NITROBENZENE
Ambient Water Quality Criteria
               Criteria and Standards Division
               Office of Water Planning and Standards
               U.S.  Environmental Protection Agency
               Washington,  D.  C.    20460

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                        CRITERION DOCUMENT



                           NITROBENZENE



CRITERIA



                           Aquatic Life



     For nitrobenzene the criterion to protect freshwater  aquatic



life as derived using the Guidelines is 480 ug/1  as  a  24-hour



average and the concentration should not exceed 1,100  ug/1 at  any



time.



     The data base for saltwater aquatic life  is  insufficient  to



allow use of the Guidelines.  The following recommendation is



inferred from toxicity data for freshwater organisms.



     For nitrobenzene the criterion to protect saltwater aquatic



life as derived using procedures other than the Guidelines is  53



ug/1 as a 24-hour average and the concentration should not exceed



120 ug/1 at any time.



                           Human Health



     For the prevention of adverse effects due to the  organo-



leptic properties of nitrobenzene in water, the criterion  is 30



ug/i.

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Introduction



     Nitrobenzene is produced for industrial use by  the  nitra-



tion of benzene with nitric and sulfuric acids.  Estimates of



annual nitrobenzene production range from 200 to over 700 mil-



lion pounds (Dorigan and Hushon, 1976; Lu and Metcalf, 1975).



The principal use of nitrobenzene is for reduction to aniline,



which is widely used as an ingredient for dyes, rubber,  and



medicinals (McGraw-Hill, 1971; Kirk and Othmer, 1967).   The



commercial applications of nitrobenzene are:  reduction  to



aniline (97 percent), solvent for Friedel-Crafts reaction,



metal polishes, shoe black, perfume, dye intermediates,  crys-



tallizing solvent for some substances, and as a combustible



propellent (Dorigan and Hushon, 1976).



     Nitrobenzene is stored in closed containers and  is  not



usually released to the open air.  Atmospheric contamination



is usually prevented in plants manufacturing or using nitro-



benzene by the use of activated charcoal absorbers or a  car-



bon dioxide blanket.  There is no industrial monitoring  of



nitrobenzene in the atmosphere.  The greatest loss of nitro-



benzene during production  (estimated as eight million pounds



annually) occurs at the acid extraction step in the  purifica-



tion of the crude reaction mixture, when nitrobenzene is lost



to the effluent wash (Dorigan and Hushon, 1976).  Thus,  the



greatest exposure to nitrobenzene occurs inside plants and



most cases of chronic nitrobenzene exposure in man are nitro-



benzene workers.  Today plant levels of nitrobenzene  are usu-



ally kept below the threshold limit value (TLV) of 5 mb/m^



(Goldstein, 1975; Am. Conf. Gov. Ind. Hyg., 1977) but much
                              A-l

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higher levels have been reported  in the past  (Pacseri  and



Magos, 1958).  Nitrobenzene may also form spontaneously  in  the



atmosphere from the photochemical reaction of benzene  with



oxides of nitrogen.



     Nitrobenzene, also known as  nitrobenzol, essence  of mir-



bane, and oil of mirbane, is a pale yellow oily liquid with an



almond-like odor (Kirk and Othmer, 1967).  The color of  the



liquid varies from pale yellow to yellowish brown depending on



the purity of the compound  (Kirk  and Othmer,  1967).  In  the



solid state it forms bright yellow crystals.  Nitrobenzene,



CgH5NC>2, has a molecular weight of 123.11 g.



     The physical properties of nitrobenzene  are as follows:



a boiling point of 210 to 211C  at 760 mm Hg, a melting point



of 6C, a density of 1.205 at 15C, a refractive index of



1.5529, and a flash point of 89C (Stecher, 1968).  It is



steam volatile (Stecher, 1968) and at 25C nitrobenzene  has a



vapor pressure of 0.340 nun Hg (Jordan, 1954).



     Nitrobenzene is miscible with most organic solvents, such



as ethanol, diethyl ether, acetone, and benzene (Kirk  and



Othmer, 1967).  It is slightly soluble in water, 0.1 per 100



parts of water (1,000 mg/1) at 20C (Kirk and Othmer,  1967).



In aqueous solutions, nitrobenzene has a sweet taste (Kirk  and



Othmer, 1967).



