in  humans, following
 ONP  administration, differs significantly from  the  situation seen in experi-
mental animal  studies.
 Synergism and/or Antagonism
    A  report  of teratogenic synergism  following  the combined  administration
of  2,4-dinitrophenol  and   insulin to  chicks  was  made  by Landauer  and  Clark
 (1964).   The  injection of 2,4-dinitrophenol at  100 yg/egg was  nontoxic  and
nonteratogenic after  96 hours  of  incubation.  However,  the  combined adminis-
tration  of  insulin  (a known  teratogen) with  100  ug   of  2,4-dinitrophenol
raised the incidence of embryo  mortality from 16  to  19  percent and shortened
the upper beak by 1.4 to 18.5  percent.
                                     C-47

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    Both  thyroid  hormones and  2,4-dinitroohenol  decrease the efficiency  of
mitochondrial oxidative  phosphorylation  in_ vivo  and  _m  vitro.   The _j_n_  yiyp
administration of  both  1-thyroxine  and 2,4-ONP results  in  larger  changes  in
metabolic rate and body temperature than are accounted for  by the  sum  of the
separate effects of each agent (Hoch, 1965).
    Other   direct    information   on   possible    synergism   between   the
dinitrophenols and other chemical compounds is not available.
Teratogenicity
    Wulff,  et  al.  (1935)  examined  the effects  of 2,4-dinitrophenol  on the
fertility,  gestation,  and  fetal  life  of  rats.   They  administered 20 mg  of
2,4-DNP/kg  body weight  to female rats  eight days  prior  to the  introduction
of males.   Oinitrophenol was  administered  intragastrically  twice  daily until
the respective  litters  were  weaned.   The  average  number  born  in  each litter
was not affected by the  use  of  dinitrophenol,  and the treatment did  not ap-
preciably affect the  body  weight gains  of  mothers during pregnancy.  Neonat-
al malformations were not detected.   Among 2,4-dinitrophenol treated rats,
however,  25 percent  of the  total  number  of young  were  stillborn  while  only
6.8 percent of the young  were stillborn in the control  group.   In addition,
the mortality during  the nursing period of  viable young born  to  mothers ad-
ministering 2,4-ONP was 30.9  percent as compared  with 13.4  percent for young
of control  mothers.   Two  possible explanations  for this  latter  phenomenon
were  offered:  treated  mothers  neglected   their  young  while in   a  febrile
state,  and  only the  more vigorous  of the offspring managed  to  reach the
mother  for  nursing;  or, a toxic  agent was  passed to the young  through the
milk.  Data to distinguish between the two possibilities are not  available.
    Intraperitoneal (7.7 or  13.6 mg/kg)  or oral  (25.5 or 38.3 mg/kg)  admin-
istration of  2,4-ONP  to mice  during  early  organogenesis  does  not  produce
                                     C-48

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morohological defects  in  the  young,  but  embryo  toxicity  occurs  at the higher
dose  levels  (Gibson,  1973).    The  higher  doses  also oroduced  overt  toxic
signs (hyoerexcitability and Hyoerthermia) in the dams,  but were not lethal.
    Bowman  (1957) has  studied  the  effect of 2,4-DNP on  the  develooino  chick
embryo j_n  vitro.   At  2,4-DNP  concentrations  of 18 mg/1  or  370 mg/1 a  syn-
drome of abnormal ities  resulted consisting  of degeneration  and  sometimes
complete absence  of  neural tissue  accompanied  by  a reduction  in  the number
of  somites.  The  2,4-ONP  concentrations  used  in  this  study are  extremely
hiqh and the  relevance of  the  experimental  findings  to  the j_n vivo situation
in mammals  is unknown.
    Malformations  such  as  hemiophthalmus  and  cross  beak  were  induced  in
chick embryos following  administration  of 0.5 uM/egg  (92 ug/egg)  into  the
yolk sack at  48 hours  of incubation (Miyamoto,  et  al.  1975).  Based on'exam-
ination  of  purified  myelin in  the  malformed  embryos the  investigators  sug-
gested that 2,4-DNP  administration resulted  in deficient  embryonic myelina-
tion.
    Based on  the  available data it appears unlikely that  the dinitrophenols
pose a teratogenic  hazard to  humans.   Further  investigations on  this  nues-
ti'on are warranted.
Mutaaenicitv
    Friedman  and  Staub (1976)  have developed an approach  to mutaoenic  test-
incj which utilizes the measurement  of  induction of  unscheduled  ONA synthesis
in testes.   These investigators  found  a  good  correlation between a reduction
in the residual  level  of cell  cycle-associated  DNA  synthesis and  the  pres-
ence of  known mutagenic  compounds.   Testicular  ONA  synthesis in mice was un-
affected  by administration  of  2,4-DNP  suggesting a lack of  mutagenic activ-
ity.
                                     C-49

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    Bacterial  mutaqenesis  of  2,4-DNP has  been  tested  by  Demerec,  et  a".

(1951), based  on  the production of  back  mutations from  streptomycin  deoen-

dence to independence in E_.  col i.  Mutations  were increased  severalfold over

control values.

    A recent study has been  conducted  on  the  effect  of various phenolic com-

pounds  including  2,4-DNP  on  chromosomes of  bone  marrow  cells  from  mice

(Mitra  and  Manna, 1971).   Mice were  injected intraperitoneally with  satu-

rated aoueous  solutions of  2,4-DNp  and the bone  marrow tissue was  collected

24  hours  after  trea:~ent.   The  results   suggest that 2,4-DNP  may  produce

chromatid type  breaks  in  bone  marrow cells.   However, there  was no  linear

relationship between the frequency of  chromosome  aberrations and  the  dose of

2,4-DNP.

    It  is  possible  to make  a rough  estimate  of  the  2,4-DNP  doses  adminis-

tered to the mice by these  investigators.  The water solubility of  2,4-ONP

at  75.8*F  is  3.01 mg/ml  (Windholz,   1976).   If  this  value  approximates  the

saturated solution used by Mitra and  Manna (1971) and a  three-to-four-month-

old mouse  weighs  approximately  40  g, the  following calculations result  in

three 2,4-DNP dose levels  expressed  as mg/kg body weight.



                      (0.25 ml)  (3.01  mq/ml) =  13.3 mg/kg
                              0.04 kg

                      (0.5 ml) (3.01  mg/ml) s 37.5 mg/|
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    Data  addressing  the  possible  mutagenicity of  the other  dinitrophenol
isomers were not found.
Carcinogenicity
    In  a  study designed  to measure  tumor  promoting activity,  Boutwell  and
Bosch,  (1959)  examined the ability  of  2,4-ONP to  promote tumor  formation
following  a  single  initiating  dose of  dimethylbenzanthracene.   Although
phenol  itself  has  a  promoting  activity  in  this  system,  2,4-DNP  failed  to
promote skin tumors  in mice under similar conditions.   In  a similar experi-
ment,  Stenback and Garcia  (1975)  examined the ability  of 2,4-DNP  to promote
skin tumor formation in mice.  No promoting activity was demonstrated.
    Spencer, et al.  (1948) failed  to detect tumor formation  during chronic
administration of 2,4-DNP  to mice (over a six month period).
    The available data suggest the  2,4-DNP does  not  possess carcingenic  pro-
perties.  Information on the other isomeric dinitrophenols  is not available.
                                     C-51

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                                TRINITROPHENOLS
Mammalian Toxicology and Human Health Effects
                                 INTRODUCTION
    Six isomeric forms of trinitrophenol exist distinguished  by  the  position
of the nitro  groups  relative to the hydroxy group  on  the  six carbon benzene
ring.  The  five  isomers  are:  2,3,4-,  2,3,5-,   2,3,6-,  2,4,5-,  2,4,6-,  and
3,4,5-trinitrophenols.  Production  volumes for  the trinitrophenols  are  not
available.  Usage  of the  trinitrophenol   isomers  is  apparently limited  to
2,4,6-trinitrophenol, other  wise  known as picric  acid.   In  fact,  a compre-
hensive search of the  literature  failed to detect a single  citation dealing
with  any  of  the  trinitrophenol  isomers  except  picric  acid.  Consequently,
the  only  information on these  isomers presented  in  this  document  are  the
chemical  and physical properties found in Table 8 and Figure  3.
    According to Matasuguma  (1967) picric  acid has  found use  as  a  dye inter-
mediate,   explosive,  analytical   reagent,  germicide,  fungicide,   staining
agent, and  tissue  fixative, tanning  agent, photochemical,  pharmaceutical,
and  a process material  for the  oxidation and  etching  of iron, steal,  and
copper surfaces.   The  extent  to  which picric  acid  finds  usage  in  any  of
these applications at the present time is unknown.
                                   EXPOSURE
Ingestion from Uater
    Monitoring  data  on  the  presence or  absence  of  2,4,6-trinitrophenol
(2,4,6-TNP) in water were  not found,  however,  a single report  of   2,4,6-TN?
contamination of  ground water  was found  (Cole,  1974).    In  1955,   2,4,6-TNP
(0.7  mg/1)  was  detected  in a  well  approximately one mile  from the former
site  of an  expolsives manufacturing  plant  in England.   The plant was engaged
in  the manufacture  of  explosives from 1914 to  1918.   The  brief  report  by
                                     C-52

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                                    TABLE  8

                        Properties of Trinitrophenols*
2,3,4-Tn'nitrophenol

  Molecular Weight
229.11
2,3,5-TrinitroDhenol

  Molecular Weight
  Melting Point
229.11
119-120*C
2,3,6-Trim'trophenol

  Molecular Weight
  Melting Point
  Water Solubility
    Room Temperature
    Hot Water
229.11
119"C

Slightly Soluble
Very Soluble
2,4,5-Trim'trophenol

  Molecular Weight
  Melting Point
  Water Solubility
    Room Temperature
    Hot Water
229.11
96'C

Slightly Soluble
Soluble
2,4,6-Trim'trophenol

  Molecular Weight
  Melting Point
  Boiling Point

  Vapor Pressure
  Density
  Water Solubility
    Room Temperature
    100'C
229.11
122-123'C
Sublimites: Explodes at
  300 *C
1 mm Hg at 195*C
1.763 g/cm3

1.28 g/1
6.7 g/1
*Source: Windholz, 1976; Weast, 1975; Matsuguma, 1967.
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              NO,
              NO,
                                    OH
                                                                OH
2,3,4-trinitrophenol         2,3,5-trinitrophenol          2,3,6-trinitrophenol
                                 OH
OH
                                                           wo
2,4,5-trinitrophenol        2,4,6-trinitrophenol          3,4,5-trinitrophenol
                                  FIGURE 3



                               Trinitrophenols
                                     C-54

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 Cole  (1974)  failed  to  describe  either  the types  of explosive manufactured by
 the  plant or  the  disposition  of  the  waste  water  during  the  period  the
 explosives were manufactured.
    Harris,  et  al.  (1946)  described an  outbreak of  hematuria which  resulted
 from  ingestion  of  2,4,6-TNP  in the drinking  water  of  U.S.  Navy  personnel
 aboard  ships anchored at  Wakayama,  Japan.   Approximately  three  weeks  prior
 to the  outbreak, more  than  100  tons  of confiscated Japanese ammunition,  (in-
 cluding  2,4,6-TNP)  had been dumped in the  immediate  vicinity of  the anchor-
 age.  2,4,6-TNP was  apparently  pumped  into the ships' drinking water  stills
 and carried  over  with the  vapor  phase  into the freshwater supply,  inducing
 hematuria  among those  who drank  the  water.   The  investigators failed  to
 detect  2,4,6-TNP in  the  sea water;  however,  analysis of  the distilled drink-
 ing water yielded 2,4,6-TNP levels to 2 to 20 mg/1.
    Although  it  is  not  possible  to  precisely estimate either the  TNP  water
 levels or duration of  exposure  necessary to  induce hematuria, Harris,  et al.
 (1946)  detected  levels of  10 mg/1  and 20 mg/1 in drinking  water aboard two
 ships  at the time of the hematuria outbreak.
    Hoffsommer and  Resen (1973) have shown that the  highly explosive  tetryl
 (N-methyl-N,2,4,6-tetranitroanil ine) dissolved in  sea water  at pH 8.1  and at
 temperature  25°C  is  largely  converted  to  2,4,6-TNP in  a few months.   Al-
 though  tetryl is no longer manufactured  in the  U.S. (Howard, et  al.  1976),
 these  experiments Indicate  that 2,4,6-TNP may be  produced  in water  as  a re-
sult of  degradation  of  other organic  compounds.   The nature of other  com-
pounds which may give rise to 2,4,6-TNP following degradation is  speculative.
    The persistence of 2,4,6-TNP  following  release to the  environment  is now
well  understood.   Fitter  (1976)  failed  to  detect degradation of  2,4,6-TNP
using  an acclimated activated sludge system with 2,4,6-TNP   as a  sole  source
                                     C-55

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of  carbon for  the  microbes  in the  inoculum.   Tabak, et  al.  (1964) on  the
other hand were able to demonstrate  95  percent  degradation of  2,4,6-TNP  (250
ma/1)  in three  to  six  days  by  acclimated cultures  of microorganisms  de-
rived from garden soils, compost, and river mud.   The  extent to  which micro-
bial populations  capable  of  degrading 2,4,6-TNP exist in the  environment  is
unknown.
    No  other  data on  possible ingestion of  2,4,6-TNP from  water by humans
were found.
Inqestion from ?
    Pertinent  data  could  not   be   located   in   the   available  literature
concerning exposure to 2,4,6-TNP via ingestion of  food.
    No measured  steady-state  bioconcentration factor (BCF)  is  available  for
any nitrophenols, but  the equation "Log  BCF  = (0.85 Log  P)  - 0.70" can  be
used  (Veith,  et  al. 1979)  to  estimate the steady-state BCF for  aauatic  or-
ganisms  that  contain about 7.6  percent  lipids  (Veith, 1980)  from the  oc-
tanol/water partition coefficient  (P).   The log P values  were  obtained  from
Hansch and  Leo (1979)  or were  calculated  by  the  method  described  therein.
The adjustment factor of  3.0/7.6  = 0.395 is used  to  adjust the estimated  BCF
from the  7.6  percent  lipids on which the  enuation is  based to the  3.0  per-
cent  lipids that  is the weighted average  for  consumed  fish and shellfish in
order to  obtain  the weighted  average bioconcentration factor for  the  edible
portion of all freshwater and estuarine  aauatic organisms  consumed by  Ameri-
cans.
Inhalation
    Pertinent  information  could not  be  located in the  available  literature
on the presence or absence of trinitrophenols in air.
Dermal
    Information on  the dermal absorption of 2,4,6-TNP  is scant  in  the  liter-
ature.   During the 1920s and  1930s, 2,4,6-TNP was  used  both  alone and  in
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 combination  with  butesin  (di-n-butyl-p-aminobenzoate trinitroohenol)   as  an
 antiseotic  surgical  dressing for the treatment of  burns.   Ehrenfried  (1911)
 remarked  on the  dangers  of poisoning  by absorption of  2,4,6-TNP  in  dermal
 ointments, but  added  that,  if  the  ointments  were  properly used,  there  was  no
 danger  of toxic  symptoms developing  in  humans.
    A serious case of central  nervous  system dysfunction following  the topi-
 cal  aoolication  of  2,4,6-TNP  was  reported  by Oennie,  et al.  (1929).   The
 patient  recovered rapidly  following cessation of  the  2,4,6-TNP  treatment.
 No  other  information on dermal  absorption of the  trinitrophenols  by  humans
 or experimental  animals was found.
                               PHARMACOKINETICS
 Absorption
    Quantitative  information on  the  absorption of 2,4,6-TNP by humans  or ex-
 oerimental animals is not available.
    Neurological   complications  following  the   topical  administration  of
 2,4,6-TNP (Oennie, et al.  1929)  indicate  that  the compound may be absorbed
 through the  skin.  Since the  compound  was  applied  to  a burned area  of the
 patient, the relevance  of  this data to the  absorption  of  2,4,6-TNP through
 intact skin in unknown.
    The occurrence of  human  cases  of  microscopic hematuria  resulting  from
 ingestion of 2,4,6-TNP in drinking water  (Harris, et  al.  1946)  and  the known
 oral toxicity of 2,4,6-TNP  in experimental  animals  indicate  that  absorption
by the gastrointestinal tract readily occurs.
 Oi stribution
    Examination  of dogs after  a  lethal  dose of  2,4,6-TNP (Dennie,  et  al.
 1929)  revealed  yellow staining of the  subcutaneous fat,  lungs,  intestines,
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and the olood vessels,  indicating tnat 2,4,6-TNP  is distributed to nany tis-
sues  in  the body.  These  investigatores  also demonstrated  the  presence  of
2,4,6-TNP in the  blood  and suggested that the compound may  be  bound to serum
proteins.    It  seems  likely that  distribution  of 2,4,5-TNP  would  occur  via
the olood.  No  other  data  on  the tissue distribution of 2,4,6-TNP following
absorption were found.
Metaoolism
    In a  review  of  the early  literature,  Burrows and Dacre  (1975) indicated
that elimination  of 2,4,6-TNP from  humans  occurs in both  the  free form and
as picramic  acid.   In  perfusion experiments with liver,  kidney and  spleen,
the liver exhibited the strongest capacity  for  reduction of  2,4,6-TNP.
    Other studies  dealing  with  the metabolism of 2,4,6-TNP in humans or  in
experimental animals were not  found.
    Decomposition of 2,4,6-TNP  by an atypical  strain of Corynebacterium sim-
plex with the production of nitrites has been reported by Gunderson ana Jen-
sen (1956).   This alternative metabolic pathway  for  2,4,6-TNP has not been
reported in mammals.
Excretion
    The presence  of 2,4,6-TNP  in blood and urine within 1.5 hours after  ad-
ministration  of   a  lethal  dose  in  dogs  was  reported  by  Dennie,   et   al.
(1929).  The presence  of 2,4,6-TNP  in  the  urine  of  humans  following  oral  ex-
posure was  reported by Harris,  et  al.  (1946).   These studies  indicate tnat
2,4,6-TNP is partially  excreted  in  the  urine following exposure.   Other data
on the excretion of 2,4,6-TNP  were not found.
                                    EFFECTS
Acute, Subacute, and Chronic  Toxicity
    According  to Windholz  (1976)  ingestion  or  percutaneous  absorption  of
2,4,6-TNP may cause nausea,  vomiting, diarrhea,  abdominal pain, oliguria,
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 anurea,  yellow staining of  skin,  pruritus,  skin eruptions,  stupor,  convul-
 sions, and death.
    Although Dennie, et  al.  (1929)  stated:  "The  application  of a solution of
 trinitrophenol to  burned  or  abraded skin is dangerous  even  for nonsensitive
 persons  since  many deaths have  been  reported  from  its  application,"  no  re-
 ports  of human fatalities  resulting  from 2,4,6-TNP  exposure were found  in
 the  literature.   Gleason,  et al. (1968) reported the  lowest  recorded  lethal
 dose for 2,4,6-TNP in humans as  5 mg/kg  body weight,  however, details  of  the
 poisoning episode  were  not  provided.   It  is  reasonable to  assume, based on
 the  known  toxicity of  2,4,6-TNP in experimental  animals,  that exposure  to
 sufficient amounts  of  the compound would  be  lethal  in  humans.   The  limited
 acute  toxicity information for experimental animals  has  been  compiled  and is
 presented in Table 9.
    Following  acutely  lethal doses of  2,4,6-TNP,  dogs  die  from respiratory
 paralysis (Dennie,  et  al.  1929).   Autopsy  results  demonstrate  the presence
 of yellow staining of the subcutaneous  fat,  the lungs, the  intestines,  and
 the blood vessels.   Swelling of the  liver and  glomerulitis  of the  kidneys
were also seen.
    The major  effect  of nonlethal  doses  of  trinitrophenol   (TNP)  appears to
be an  allergic or  irritative dermatitis  (Anon.  1937;  Ehrenfried, 1911).   Ac-
cording  to Dennie,  et  al.  (1929)  about four percent of people treated  with
TNP are sensitive and develop a  local dermatitis.   Reactions  may also  appear
 in unexposed  areas.   An  intense itching and  burning,   pruritis,  skin  erup-
 tions,  and irritability are  common.  Skin eruptions  are  characterized  by  ir-
 regular-shaped macules,  papules, vesicles,  blebs,  excoriations,  and  edema,
 as well  as  dried  yellow crusts  which  are sources  of reabsorption.   In  the
maculopapular stage, a purplish-yellow color is characteristic.
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                                        TABLE  9