     Nitrobenzene undergoes substitution reactions but re-



quires more vigorous conditions than does benzene.  Substitu-



tion takes place at either the meta (3) position or the  ortho-



(2) para-(4) positions depending  on the physical conditions
                             A-2

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(Kirk and Othmer, 1967).  Nitrobenzene undergoes photoreduc-



tion when irradiated with ultraviolet light in organic sol-



vents that contain abstractable hydrogen atoms (Barltrop and



Bunce, 1968).



     Nitrobenzene is a fairly strong oxidizing agent  (Kirk and



Othmer, 1967; Millar and Springfield, 1966).  Since the com-



pound can act as an oxidizing agent in the presence of aqueous



solutions of alkali hydroxides, it has the capability of oxi-



dizing compounds containing free phenolic hydroxyl groups



without effectively changing these groups (Millar and Spring-



field, 1966).  Nitrobenzene is reactive and will undergo



nitration, halogenation, and sulfonation by the same methods



used for benzene.  However, these reactions are unlikely to



occur in environmental conditions.



     The reduction of nitrobenzene to aniline probably out-



ranks all other uses of nitrobenzene as an industrial chemical



(Kirk and Othmer, 1967).  The di- and the trinitrobenzenes are



used in military and industrial explosives.  The great toxi-



city of nitrobenzene impairs its usefulness as an organic sol-



vent.  It is readily absorbed by contact with the skin, inha-



lation of the vapor, or by ingestion.  The absorption of



nitrobenzene into the body produces cyanosis (Kirk and Othmer,



1967).



     Nitrobenzene has been found to have a metabolic  turnover



slow enough to result in distinct accumulation under  condi-



tions of daily exposure (Piotrowski, 1967).  Complications



could arise from the possible accumulation of either  nitro-
                             A-3

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benzene or p-nitrophenol.  The half-life of  the excretion  of



the p-nitrophenol  is approximately 60 hours  (Salmowa,  et al.



1963).



     The toxicological data on the effects of nitrobenzene  are



limited primarily  to mammalian, especially human,  studies  and



case histories.  There are few data on the toxic effects of



nitrobenzene to aquatic organisms.  A freshwater fish  acute



value for nitrobenzene was found to be 42,600 ug/1 with a



chronic value of more than 16,000 ug/1.  A saltwater acute



value was 58,539 ug/1.  In the case of mammals, nitrobenzene



is highly toxic when ingested, inhaled, or absorbed through



the skin.  Exposure by any of these routes can result  in head-



aches, drowsiness, nausea, vomiting, and methemoglobinemia



with cyanosis.
                             A-4

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                          REFERENCES







American Conference of Governmental Industrial Hygenists.



1977.  Documentation of the threshold limit value for sub-



stances in workroon air.   Cincinnati, Ohio.







Barltrop, A.J.,  and N.J.  Bunce. 1968.  Organic photochemis-



try, Part 4.  The photochemical reduction of nitre-compounds.



Jour. Chem. Soc.  Sec. C.  12: 1467.







Dorigan, J., and  J. Hushon. 1976.  Air pollution assessment



of nitrobenzene.   U.S. Environ. Prot. Agency.







Goldstein, I. 1975.  Studies on MAC values of nitro- and



ammo-derivatives of aromatic hydrocarbons.  Adverse Effects



Environ. Chem. Psychotropic Drugs 1: 153.







Jordan, T.E. 1954.  Vapor pressure of organic compounds.



Interscience Publishers,  Inc., New York.







Kirk, R.E., and  D.F. Othmer. 1967.  Kirk-Othmer Encyclopedia



of Chemical Technology.  2nd ed. John Wiley and Sons, Inc.,



New York.








Lu, P.Y., and R.  Metcalf. 1975.  Environmental fate and bio-



degradability of  benzene  derivatives as studies in a model



aquatic ecosystem.  Environ. Health Perspect. 19: 269.
                            A-5

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McGraw-Hill. 1971.  Encyclopedia of science and  technology.



McGraw-Hill Book Co., New York.








Millar, I.T., and H.D. Springfield, eds. 1966.   Sidgwick's



organic chemistry of nitrogen.  3rd ed.  Clarendon Press,



Oxford.







Pacseri, I., and L. Magos. 1958.  Determination  of the mea-



sure of exposure to aromatic nitro and amino compounds.



Jour. Hyg. Epidemiol. Microbiol. Immunol. 2: 92.







Piotrowski, J. 1967.  Further  investigations on  the evalua-



tion of exposure to nitrobenzene.  Br. Jour. Ind. Med.   24:



41.