                       Acute  Toxicity  of Trinitrophenol  Isomers3
Species
Dog
Dog
Dog
Rabbit
Frog
Frog
Cat
Human
Dose
(mg/kg)
100-125
60
60
120
200
200-300
500b
5
Route of
Administration
2,4,6-Trinitroohenols
s.c.
s.c.
UNK
Oral
s.c.
s.c.
Oral
Oral
Effects
Lethal Dose
MLD
MLD
Lethal Dose
Lethal Dose
MLD
Lethal Dose
Lethal Dose
References
Dennie, et al .
Spector, 1956
von Oettingen,
von Oettingen,
Windholz, 1976
Spector, 1956
von Oettingen,
Gleason, et al .

1929

1949
1949


1949
1968
3Acute toxicity data for trinitrophenol isomers other than 2,4,6-TNP were not
 found.

bTotal dose in milligrams.

MLO = Minimum Lethal Dose

UNK = Unknown
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    More  severe reactions  can  lead  to  diffuse, often  severe erythema  and
desiuamation  of affected areas  (Sulzburger  and Wise,  1933;  Am.  Conf.  Gov.
!nd,  Hyg.,  (ACGIH)  1971).  The  reaction  may last from several weeks  to  al-
most  a year (Sulzburqer and Wise, 1933).
    Effects  on the  skin are  apparent  at  concentrations  well  below  those
necessary for  oral systemic poisoning.  Of 71  individuals  exposed at  concen-
trations  of  0.0088 to  0.1947  mg/m ,  dermatitis  developed only among  those
exposed  to  the  lower  concentrations.  Desensitization  or adaptation  reac-
tions may occur (ACGIH, 1971).
    Guinea  pigs tested  for  allergic  reactions  gave  similar  results  (Land-
steiner and OiSomma,  1940; Maguire and Chase,  1972; Maguire,  1973;  Chase  and
Maguire,  1972).   Using a split-adjuvant  method of sensitization,  reactions
have been noted at concentrations of  less than  one percent, with weaker sol-
utions often giving stronger reactions.  A boosting effect was also noted on
subsequent tests with sensitized animals  (Maguire and  Chase, 1972).
    Sub-lethal  doses of less  than or enual  to 50 mg/kg body  weight  in dogs
have resulted  in transitory changes in the kidney which  include glomerulitis
and involvement of TNP  ranging  from cloudy swelling  to  gelatinous  degenera-
tion.   The  liver also  showed  cloudy  swelling with  no  staining  while  the
lungs  were stained brownish-yellow in  some animals (Dennie, et al.  1929).
    Other reactions in humans include central  nervous system  effects  result-
ing  in  temporary  impairment   of  speech,   memory,   walking,  and   reflexes
(Dennie,  et al.  1929)  and microscopic hematuria  caused  by ingestion of  TNP
(2 to 20 mg/1) in water distilled from sea water (Harris,  et al.  1946).
    Althouah it is not  possible to estimate precisely  either the  TNP  water
levels or duration of exposure necessary to  induce hematuria,  Harris,  et  al.
(1946) detected levels  of 10 mg/1 and 20 mo/1  in  drinking water aboard  two
ships at the time of  the hematuria outbreak.
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    Crenation  of  intact  erythrocytes  by  1  mM 2,4,6-TNP  has  been noted  i-j
Sheetz  and  Singer  (1976)  with  lysing  occurring  at higher  concentrations.
2,4,6-TNP has  also  been  observed  to  affect  glycolysis  in human  red  blood
cells u^ vitro by affecting ATP  production  and consumption, although  the ex-
act mechanism  has not  been  determined  (Vestergaard-Bogind and  Lunn,  1977).
Pugh and Stone  (1968)  noted  that from 6  to 11 mg/kg  2,4,6-TNP  administered
intravenously  to  anesthetized dogs  results  in a  moderate increase  in  bile
flow and a rise in body temperature.  The  clinical  significance  of these ef-
fects is unknown.
Synergism and/or Antagonism
    Information on  synergistic  or  antagonistic effects  involving 2,4,6-TNP
is scant in the literature.   An interesting study  by  Huidobro  (1971)  demon-
strated  that   administration  of 50  mg  of 2,4,6-TNP  kg  of  body  weight  30
minutes before the administration of a number  of  analgesic drugs resulted in
a  significant  increase in the  area of  analgesia  induced  by  the opioids and
the minor analgesics employed.   2,4,6-TNP did not,  itself,  evoke analgesia.
The compounds  tested  included:  morphine,  meperidine, methadone,  pentazocine,
aminopyrine, sodium salicilate,  and etonitazene.   The  investigators suggest-
ed that  the  elimination or metabolism  of the analgesics  may be modified by
an effect of 2,4,6-TNP on  enzymatic  systems.   However,  further   investigation
is needed to definitively answer this question.
Teratogenicity
    Pertinent  information  could not be  located in  the  available  literature
on possible teratogenic effects of 2,4,6-TNP.
Mtitagenicity
    Streptomycin-independent  mutants were  induced  in  streptomycin-requiring
E_. col i B/Sd-4 after preincubation  in the  presence of  0.18 percent 2,4,6-T'i?
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for  three  hours before  olating  on streptomycin  free agar (Demerec,  et  al.
1951)  Yoshikawa, et  al.  (1976)  reported that  2,4,6-TNP  was  capable  of  in-
ducing mutations in  Salmonella,  when  tested in a  system  which  included  mic-
rosomal  activation.    In  contrast,  Auerbach  and Robson  (1947)  failed  to
demonstrate  sex-linked  lethals  in  Drosophila  after  bathing  the eggs in  an
aoueous solution of  2,4,6-TNP.   Other data  on possible mutagenic  properties
of 2,4,6-TNP were not found.
Carcinoaenicity
    Pertinent data could  not  be  located in the available  literature  on  pos-
sible carcinogenic  effects of 2,4,6-TNP.
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                                DINITROCRESOLS
Mammalian Toxiciloqy and Human Health Effects
                                 INTRODUCTION
    Dinitro-ortHo-cresol is  a  yellow crystalline  solid  derived from  o-cre-
sol.  There  are  six  possible isomers but the 4,6-dinitro-o-cresol  isomer  is
the only one of  any  commercial  importance.   In  fact, a comprehensive  search
of  the  literature failed  to reveal  information on  any  of  the  other  five
dinitrocresol isomers.
    4,6-Oinitro-o-cresol (hereafter  referred to as ONOC)  is  produced  either
by  sulfonation  of o-cresol  followed by  treatment with  nitric acid  or  by
treatment of o-cresol in glacial acetic  acid with  nitric  acid at  low temper-
ature.  Some important chemical and physical properties of DNOC are  shown  in
Table 10 and Figure 4.
    An  excellent  review of  the  toxicological  effects of  ONOC on  human  and
labortory animals has recently  been  published  by the National  Institute  for
Occupational Safety  and  Health  (NIOSH,  1978).   In view of the comprehensive
coveraae of both English and foreign  language literature, no  attempt will  be
made  to duplicate  this impressive  effort  within  this  criterion  document.
Key papers and frenuent  reference to the NIOSH review will be used  where the
available literature  does  not contain information directly relevant  to  cri-
teria formulation.
    ONOC usage in the U.S.  has  declined  in  recent  years  because the compound
is highly toxic to plants  in the growth stage and nonselectively kills  both
desirable and  undesirable  vegetation.  Additionally, the compound  is  highly
toxic to  humans  and  is  considered  one  of  the  more dangerous  agricultural
pesticides.
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                         TABLE 10
            Prooerties of 4,6-Dinitro-o-cresol
Molecular Weight                   198.13
Appearance                         Yellow Solid
Melting Point                       85.8*C
Vapor Pressure                       0.000052 mm Hg at 20*C
Water Solubility                   100 mg/1 at 20*C
pKa                                  4.46
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4,6-Dinitro-o-cresol (ONOC)
          FIGURE  4
       Oinitrocresols
             C-66

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     The Environmental  Protection  Agency has  no record of DNOC being current-
 ly manufactured in  the  United  States  for use as an  agricultural  chemical.
 Imports of DNOC  have  also decreased  in  recent  years; from  217,899  Ibs.  in
 1972  to 146,621 Ibs. in  1973  and  then to 30,442  Ibs.  in  1976 (NIOSH,  1978).
 Since  DNOC  is  not  manufactured  in the U.S.,  pesticide formulators and spray-
 ers are the major groups with potential occuoational  exposure to DNOC.
    DNOC  is  used  primarily as  a blossom-thinning agent on fruit trees and as
 a  fungicide,   insecticide,  and  miticide  on  fruit trees  during  the  dormant
 season.   NIOSH (1978)  estimates  that  3,000  workers  in the  U.S.  are  potent-
 ially  exposed  to DNOC.   In view of  the small  amount of DNOC  used   in  the
 U.S.,  exposure  of the general  public is expected to be minimal.
                                   EXPOSURE
 Ingestion from Water
    Monitoring  data  on the presence of DNOC  in  ambient  water are not  avail-
 able.    An  unspecified  amount of  DNOC  was detected  in  the  waste waters  of
 Fison's  Pest   Control  Limited   in Harston,  Cambridge,  England (Jenkins  and
 Hawkes, 1961).   Webb, et  al. (1973) detected  18  mg DNOC/1  in  the  waste water
 of a  specialty chemical  plant.   The  extent  to which  human exposure  to DNOC
 results from the ingestion of  contaminated water is unknown.
 Inqestion from Food
    No  data  are available  on  the  presence  or  absence  of DNOC  residues  in
food for  human consumption.  Since  the  primary usage of the compound  in-
volves  treatment  of fruit  trees  during  the  dormant  season,   it  appears  un-
 likely that contamination of human  food  stuffs would  occur to any  large  ex-
tent.
    No  measured steady-state bioconcentration factor  (BCF) is  availahle  for
any nitrophenols,  but  the  equation  "Log  BCF  = (0.85  Log  P)  - 0.70"   can  be
                                     C-67

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used  (Veith,  et  al.  1979) to  estimate  the steady-state BCF for aouatic or-
ganisms that  contain  about  7.6  percent lipids  (Veith,  1980) from  the oc-
tanol/water partition coefficient  (P).  The  log P values were obtained from
Hansch and  Leo  (1979)  or were  calculated  by  the  method described  therein.
The adjustment factor of  3.0/7.6 = 0.395 is  used  to  adjust  the estimated BCF
from the 7.5  percent  lipids  on which the  eouation is  based to  the  3.0 per-
cent  lipids that  is  the  weighted average  for  consumed  fish  and  shellfish  in
order to obtain  the weighted average bioconcentration factor for  the  edible
portion of  all freshwater and  estuarine anuatic organisms consumed by  Ameri-
cans.
Inhalation
    An evaluation of the  literature  (NIOSH,  1978)  indicates that occupation-
al  injury  and disease  associated  with  exposure  to  DNOC  results  primarily
from  inhalation of, and skin contact with, the aerosol  form. A  large  number
of human  intoxications,  including  fatalities,  have  been reported  resulting
from such DNOC exposure.  Persons  at risk  include  those manufacturing,  form-
ulatina,  or applying the compound as an aerosol.   Inhalation exposure  to the
general public is expected to  be minimal although  data addressing  this point
are not available.
Dermal
    As mentioned  in the preceeding  section,  occupational  intoxication  by ex-
posure to  DNOC  has occurred as  a  result  of  inhalation and  dermal  exposure
where  the  compound  is manufactured, formulated or applied.   Dermal  exposure
of the general  public is considered  unlikely, however, direct  data bearing
on this point were not found.
                               PHARMACOKINETICS
Absorption
    DNOC is readily and rapidly  absorbed through  the skin,  the  gastrointest-
inal  tract and  respiratory  tract  in  humans  (NIOSH,  1978).  Although most
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 cases  of occupational  intoxication  resulting from D'iOC exposure contain both
 a  respiratory and a dermal  component,  human  intoxication  has  been reportsd
 as  a result of dermal contact with DNOC alone.
    In  a report  from the Russian literature  (3uchinskii,  1974)  a four-year-
 old boy was fatally intoxicated after  a  rash had been treated with  50 g of
 an ointment to which 25  percent  DNOC  was  added by mistake.   Stott (1956) re-
 ported  two  cases of DNOC poisoning resulting  from skin absorption.  The two
 men were involved  in the cleaning  and maintenance of  aircraft  booms  used to
 spray  solutions  of DNOC.   Since neither man  worked near  the  actual  opera-
 tion, and both denied blowing into the  spray jet  to  clean them,  Scott (1956)
 concluded that the major route of exposure was skin contact.
    Work by Harvey, et al.  (1951) indicates  that  DNOC  is  rapidly absorbed by
 the human  gastrointestinal  tract.   These  investigators described the effects
 of DNOC  taken orally by  five  male  volunteers.  It was  noted  the  DNOC levels
 in  the  blood  increased  steadily after  administration   and  were  maximal  from
 two to  four hours after  ingestion.   Van Noort,  et al.  (1960)  investigated
 the effectiveness  of personal  protective equipment used  by 24  sprayers  in
 Holland.  Serum  DNOC levels  and the  quanitity of DNOC used  were determined
 in a three-week  spraying period.  Their findings  indicated  that both inhala-
 tion and dermal  contact with  DNOC can lead  to an  appreciable absorption into
 the blood stream.
    Experimental   animal  studies, reviewed  by  NIOSH  (1978), also  have con-
firmed the  toxicity  of  DNOC in humans  exposed by the  oral,  inhalation, and
dermal  routes.
Distribution
    Whether  absorption   of  DNOC occurs  through  the  skin,  gastrointestinal
 tract,  or respiratory tract,  the compound is  transported  in and  distributed
 by  the  blood  (NIOSH,  1978).  Harvey, et  al.  (1951) described  the  effect of
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DNOC  taken  orally  by  five male  volunteers.   Caosules containing  75  T,q Of
oure DNOC were  administered  daily for five consecutive days  amounting to a
total dose of from  0.95  to  1.27 mg/kg/day.  The  concentration  of  ONOC  in  the
blood increased  in  the  first three to  four  days and  reached  concentrations
of  15  to 20  mq/kg.  After  concentrations  of 15  to  20 mg/kg  had been  ob-
tained,   additional  doses aopeared to cause temporary  high blood  concentra-
tions which were associated with toxic symptoms.
    Blood analysis of humans displaying symptoms of  ONOC toxicity  has  invar-
iably revealed concentrations exceeding 10 mg/kg  (NIOSH,  1978).
     In studies conducted to determine the kinetics of  absorption  and distri-
bution,   DNOC has not been  shown to  accumulate  in the blood of  .-arious  animal
species  (King and Harvey,  1953a;  Parker,  et  al.  1951).  In rats  and rabbits
that were  given two or  more  daily  injections of DNOC subcutaneously,  serum
levels on succeeding days  were  no higher than they  were  24 hours  after  the
first dose  (Parker, et  al.  1951).  Serum levels in  dogs  rose for  the first
three days  but  then decreased despite  the  administration of  two  additional
doses.
    ONOC is more rapidly eliminated from the blood  of animals  than from the
blood of humans  (King and Harvey, 1953b;  Parker, et  al.  1951;  Harvey,  et al.
1951).   Within  a 24-hour period following a  single subcutaneous  injection of
ONOC, elimination from the  serum  of rabbits  was nearly complete.   Four days
were necessary  for  serum clearance  in  rats and cats, while six days were re-
nuired for  elimination from the serum of dogs  (Parker,  et al.  1951).   DNOC
accumulated only slightly  in  the  blood  when  given  to rats by stomach tube or
i.p. injection and did not  accumulate  in  the  blood of  rabbits  after adminis-
tration  by stomach tube  (King and Harvey, 1953a).
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     The  accumulation  of DNOC in the blood  of  humans  following DNOC exposure
 has  been  we:l  documented  (^arvey,  et  al. 1951; Bidstruo, et  al.  1952).   The
 accumulative  effect  may  reflect  the  binding  of  DNOC  with  albumin  in  the
 blood  and a  subsenuent slow  rate of excretion  in  humans  (Harvey, et  al.
 1951).
     ONOC  is slowly  eliminated  from humans.   The investigations by Harvey,  et
 al.  (1951)  indicated  detectable amounts  of DNOC  in  the  blood (1  mg/kg)  as
 long as  40 days  following  the  last of five consecutive  daily oral  doses  in
 human volunteers.   Another  study (Van  Noort, et al„  1960; reviewed  by NIOSH,
 1978) showed that  it  took  two  to eight weeks for  ONOC to be cleared from the
 serum.
     Parker, et  al.  (1951)  studied  the tissue  distribution  of  ONOC  following
 subcutaneous  injection  in  the  rat.   They  noted  that  a single  dose of  10
 mg/kg DNOC  produced very high  levels  in  the serum (100 mg/1  at  30 min)  but
 no  accumulation  in other  tissues  was detected.   The  lungs  and  heart  con-
 tained  high  levels of  DNOC  but  the  investigators   postulated  that  these
 levels were the  highest due to  the h.igh  blood  content of these organs.   The
 investigators  calculated that within 30  minutes of  the injection,  83 percent
of the DNOC that could be accounted for was  present in  the  blood.   Six  hours
 after the  injection,  0.37 mg of the  1.5 mg dose  of  ONOC could be  accounted
for, of which  72 percent was in the blood.
    ONOC content  of a number  of tissues was determined  in rats  receiving  a
single subcutaneous injection  of  the  compound (Parker,  et al.  1951).   The
 results,  presented  in Table 11, clearly   indicate the  ONOC  failed to accumu-
 late in the tissues.
     In another experiment Parker,  et al.  (1951) failed to detect  significant
 DNOC  accumulation   in  liver or kidney tissue   of  rats  after  40  successive
 daily injections of 20 mg/kg DNOC.
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                                             TABLE 11*