Salmowa, J., et al. 1963.  Evaluation of exposure to nitro-



benzene.  Br. Jour. Ind. Med.  20: 41.








Stecher, P.G. , ed. 1968.  The  Merck Index. 8th ed. Merck and



Co., Inc., Rahway, N.J.
                             A-6

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AQUATIC LIFE TOXICOLOGY*
                       FRESHWATER ORGANISMS
Introduction
     Static tests with the bluegill, Daphnia magna,  and  the  alga,
Selenastrum capricornutum, indicate little difference  in  sensi-
tivity with no 50 percent effect concentrations lower  than 27,000
ug/1.  An embryo-larval test with the fathead minnow demonstrated
no adverse effects at the highest test concentration.
Acute Toxicity
     The 96-hour LC50 for the bluegill is 42,600 ug/1  (Table  1)
and, after adjustment for test methods and species sensitivity,
this result provides a Final Fish Acute Value of 6,000 ug/1.   The
Final Invertebrate Acute Value (1,100 ug/l)f based on  a  48-hour
EC50 of 27,000 ug/1 for Daphnia magna, is lower and, therefore,  it
becomes the Final Acute Value.
*The reader is referred to the Guidelines for Deriving Water
Quality Criteria for the Protection of Aquatic Life  [43  FR  21506
(May 18, 1978) and 43 FR 29028 (July 5, 1978)] in order  to  better
understand the following discussion and recommendation.  The  fol-
lowing tables contain the appropriate data that were  found  in  the
literature, and at the bottom of each table are the calculations
for deriving various measures of toxicity as described in the
Guidelines.
                             B-l

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Chronic Toxicity



     No adverse effects were observed during  an  embryo-larval  test



with the fathead minnow at test concentrations of  nitrobenzene  as ,



high as 32,000 ug/1  (Table 3).  After division by  the  sensitivity



factor  (6.7) a Final  Fish Chronic Value of greater than  2,400  ug/1



is obtained.  This also becomes the Final Chronic  Value  since



there are no reported results with invertebrate  species  and  EC50



results with an alga  are higher.  A criterion may  be derived in



the absence of a precise chronic value by using  0.44 times the



Final Acute Value  (1,100 ug/1) at which concentration  no adverse



effects were observed with the fathead minnow enbryo-larval  test.



Plant Effects



     The 96-hour EC50 values for reduction of cell numbers and



inhibition of chlorophyll a_ in the alga, Selenastrum capricornutum



are 42,800 and 44,100 vg/1, respectively (Table  4).



Residues



     No measured steady-state bioconcentration factor  (BCF)  is



available for nitrobenzene.  A BCF can be estimated using the



octanol-water partition coefficient of 71.  This coefficient is



used to derive an  estimated BCF of 15 for aquatic  organisms  that



contain about 8 percent lipids.  If it is known  that the diet  of



the wildlife of concern contains a significantly different lipid



content, an appropriate adjustment in the estimated BCF  should  be



made.
                              B-2

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CRITERION FORMULATION

                     Freshwater-Aquatic Life

Summary of Available Data

     The concentrations below have been rounded  to  two  significant

figures.

     Final Fish Acute Value = 6,000 ug/1

     Final Invertebrate Acute Value = 1,100 ug/1

          Final Acute Value = 1,100 ug/1

     Final Fish Chronic Value = greater than  2,400  ug/1

     Final Invertebrate Chronic Value = not available

     Final Plant Value = 43,000 ug/1

     Residue Limited Toxicant Concentration = not available

          Final Chronic Value = greater than  2,400  ug/1

          0.44 x Final Acute Value = 480 ug/1
                                *
     The maximum concentration of nitrobenzene is the Final  ^cute

Value of 1,100 ug/1 and the 24-hour average concentration  is  0.44

times the Final Acute Value.  No important adverse  effects on

freshwater aquatic organisms have been reported  to  be caused  by

concentrations lower than the 24-hour average concentration.