               ONOC Content of Blood and Tissues** of Rats  Killed  at  Intervals After
                        Subcutaneous  Injection of One Dose of 1.5 mg DNOC*
Time
After Injection
30 min.
1 hr.
2 hrs.
3 hrs.
4 hrs.
5 hrs.
6 hrs.
Serum
(rag/1)
100
89
97
93
79
76
45
Brain
1.5
3.5
2.0
4.0
3.5
2.0
3.0
Spleen
4.0
4.0
4.5
8.0
3.0
4.0
1.5
Kidney
7.5
7.5
11.0
11.0
4.5
4.5
7.5
Liver
14.0
12.0
10.5
11.5
13.5
8.5
8.5
Muscle
0.5
2.0
0.0
3.5
0.5
2.0
1.5
Heart
98.0
13.5
19.0
14.0
13.0
14.0
10.5
Lung
18.0
20.0
20.5
15.5
14.0
14.5
30.0
 *Source: Parker,  et al.  1951.

**DNOC content of  tissue  mg/kg  net  weight.
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     In  a single  study reviewed  by  NIOSH  (1978)  Sovljanski,  et  al.  (1971)
discussed  tissue  distribution  of DNOC  in  humans.  Autopsy  results  of  two
victims, who had  commmitted  suicide  by ingestion  of  DNOC,  yielded  detectable
DNOC  in  the  stomach,  intestines,  liver, kidneys,  heart, and brain, with  the
stomach  containing the greatest amount.   Neither  blood  DNOC  levels nor  quan-
titative data on tissue levels were reported.
     Steer  (1951), on  the other  hand,  demonstrated  that the  tissues  of  a
fatal case of DNOC poisoning contained no more than  5 mg/kg  of  DNOC and many
contained 1 mg/kg or less.
    According to  King  and  Harvey  (1953b)  the accumulation  of DNOC  in  man can
be explained  in  two  ways; either  the  detoxification and excretion are very
slow  or  there  is  some storage of  DNOC  in body tissues.   Based  on  their cal-
culation of  excretion  kinetics  in man, the  investigators  suggested that  de-
toxification and excretion of DNOC are inefficient and slow in humans.
    None of  the  available data suggest  significant  accumulation  of  DNOC  in
specific tissues of humans or experimental animals (NIOSH, 1978).
Metabolism
    The metabolism of  DNOC  in humans  has not been studied.  However,  several
investigators have conducted experiments  to  determine the  rate  of  DNOC  after
its administration to animals.
    Truhaut and De Lavaur (1967)  reported on the  metabolism of DNOC  in rab-
bits.  Following  the administration of  DNOC  by gastric  intubation, both DNOC
and  6-amino-4-nitro-o-cresol were detected in  liver,  kidney,   brain,  and
urine  of animals.  4-Amino-6-nitro-o-cresol  was  not  detected  in the  ani-
mals.   It  was concluded  by the  investigators that  the ratio  of  6-amino-4-
nitro-o-cresol to DNOC in the tissue and  urine was a  function of the  dose of
DNOC  administered to  the  animal.   When  a low dose of DNOC was  administered,
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ve*-y  litt'e 5-amino-d-nitro-o-cresol  *as  detected  in eitner  the urine  or
tissues.  The  authors  considered  the metabolism of ONOC  to  6-amino-4-nitro-
o-cresol a  detoxification mechanism  that plays  an  important  role only when a
toxic dose  of  DNOC  is administered.   They  further suggested that  the ratio
of  5-amino-4-nitro-o-cresol  to DNOC might be  a useful indicator  in  evalua-
tion of the severity of exposure to DNOC.
    The metabolic fate of DNQC in  rabbits  was  also investigated by Smith, et
al, (1953).  Following  administration  of 20 to  30 mg/kg  ONOC  to  rabbits  by
stomach  tube,  urinary metabolites  were identified  by paper  chromatography
and spectrophotometry.   Less than 20  percent  of  the  dose was  recovered  in
the urine in two  days.   Between  5  and  5.5  percent was detected as free DNOC,
and 0.7  percent  as DNOC conjugates.   The  conjugates were not  characterized
by  the  investigators.  Most of the urinary metabolites  (about  12 percent of
the dose) were derivatives  of 6-amino-4-nitro-o-cresol.   About  1.5  percent
of  the dose was excreted as 6-acetamido-4-nitro-o-cresol, and  9 to 10.5 per-
cent as  the hydroxyl group conjugate.  Traces  of 6-amino-4-nitro-o-cresol,
4-amino-6-nitro-o-cresol,  and 3-?amino-5-nitrosal icyl ic   acid  were also  de-
tected.
    Since the  detoxification  and excretion of  DNOC in man are very slow com-
pared to rats  or  rabbits (King and Harvey, 1953b), the applicability of the
experimental animal  detoxification mechanism  to the human  situation  is un-
known.   The  elucidation  of  DNOC  detoxification mechanism in  humans  awaits
further investigation.
Excretion
    Available  data  indicate that  DNOC  is rapidly excreted following adminis-
tration to experimental  animals.   Parker,  et al.  (1951)  found  that DNOC in-
jected subcutaneously disappeared from the blood at various  rates in differ-
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 ent  species.   Single  10 mg/kg doses of DNOC were administered subcjtaneously
 to  an unspecified number  of  dogs,  cats, rabbits,  and  rats.  DNOC  given  in
 one  injection  was completely eliminated from  the  serum of rabbits within 24
 hours,  while  blood DNOC  levels were  between  30  and  40  mg/1  in  the  rats,
 cats,  and dogs  at  this time.   It  took four  days  for DNOC  blood  levels  to
 fall  to zero  in  rats  and  cats,  and  six  days in  dogs.   The half-time  for
 elimination of  DNOC from  the  blood  after a single  injection of 10 mg/kg 3NOC
 was  approximately three hours in the  rabbit,  15  hours  in the rat,  20  hours
 in the  cat, and 36 hours in the dog.
    Lawford, et  al.  (1954)  reported  that  animals  eliminated DNOC  from  the
 blood  in  the following  descending order of  efficiency:  mouse, rabbit, guinea
 pig,  rat, and monkey.
    DNOC  is  eliminated in  the  blood of animals  faster than it  is  from  the
 blood  of  humans  (King  and Harvey,   1953b;  Parker,  et  al. 1951).   King  and
 Harvey  (1953b)  calculated  the  half-time  for  elimination  of DNOC  from  the
 blood of rats,  rabbits, and  humans.   The values were 28.5 hours,  6.6 hours,
 and 153.6 hours,  respectively.
    Pollard and Filbee  (1951) reported on  the  urinary  excretion  of DNOC from
 a seriously poisoned man in Great Britain.  The man was admitted  to the hos-
 pital  and  full  biochemical  investigations  were performed immediately  after
 admission.  The  man  recovered   almost totally  from  the poisoning  episode
within five days.  However, DNOC  levels of  4  mg/1  were still detected in  the
blood one month following the  exposure.   Blood DNOC  level  was  reported  to
 fall   in an exponential fashion.
    Van Noort,  et al.  (1960)  measured  the  serum  DNOC  levels in  ten spraymen
 on a  weekly basis  for two  months   after  the spraying  period ended.   They
 found the ONOC  was eliminated from  the serum  slowly and that the rate varied
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from  individual  to  individual.   TA/O to  e^gnt  weeks elapsed before DNOC
cleared  comoletely  from the  serum of  these  workers.   The amount  of  tir^.e
needed for  QNOC  to  be  totally  eliminated  was  directly related to the  quan-
tity of DNOC in the  serum on the last  day of exposure.
    In experiments whe^e  DNOC was orally  administered  to five human  volun-
teers, Harvey, et  al.  (1951) demonstrated  that  DNOC,  absorbed by  ingestion
at  24-hour  intervals  accumulates in the  human  body and  is excreted  slowly.
Forty days  after  the  last  dose  of DNOC was orally  administered by mouth,  1
to 1.5 mg/1 ONOC was still  present in  the blood.
    The  experimental evidence  suggests, therefore,  that  a substantial  dif-
ference  in  the  excretion  patterns of  humans  vs.  experimental animals  ex-
ists.  Since storage of DNOC in the tissues of humans has  not  been  reported,
it  is  concluded  that  slow  and  inefficient  detoxification  or excretion  prob-
ably occurs in humans.
    Occupational  studies  (NIOSH,  1978)  have  long  utilized serum levels  of
DNOC in  order  to  assess exposure  of humans to  dangerous amounts of the  com-
pounds.   A  review of  the  literature  (NIOSH,  1978)  indicates that  workers
with DNOC concentrations of 40  mg/kg of  whole  blood (approximately  80  mg/1
of  serum)  or  greater will  most likely  develop toxic effects.   In  addition,
in the concentration range  between  20  and 40  mg/kg of whole blood  (probably
because  of  variation in  individual  susceptibility)  some  workers are  affected
and others show no adverse  effects.  Most  workers with  blood DNOC  levels be-
low  20 mg/kg  are not  affected, although because of  individual susceptibil-
ity, some exhibited mild effects.  The  blood level  of  20  mg/kg has  been used
as  a  maximum  permissible level  for  industrial  or  agricultural workers  uti-
lizing the compound during employment.
                                     C-76

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    Bidstruo, et  al.  (1952)  recommeded  that a person should be  removed  fron
further contact with  ONOC for at  least  six weeks  if  the  blood level eight
hours after the last exposure was 20 mg/kg or higher.
    Other data on  the elimination of DNOC from humans  were  not  found.
                                    EFFECTS
Acute. Subacute, and Chronic Toxicity
    The acute  toxic dose of DNOC with  different  routes of administration,
has been  determined for a number  of different experimental animal species.
These data have been compiled and presented in Table 12.
    Although the available human toxicity data do  not permit the calculation
of  the  acute lethal dose for  ONOC  in humans,  it  has been estimated  (Fair-
child, 1977) that  5 mg/kg may prove lethal  to humans.
    A  large  number of occupational  and  nonoccupational  poisonings  of  humans
by DNOC have  been  reviewed  by NIOSH (1978).   The available literature  con-
cerning humans  indicates  that  DNOC may be  absorbed  in  acutely toxic  amounts
though the respiratory and gastrointestinal  tracts  and  through  the  skin,  and
that  it  accumulates  in the  blood.   Individuals  exposed   to  DNOC  by  these
routes usually  demonstrate  signs of  increased metabolic rate.  Symptoms  of
poisoning  include  profuse   sweating,  malaise,  thirst,  lassitude,  loss  of
weight, headache,  a  sensation  of  heat,  and  yellow  staining  of  the  skin,
hair, sclera, and  conjunctiva.
    In additon  to  the  effects  associated  with  increased   metabolism,  other
effects occasionally  reported in  humans  poisoned  by  DNOC included  kidney
damage, diarrhea,  unspecified  changes in the  gastrointestinal  tract,  in the
cardiovascular  system,  and   in the peripheral vascular  and central  nervous
systems.
                                     C-77

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               TABLE 12




Acute Toxicity of 4,6-Dinitro-o-cresol
Dose
Species (-rig/kg)
Mouse 187
Ratbit 1000
Guinea Pig 500
Rat 85
Rat 30
Rat 40
Rat 30
Mouse 47
Mouse 16.4
Hare 24.8
Cat 50
Pheasant 8.4
Partridge 8.3
Rat 26-39
Rat 20
Mouse 24.2
Rat 24.6
Goat 50
Ooq 15
Dog 5
Ooq 10
Pigeon 5
MLD = Minimum Lethal Dose
s.c. = subcutaneous
i.m. = intramuscular
i.v. = intravenous
i .p. = intraperitoneal
Route of
Administration
Dermal
Dermal
Dermal
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
s.c.
s.c.
s.c.
s.c.
s.c.
i.v.
i.m.
i.p.
i.m.






Effects
LD50
LD50
100% Lethal
LD50
MLD
100% Lethal
I-DSO
LD50
LD50
L050
L050
LD50
LD50
LD50
MLD
LD50
LD50
LD50
LD
LD
LD
LD






References
Arustamyan, 1972
Burkatskaya, 1965
Spencer, et al . 1948
Burkatskay, 1965
Ambrose, 1942
Ambrose, 1942
Spencer, et al. 1948
Burkatsukaya, 1965
Arustamyan, 1972
Janda, 1970
Burkatsukaya, 1965
Janda, 1970
Janda, 1970
Harvey, 1952
Ambrose, 1942
Parker, et al. 1951
Spector, 1956
Ambrose, 1942
Soector, 1956
Spector, 1956
Soector, 1956
Spector, 1956






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     It  is  gene^i1;/ relieved that the  toxic  effects  of D'.CC ^esjlt f'-om its
ability  to  uncouple the oxidative phosphorylat ion  process.   DNOC  is  an ex-
tre^ely  potent  uncoupler of oxidative  phosphorylation.   This  effect results
in  tie  decreased for-nation  of  adenosine  triphosphate  (ATP) and  a resulting
inhibitory effect of enzyme  reactions  requiring  ATP.   Such a toxicant is ex-
pected to have extreme and profound effects  on  all  tissues where  the concen-
tration  of  the  chemical  is  high enough  to severely  affect oxidative phos-
ohorylation.  Since energy generated  in the body cannot  be  converted  to its
usual  form  (ATP) in the  presence of DNOC,  it  is released  as  heat instead,
causing many of  the commonly observed signs and symptoms of DNOC toxicity.
     Several  investigators  have  correlated blood  DNOC  levels with  the sever-
ity  of toxic effects in  humans  (Harvey, et al. 1951;  Bidstrup, et al. 1952;
Pollard  and  Filbee,  1951)  and  have shown  that,  unlike the situation in ani-
mals, DNOC accumulates in  the  blood  of humans.   Accumulation  is  believed to
occur as  a  result of  DNOC  binding  to  albumin  in the  blood (Harvey,  et al.
1965).   In one of the  few  cases  where  DNOC in the blood  of  a  poisoned human
was monitored throughout his recovery  period,  (Pollard and Filbee, 1951) the
severity of the  symptoms decreased as  blood levels of DNOC  decreased.  Data
on blood DNOC levels in  humans  and the accompanying effects are compiled and
presented in Table 13.   The  data show  that workers with  DNOC  concentrations
of 40 mg/kq  of  whole blood  (approximately 80 mg/1  of serum) or greater will
Tiost  likely  develop toxic effects.   In  the  concentration range  between 20
and  40 mg/kg of whole blood,  some  workers  are  affected  and others  show no
adverse effects  (probably  because of  differences in  individual  susceptibil-
ity).  fj1ost  individuals  with blood  levels of DNOC  below 20 mg/kg  were not
affected, although some exhibited mild  effects.   As the data in  Table 4 sug-
gest, most  investigators  have  concluded  that  blood DNOC  levels  are associ-
ated with the severity  of intoxication in humans (NIOSH, 1978).
                                     C-79

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                                          TABLE 13

                  Relationship to Blood DNOC Levels and Ejects in Humans*
Route
Exposure
Inhalation, Dermal
Inhalation, Dermal

Inhalation, Dermal
Inhalation, Dermal

Inhalation, Dermal

Inhalation, Dermal
Inhalation, Dermal
Oral

Inhalation, Dermal
Inhalation, Dermal
Inhalation, Dermal


Inhalation, Dermal

Inhalation, Dermal
Inhalation, Dermal
Inhalation, Dermal
Inhalation, Dermal
Inhalation, Dermal
Inhalation, Dermal
Inhalation, Dermal
Inhalation, Dermal
Inhalation, Dermal
Oral

No. of Individuals
and Occupation
1 Agricultural Worker
1 Agricultural Worker

1 Agricultural Worker
1 Agricultural Worker

1 Agricultural Worker

1 Agricultural Worker
2 Agricultural Workers
5 Experimental Subjects

4 Agricultural Workers
5 Agricultural Workers
6 Agricultural Workers


32 Agricultural Workers

1 Agricultural Worker
16 Agricultural Workers
1 Agricultural Worker
21 Agricultural Workers
149 Agricultural Workers
4 Agricultural Workers
23 Agricultural Workers
1 Agricultural Worker
2 Manufacturing Workers
5 Experimental Subjects

Blood DNOC
Level (mg/kg)
10003, b
2003, b

75
60

60a»b

55
44-55
40-48

20-403
30-40
21-403


7-3?a

303
20-30
253
10-20
<10
4_ga,b
l-8a»b
<53,b
10-20
20

Effects
Death
Sweating, labored
breathing, vomiting
Death
Headache, lassitude,
BMR 275%
Sweating, headache,
labored breathing, fatigue
Unconsciousness
Acute Poisoning
Headache, lassitude,
malaise
Liver damage
No effects
Moderate poisoning;
recovery period longer
than 8 days
Mild poisoning; recovery
within 8 days
Fever
No effects
Kidney damage
No effects
No effects
Sweating, thirst
No effects
Fatigue
No effects
Exaggerated feeling of
well-being
*Source: NIOSH, 1978
Reported as mg/1
bSerum or Plasma DNOC Level
BUR = Basal  Metabolic Rate
                                          :-ao

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     In  conparing  studies  on blood  DNOC  levels,  certain orecautions must  3e
taken when  correlating the  results.   It  has  been reported that over 90  per-
cent  of the DNOC  detected  in  the  blood is  found  in  serum (Parker, et  al.
1951) and that most of this amount  is bound  to albumin  in  humans  (Harvey,  et
al.  1951).  A  comparison  of numerically similar blood  DNOC  levels  expressed
as  weight/volume  of  serum  with  those  expressed as  weight/weight  of whole
blood can therefore only  be done  by approximate  conversions.   Any given  O.NOC
serum level will have a lower value when expressed  per unit of  whole blood.
     It  is  impossible  to  develop  a  dose-response  relationship  for occupa-
tional DNOC poisoning  in  humans  since air concentrations of DNOC are  rarely
reported  and  the  exposure time of  poisoned  individuals is  highly  variable.
In  most  cases  of human poisoning  total  exposure amounts  can  only be esti-
mated.   This   lack  of data  make's  assessment  of  a minimum  toxic  dose  for
humans  extremely  difficult.   Several studies  however, where  the  oral  tox-
icity of ONOC has been assessed in humans, shed some light on this question.
    Harvey, et  al.  (1951) orally administered DNOC to five male volunteers
and studied both the  resulting  blood, levels  and  toxic  effects.  Each man was
given capsules  containing  75  mg of pure DNOC  daily  for five  consecutive
days, amounting  to  a total dose  of 0.92 to 1.27 mg/kg/day.   The men  exper-
ienced an  exaggerated sense of well-being  when  blood  levels  were about  20
mg/kg.   Headache,  lassitude,  and  malaise  were associated  with DNOC  blood
levels  of  40  to  48  mg/kg.   Although  individual  variation was  evident  in
these experiments,  it  is  obvious  that chronic administration  of  1  mg/kg/day
DNOC  to  healthy humans may  result  in  signs of  toxicity.  The  exaggerated
sense of well-being  described  by Harvey, et  al. (1951)  is  a  typical sign of
impending toxic effects among agricultural workers  exposed to DNOC.
                                     C-81