     CRITERION:  For nitrobenzene the criterion  to  protect

freshwater aquatic life as derived using the Guidelines is 480

ug/1 as a 24-hour average and the concentration  should  not exceed

1,100 ug/1 at any time.
                             B-3

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              Table  1   Freshwater fifah acute values for nitrobenzene  (U.S. EPA,  1978)


                                                                Adjusted
                        Bloaseay  Teat      Tine      LC50      LC50
                        Method*   Cong.**   (hrtQ     (uq/l>    (uq/1)


Blueglll,                  S        U        96       42.600    23.300
Lepomis macrochlrua
*  S = static

** U " unmeasured

   Geometric mean of adjusted  values - 23,300 pg/1    33,300 m fi(0oo ,lg/i

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CO
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                    Table   2.    Freshwater Invertebrate acute values for nitrobenzene  (U.S.  EPA,  1978)
                               fiiodssay  Test      rime
                               Hctnou*   Cone,**   (firs')
                                                             LC50
Adjusted
LCbO
Cladoceran,
Daphnia ma en a
S U 48 27.000 22,900
      *  S = static

      ** U = unmeasured


         Geometric mean  of adjusted   values  22,900 tig/1   22A?Q - 1,100 t>g/l

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>  7
   CTi
                       Table  3.   Freshwater fish chronic values for nitrobenzene (U.S. EPA, 1978)


                                                          Chronic
                                                Limits    Value
         organism                     Test*     lug/i)    (uq/1)


         Fathead minnow.               E-L      >32.000   >16,000
         Pimephales promelas
         * E-L = embryo-larval

           Geometric mean of cl

           Lowest chronic value  >16,000 Mg/1
Geometric mean of chronic values - >16,000 pg/l     &~T~   >2,AOO Mg/1

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              Table 4.  Freshwater plant effects for nitrobenzene  (U.S. EPA, 1978)


                                       Concentration
Organism                Eftect         (ug/l>	


Alga.                   EC50 96-hr         44.100
Selenaatrum             chlorophyll a
capricornutum

Alga.                   EC50 96-hr         42.800
Selenastrum             cell numbers
caprtcornutum
Lowest plant value  42,800 Mg/1

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                       SALTWATER ORGANISMS



Introduction



     Static acute  tests with  the sheepshead  minnow  and Mysidopsis



bahia indicate the latter  is  much more sensitive  to nitrobenzene.



Adverse effects were observed on the saltwater  alga at concentra-



tions slightly higher  than the LC50 for  the  mysid shrimp.



Acute Toxicity



     The 96-hour LC50  value for the sheepshead  minnow is  58,539



ug/1 and, after adjustment for test methods  and species  sensi-



tivity, this results in the Final Fish Acute Value'of 8,700   ug/1



(Table 5).



     As stated earlier, the mysid shrimp  is  more  sensitive with  a



96-hour LC50 of 6,676  ug/1 (Table 6).  The Final  Invertebrate



Acute Value derived from this test  is 120 ug/1; this concentration



also becomes the Final Acute  Value.



Chronic Toxicity



     No chronic tests  have been reported  on  the adverse  effects  of



nitrobenzene on saltwater organisms.



Plant Effects



     The cell numbers  of Skeletonema costatum were  reduced by 50



percent at a concentration of 9,650 ug/1  (Table 7).  Chlorophyll a_



was equally inhibited  at a concentration  of  10,300  ug/1-  The



lower of these two results is the Final Plant Value.



Residues



     No measured steady-state bioconcentration  factor (BCF)  is



available for nitrobenzene.   A BCF  can be estimated using the



octanol-water partition coefficient of 71.   This  coefficient is
                              B-8

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used to derive an estimated BCF of 15 for aquatic organisms  that
contain about eight percent lipids.  If it is known that  the diet
of the wildlife of concern contains a significantly different
lipid content, an appropriate adjustment in the estimated BCF
should be made.
                             B-9

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CRITERION FORMULATION
                      Saltwater-Aquatic Life
Summary of Available Data
     The concentrations  below have been rounded  to  two  significant
figures.
     Final Fish Acute Value = 8,700 ug/1
     Final Invertebrate  Acute Value = 120 ug/1
          Final Acute Value = 120 ug/1
     Final Fish Chronic  Value = not available
     Final Invertebrate  Chronic Value = not available
     Final Plant Value = 9,700 ug/1
     Residue Limited Toxicant Concentration = not available
          Final Chronic  Value = 9,700 u9/l
          0.44 x Final Acute Value = 53 ug/1
     No saltwater criterion can be derived for nitrobenzene  using
the Guidelines because no Final Chronic Value for either  fish  or
invertebrate species or  a good substitute for either value is
available.
     Results obtained with nitrobenzene and freshwater  organisms
indicate how a criterion may be estimated.
     For nitrobenzene and freshwater organisms 0.44 times the
Final Acute Value is less than the Final Chronic Value  which is
derived from an embryo-larval test with the fathead minnow.
Therefore, it seems reasonable to estimate a criterion  for nitro-
benzene and saltwater organisms using 0.44 times the Final Acute
Value.
     The maximum concentration of nitrobenzene is the Final  Acute
Value of 120 ug/1 and the estimated 24-hour average concentration
                              B-10

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is 0.44 times the Final Acute Value.  No  important  adverse  effects



on saltwater aquatic organisms have been  reported  to  be  caused  by



concentrations lower than the 24-hour average  concentration.