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    ONOC was  introduced  in 1933 as  an  alternative to dinitrophenol for  tie
treatment of  obesity  (NIOSH,  1978).  Many  poisonings,  and  some deaths,  re-
sulting  from  overdoses  were reported, as well  as the development  of  catar-
acts  in  some patients,  months  after  they  had  stopped  taking  ONOC.    Some
patients developed  symptoms  of DNOC  poisoning  at  the  accepted  theraputic
dose  level.   Signs  and  symptoms   of DNOC  intoxication  including thirst,
fatigue, excessive sweating, decreased appetite,  and  elevated  basal  metabol-
ic rates, appeared  in  three persons who had taken as little  as 0.35  to  1.5
mg/kg/day of  ONOC for  up  to  9 weeks  (Plotz,  1936).   Hunter (1950)   noted
that, although,  less than one percent of those  individuals treated  with  ONOC
developed complications, he considered the  difficulty of  setting  a  safe  dose
for each individual to  be the reason that  its  use as an aid  to weight  loss
was discontinued.
    Although ONOC is considered a cumulative poison  in  humans, probabl-y  as  a
result of  slow  metabolism  and  inefficient  excretion,  true  chronic or  sub-
acute effects (with the possible exception  of cataract  formation)  have never
been reported in  either human  or experimental  animals.  Signs and symptoms
of toxicity occur when the  total body burden exceeds  a  threshold  level.   The
toxic effects noted after either acute or chronic administration  are similar
in  quality  and  their  severity appears  to be correlated with  DNOC  blood
levels (and by  inference,  total body burden).   It is generally  agreed  that
the toxic manifestations  of DNOC  result from its  potent  effects  on metabol-
ism (NIOSH,  1978).
    Several  long-term  studies designed to  determine  dietary  levels of  DNOC
necessary to  cause toxic  symptoms   in  experimental   animals  have  been  con-
ducted.  Spencer,  et  al.  (1948)  maintained rats  on  a diet  containing  DNOC
for  six  months.  Growth  curves,  periodic  blood  counts,  analyses  of  urea-
                                     C-82

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 nitrogen,  organ  weights,  and nistrcpathological  examinations  were  performed
 on  a:l  animals.   'Jo  adverse effects on these parameters were  detected  among
 rats  fed  diets containing  100 mg  3NOC/"
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health effects  in  humans.   Exposure  to airborne ONOC at  concentrations  that
averaged  0.9  mg/m  produced  unspecified  changes  in the  cardiovascular  sys-
tem, the  central  and  autonomic  nervous systems, the gastrointestinal  tract,
and the cell  pattern  of  the  peripheral  blood  of workers involved in manufac-
turing and  applying DNOC.   In  agricultural  workers  exposed to  ONOC at  an
average  concentration  of  0.7  mg/m  ,  slight  unspecified  changes  in  the
blood and autonomic nervous system were observed.
    Another study  (Batchelor, et  al.  1956) revealed that  agricultural spray-
ers exposed to  an airborne  ONOC  concentration about  0.23  mg/m   failed  to
demonstrate adverse effects  of  the compound.   No symptoms of poisoning  were
observed  and  blood ONOC  levels were  well  below those  associated  with  toxic
effects.
    In the  study by  Burkatskaya  (1965)  the  effect of  airborne  ONOC  on  cats
was  examined.   Cats  exposed at  0.2  mg/m   for   two  or three months  had
slightly  increased body  temperatures  and  leucocyte  counts  and decreased  hem-
oglobin concentrations,  erythrocyte  counts,  and catalase  and peroxidase ac-
tivities.   The  changes, which  were  characterized  as  slight and  transient,
occurred  after  one to two weeks but  further  exposure  produced  no additional
effects.
    The  report  by  NIOSH (1978)  concludes  "since  only slight   effects  were
seen  in workers exposed to  ONOC  at  an average  concentration  as  low  as 0.7
mg/m   for  an  unspecified  duration,  and  since short-term  exposure  at 0.2
mg/m   had no  lasting effect on  cats."  NIOSH  recommends  that  the current
federal workplace environmental  limit of 0.2 mg/m   be retained.
    It is  possible to calculate  the  anticipated daily exposure of  a  70  kg
human  male  exposed to ONOC  at  0.2 mg/m  for  an 8-hour  period.   If one as-
sumed  the  average  minute volume was  28.6  1  of  air/minute (NIOSH,  1973) the
anticipated daily exposure  is 39 ug/kg/day.
                                     C-84

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     If  one assures  that  abso^ot'on  of 3NGC across  the  respiratory  tract,  -s
 identical  to  gastrointestinal  absorption,  and that  a  70 kg human male  con-
 sumes  2.0  liters  of water  daily,  the  following  calculation  indicates  the
 maximum  allowable  levels of  DNOC  in drinking  water  based   on  the  NIOSH
 recommendation  for workplace air.
                      39  ug/kg/day x 70 kg = 2.73 mg/day
                            2.75  mq/day m  1.33  mg/i
                            2 I/day
    Although  NIOSH  (1978)  states  "the  standard was  not designed for  the  pop-
 ulation-at-large,  and  any extrapolation beyond the  occupational  environment
 is  not warranted,"  development of a  baseline  level  for chronic human effects
 using the same data used  by NIOSH appears  to be  a  reasonable  approach to the
 development of a water criterion.
    In summary,  daily  human  exposure to 0.35  mg/kg  ONOC may  result  in signs
 of  intoxication  in humans.  Some  persons  develop cataracts  as a result  of
 chronic  exposure to DNOC,  but  the  no-effect-level  for cataracts cannot  be
 calculated.   Although  true "chronic" effects  of  DNOC  have never  been docu-
 mented,  the compound accumulates  in the  human  body and  toxic symptoms may
 develop when blood levels  exceed  20  mg/kg.  Such  symptoms  have been  observed
 in  humans  receiving as  little  as 0.35 mg/kg/day over  a period  of several
weeks.  The no-observable-effeet-level  for  rats  in  long-term  feeding studies
has been variously reported  as  63 mg/kg food, 100 mg/kg food, and 200 mg/kg
 food.  Based  on  the available  human  and experimental  animal data,  NIOSH
 (1973)  has recommended   a  federal   workplace  limited  of 0.2 DNOC/m   air.
 Based on an estimate of   iman exposure for an eight-hour work  shift,  it was
calculated  that  a  drinking   ••ter  level  of  1.4  mg/1  would result in a similar
exposure to the general  population.
                                     C-85

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    De*-tinent  information  could not  be  located in the  available  literature
describing synergistic or antagonistic effects associated with DNOC.
Ta>-atocem'ci ty
    Pertinent  information  could not  be  located in the  available  literature
regarding the presence or absence of teratogenic properties of DNOC.
Mutagenicity
    Andersen,  et  al.  (1972)  reported an  evaluation  of  the ability  of  110
herbicides,  including  DNOC,  to  produce  point mutations  in  histidine-depen-
dent  mutants  of  Salmonella  typhimurium,  bacteriophage  T4,   and  in   two  RII
mutants  of  bacteriophage  T4.    The  culture  media  were  prepared  by  mixing
freshly  grown  cultures  of  the  mutants with soft  agar  and pouring  into petri
dishes.  After the  agar  solidified,  DNOC was  applied  to  the  surface  of each
plate.   They  found  that the mutation  frequency rates produced  by ONOC were
no greater than the spontaneous rates.
    Nagy, et al.  (1975)  tested  ONOC  for  its ability to induce back-mutations
of her*  and her" derivatives of E_.  col i WP2 Try-  bacteria.   DNOC failed to
induce reverse mutations in this system.
    The  difference  in  growth  inhibitions of wild type Proteus  mirabilis  and
the corresponding repair-deficient  strain  has been  used by Adler,   et  al.
(1976) as an  indication of DMA  damage.   Evidence of  DNA damage in  the pres-
ence of ONOC was reported.
    Information  on  the  potential  mutagenicity of  DNOC  for mammals  is  not
available.
                                     C-86

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Carcinogenicity
    Spencer, et  al.  (1948)  failed  to report  tumor formation  in  rats  main-
tained on  diets  containing ONOC  for  six moths.   Similarly,  no tumors  were
reported in  rats  maintained  on diets containing DNOC  for  105 days (Ambrose,
1942)  or 126 days (Parker, et al.  1951).
    No further  information was found  regarding the  presence or  absence  of
carcinogenic properties of DNOC.
                                     C-87