     CRITERION:  For nitrobenzene the criterion  to  protect



saltwater aquatic life as derived using procedures  other than the



Guidelines is 53 ug/1 as a 24-hour average  and  the  concentration



should not exceed 120 ug/1 at any time.
                             B-ll

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CO
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                      Table  5.   Marine fish acute values for nitrobenzene  (U.S. EPA,  1978)


                                                                        Adjusted

                                Bioaaeay  Teat      Time      LC50      LC50
                                Method*   gone.**   (hre)     tug/l>
        Sheepshead minnow,        S        U         96      58,539     32,004
        Cyprinodon variegatus
        *  S = static


        *A II = unmeasured


           Geometric mean of adjusted  values  32,004    ~TL7 " 8,700 Mg/1

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00
I
M
U)
                       Table  6    Marine Invertebrate acute values for nitrobenzene  (U.S.  EPA,  1978)


Soi^aisa
Mysid shrimp,
Mysldopaia bahia


bioassay Test Time
Metiiou* Cone.** (nrs.)
S U 96


LCSO
(uq/i)
6,676

Adjusted
LCbO
lun/ll
5.654

         *  S = static

         ** U  unmeasured


            Geometric mean of adjusted  values  5,654 vg/l
- 120 Mg/1

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                        Table  7   Marine plant effects for nitrobenzene  (U.S  EPA, 1978)


                                                 Concentration
          Organism                Effect         fug/lj	


          Alga.                    EC50 96-hr         9.650
          SUcletonema costaturn    cell numbers

          Alga,                    EC50 96-hr        10.300
          Skeletonema coatatum    chlorophyll a
          Lowest plant value - 9,650 pg/1
00
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                        NITROBENZENE



                         REFERENCES








U.S. EPA. 1978.  In-depth  studies on health and environmental



impacts of selected water  pollutants.   Contract No. 68-01-4646,
                              B-15

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                         NITROBENZENE



Mammalian Toxicology and Human Health Effects



                           EXPOSURE



Introduction



    Nitrobenzene, a pale yellow liquid at room temperature



with a characteristic bitter almond aroma, is also known  as



oil of mirbane, nitrobenzol, and artificial bitter almond



oil.  It is produced for industrial use by the nitration  of



benzene with nitric and sulfuric acids.  Estimates of  annual



nitrobenzene production range from 200 to over 700 million



pounds (Dorigan and Hushon, 1976; Lu and Metcalf, 1975).  The



principal use of nitrobenzene is for reduction to aniline,



which is widely used as an ingredient for dyes, rubber, and



medicinals.  The commercial applications of nitrobenzene  are:



reduction to aniline (97 percent), solvent for Friedel-



Crafts reaction, metal polishes, shoe black, perfumes, dye



intermediates, crystallizing solvent for some substances, and



as a combustible propellant (Dorigan and Hushon, 1976).



     Nitrobenzene is stored in closed containers and not



usually released to the open air.   Atmospheric contamination



is usually prevented in plants manufacturing or using  nitro-



benzene by the use of activated charcoal absorbers or  a car-



bon dioxide blanket.  There is no industrial monitoring of



nitrobenzene in the atmosphere.  The greatest loss of  nitro-



benzene during production  (estimated as eight million  pounds
                             C-l

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annually) occurs at the acid extraction step  in  the purifica-



tion of the crude reaction mixture, when nitrobenzene  is  lost



to the effluent wash  (Dorigan and Hushon, 1976).  Thus  the



greatest exposure to  nitrobenzene occurs inside  plants  and



most cases of chronic nitrobenzene exposure in man are  nitro-



benzene workers.  Today plant levels of nitrobenzene are  us-



ually kept below the  threshold limit value  (TLV) of 5 mg/m3



(Goldstein, 1975; TLV, 1978) but much higher  levels have  been



reported in the past  (Pacseri and Magos, 1958).  Nitrobenzene



may also form spontaneously in the atmosphere from the  photo-



chemical reaction of  benzene with oxides of nitrogen; the



symptoms of nitrobenzene poisoning are similar to the symp-



toms experienced by victims of Japanese photochemical smog



(Dorigan and Hushon,  1976).