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Existing 3ui3e1ines and Standards
    U.S.  standards  for  exoosure to  the  nitrophenols  or dinitrocresols  in
drinking or ambient water have not been set.
    The  following  limits for  toxic  substances  in  drinking  water  have  been
set in the USSR (Stofen, 1973):
                 2-nitrophenol                0.06 mg/1
                 3-nitrophenol                0.06 mg/1
                 4-nitrophenol                0.02 mg/1
               2,4-dinitrophenol              0.03 mg/1
    Based  on  organoleptic  considerations,  a  limit  of 0.5  mg/1  for  2,4,6-
trinitrophenol has been set by the USSR (Stofen, 1973).
    The  maximum  air  concentration established by the American  Conference  of
Governmental  Industrial  Hygienists  (ACGIH,  1971)  is  0.1  mg/m  for  2,4,6-
trinitrophenol  and  0.2  mg/m   for  4,6-dinitro-o-cresol   for  an  eight-hour
exoosure (TLV).
    The  Code  of  Federal  Regulations  (40  CFR Part  180) establishes a  toler-
ance of  0.02  rug/kg for residues of 4,6-dinitro-o-cresol  and  its  sodium  salt
in  or  on  apples  resulting  from  applications  to apple  trees at  the blossom
stage as a fruit-thinning agent.
Current Levels of Exposure
    Human  exposure  to  the nitrophenols  or  dinitro-o-cresols  has  not  been
monitored.  Unspecified  amounts  of  4-nitrophenol have  been detected  in  sam-
ples of urban ambient particulate matter.
    The  photochemical  reaction between benzene  vapor  and nitrogen monoxide
results  in the production  of  2-nitrophenol,  4-nitrophenol,  2,4-nitrophenol,
and 2,6-dinitrophenol under laboratory  conditions  and 4-nitrophenol has  been
                                      :-88

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 detected  in rainwater  in  Japan.   Available  data  indicate that  the  general

 public  may  be  exposed to  nitrophenols  in the atmosphere  when  severe  photo-

 chemical  fog conditions develop.   Quantitative  estimates of  such exposures
 are not possible at the present time.
    4-Nitrophenol  has  been detected in the urine  of  1.0 percent of the gen-

 eral  population  at  levels  as  high  as 0.1  mg/1 (with a mean urinary level  of

 10 ug/1).
    If  it is assumed  that urinary residues  of  4-nitrophenol  reflect  direct
 exposure  to the compound,  a  pharmacokinetic  estimate  of exposure based  on
 steady-state conditions can  be  made.  The exposure level  leading  to  the 1.0
 ug/1  residue can be calculated as follows.

          Exposure = (10 ug/1) (1.4  1 of urine/day) = o.02 ug/kg/day
                            (70 kg/man)
    A  similar  calculation using  the  maximum urine  residue   level  observed
 (113.  ug/1) gives an exposure of 2.26 ug/kg/day.

    However, these urine levels are  not oelieved to result from direct expo-
 sure  to 4-nitrophenol.  A  number  of widely used  pesticides,  including para-
 thion,  are  readily metabolized  to 4-nitrophenol  in  the human  body and are

 believed to be the source of 4-nitrophenol residues in human urine.
    Current  levels  of human  exposure  to  the  nitrophenols  or  dinitrophenols
 (with the possible exception  of 4-nitrophenol) are either very low, nonexis-
 tent,  or  have gone  undetected.   In the  absence  of data  any of  the above
could be operative.
 Special  Groups  at Risk
    The only individuals  expected  to be at   risk  for  high  exposure  to the
 nitrophenols are industrial workers  involved  in  the manufacture of compounds
 for which the m'trophenols are  intermediates.  Since  picric  acid (2,4,6-tri-
                                     C-89

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chloroohenol)  ~nay  find some jse  as  an explosive, germicide,  tanni-ng  agent,
fungicifle,  tissue  fixative, or  industrial  process  material,  a  higher  ris<  of
exposure exists among personnel engaged in such operations.
    Although  4,6-dinitro-o-cresol  (DfJOC)  is  no  longer  manufactured   in  the
U.S.,  a  limited quantity  is  imported  and  used as a blossom-thinning agent on
fruit  trees and as  a fungicide,  insecticide,  and miticide  on  fruit  trees
during  the  dormant season.  Hence,  individuals formulating or  spraying  the
compound incur the highest risk of exposure to the compound.
3asis  and Derivation of Criterion
    The   -ganoleptic   thresholds  for  mononitrophenols  in  water range  from
0.24 to  339  mg/1.   These  levels,  extracted from the  Russian  literature,  are
detection thresholds;  acceptability  thresholds from  the  standpoint of human
consumption are not available.
    With the exception of  a single study  abstracted  from the Russian  litera-
ture,  data  on  chronic mammalian  effects  of the mononitrophenols  are  absent
from the 1iterature.
    The  Russian investigation  (Makhinya,  1969)  was reported in abstract form
only.  Attempts to obtain  the  full  report  proved  fruitless.   The investiga-
tors reported  distinct cumulative  toxic  properties  of  the  mononitrophenol
isomers  in  mammals.   Threshold  levels  for effects  of  mononitrophenols  on
conditioned  reflexes  were  reported,  but details of  the  experiment  including
animal  species, mode  of  administration, duration  of  the  experiment,  and  the
exact  parameters measured  are  not available.   Hence, it  does  not  seem pru-
dent to develop a criterion based on  these results.
    In  the  absence of data on  chronic mammalian  effects no water  criterion
for human health can  be established  for  any of th .•  mononitrophenol  isomers
at this  time.
                                     C-90

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     Information  on  the  dinitrophenol  isomers  is  limited  to  2,4-dinitro-
phenol.   Spencer, et  al.  (1943),  in  a six-month feeding study with  rats  ad-
ministered  5 dietary  levels  of  2,4-dinitrophenol,  demonstrated the  no-ob-
served-effect-level (NOEL) to be  between  5.4 mg/kg and 20 mg/kg.   Using  the
lower  of  the  two  figures  and  assuming a 70 kg man consumes 2 liters of water
daily  and 6.5 grams  of contaminated fish  having  a BCF  of  1.51 the  corre-
sponding   NOEL   for   humans   based  on   the   results   obtained   in   rats
may be calculated as follows:
                          5.4 mg/kg x 70 kg = 378 mg
                   	378 mg	 = i88 mg/1
                   2 liters + (1.51 x 0.0065) x 1.0
    Based on  these  calculations,  no  biological effect would be  predicted  in
a man drinking water containing 2,4-DNP at 188 mg/1.
    Experience with the use of 2,4-DNP  as  an  anti-obesity  drug  in the  1930's
indicates that  adverse effects,  including cataract formation,  may  occur  in
utimans exposed to as little as 2 mg/kg/day.   The drug  was  frequently used  in
an uncontrolled manner and the available  data do  not  allow the calculation
of a  no-adverse-effect-level  in humans.   It  is clear, however,  that  inges-
tion of 2,4-ONP at  2  mg/kg/day  for a protracted period may result in adverse
effects,  icluding cataracts,  in  a small proportion of  the population.   This
dietary   intake   level   consitutes   a   low-observed-adverse-effect-level
(LOAEL).  Assuming a  70 kg man  consumes 2  1  of water daily and  6.5  grams  of
contaminated  fish   having  a  BCF   of   1.51   and  assuming   100   percent
gastrointestinal  absorption  of   2,4-ONP,  a   2   mg/kg   dose   of   2,4-ONP
would result if drinking water contained 2,4-ONP at 69.7 mg/1.
            	2mq/kg/day x 70 kg	= 69.7 (or - 70  mg/1)
           (2 liters + (1.51 x 0.0065) x 1.0
                                     C-91

-------
    According to current guidelines, extrapolation from a  LOAEl  requires  ap-
plying  an  uncertainty factor of  10.   Furthermore,  in  light  of the demon-
strated  bacterial  mutagenicity of  2,4-DNP (Oemerec, et   al .  1951)  and  the
suspected  ability  of the  compound  to  induce  chromosomal  breaks in mammals
(Mitra,  and  Manna, 1971),  an  additional  uncertainty factor of 100 must  oe
used in the criterion formulation.
    The suggested water criterion for 2,4-DNP  is, therefore:
                               70 mg/1  =» 70 ug/1
                              10 x 100
If exposure  is  assumed to result  from the consumption of  contaminated  fish
or shellfish only, the criterion is 14.3 mg/1.
    The available data  are  insufficient to enable calculation of  water  cri-
terion levels for the  remaining dinitrophenol  isomers.  For the present,  it
seems reasonable to assume that  the  2,4-dinitrophenol  criterion  would  be ap-
propriate for the other isomers.
    Chronic  mammalian   toxicilogy  data  for  the  trinitrophenols  are  absent
from the  literature.   An outbreak of  microscopic hematuria among shipboard
U.S.  Navy personnel exposed  to 2,4,6-trinitrophenol  in  drinking water has
been reported, however.  It is not possible to precisely  estimate  either the
2,4,6-trinitrophenol water  level  or  duration of  exposure  required for the
development of  hematuria.   Consequently, criteria for  trinitrophenol  cannot
be derived.
    Although  4,6-dinitro-o-cresol  (DNOC)  is  considered a  cumulative  poison
in humans,  probably as a result of slow  metabolism and inefficient  excre-
tion, true  chronic  or  subacute effects have  never  been reported  in  either
humans or  experimental  animals.  Since  DNOC  is   not  a cumulative poison  in
experimental  animals,  extrapolation  to humans from  long-term  animal  studies
is of questionable value.
                                     C-92

-------
    The  no-ODservab'e-effee'-level  i'JCEL)  for  3'
-------
    Sufficient data  is  not available  with  which  to  derive



teria for other dinitro-o-cresol  isomers.
                                         C-94

-------
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'•!un"ec
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Kokuritsu Eisei  Shikenjo.  94: 28.
                                              U S OOV CRN WENT PRINTING OFFICE :"' 'CJ-Jlc
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