     Nitrobenzene can be detected for monitoring purposes by



colorimetric reaction, or by collection on a  charcoal filter,



extraction, reduction to aniline, and production of a colored



product by diazotization of the aniline.  These  methods can



detect 1.0 to 500 mg/m3 (0.2 to 100 ppm) of nitrobenzene



(Dorigan and Hushon,  1976).  Nitrobenzene in  waste water  can



be measured by gas chromatography (Austern, et al.) 1975).



Exposure of workers to nitrobenzene is monitored by urinary



levels of p-nitrophenol (Piotrowski, 1967) and p-aminophenol



(Pacseri and Magos, 1958).



     The liquid nitrobenzene has a very low solubility  in



water, although it is a good organic solvent.  It also  has a



low volatility, does  not readily react with light, and  is



non-corrosive.  Nitrobenzene has a low volatility but a high
                             C-2

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specific gravity, so that the fumes can accumulate at  floor
level in production plants.  Some of the common derivatives
of nitrobenzene (besides aniline) are dinitrobenzene,  nitro-
benzene-sulfonic acid, and nitrochlorobenzene.  There  are
many other derivatives of nitrobenzene, and many of  them are
very hazardous to man as toxic agents, mutagens, and car-
cinogens.
     Some of the physical and chemical properties of nitro-
benzene are summarized in Table 1.
                             C-3

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                           TABLE 1

    Properties of Nitrobenzene  (Dorigan and Hushon, 1976)

Formula:                     C6 H5 NO2 r/JJ\.N02
Molecular weight:

Freezing point:

Boiling point:

Water solubility:




Soluble in:


Vapor pressure:


Vapor density:

Log partition co-efficient:

Density:

Flash point:

Autoignition temp:

Fire hazard  (N.F.P.A.):


Viscosity:

Detection level of charac-
  teristic bitter almond
  odor:
123.11

5.6 - 5.7C

210.9C at 760 torr

0.1 - 0.2 gm/100 ml at 20C

1.0 gm/100 ml at 100C


ethanol, diethyl ether, acetone,
  benzene, lipids

0.284 mmHg at 25C
600 mmHg at 200C

4.24 (air = 1.0)

hexane/water - 3.18 at 24.4C

1.199 gm/ml at 25C

87.8C

482.2C

medium; fire can be extinguished^ by
  water, foam, CO^r or dry chemicals

1.682 cp at 30C



10~4 mmoles/1
                             C-4

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Ingestion from Water



     Nitrobenzene can be released into waste water  from  pro-



duction plants as the result of losses during the production



of nitrobenzene, aniline, and dyestuffs.  The solubility of



nitrobenzene is low, and it produces a detectable odor in



water at a concentration as low as 0.03 mg/1  (Austern, et  al.



1975), so that large amounts can not readily accumulate  un-



noticed.  Levels of nitrobenzene in waste water are monitored



by plants producing and using the chemical but nitrobenzene



levels in city water systems are usually too low to measure



(Pierce, 1979).  Nitrobenzene in water from an industrial



spill is removed by treatment with activated charcoal.



     There are no data available on mammalian toxicity Of



nitrobenzene ingested in drinking water.



Ingestion from Foods



     There are reports of nitrobenzene poisoning resulting



from its uses as false almond oil in baking, rubbing on  the



gums to ease toothache, contamination of alcoholic  drinks,



and contamination of food (Nabarro, 1948).  Leader  (1932)



reported a case of nitrobenzene poisoning in a child who was



given "oil of almonds" for relief of a cold.  Acute nitro-



benzene poisoning has occurred from ingestion of denatured



alcohol (Donovan, 1920; Wirtschafter and Wolpaw, 1944).



These cases are typical of accidental nitrobenzene  ingestion.



Nitrobenzene is not an approved food additive (Dorigan and



Hushon, 1976).



     A bioconcentration factor (BCF) relates the concentra-



tion of a chemical in water to the concentration in aquatic
                             C-5

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organisms, but BCF's are  not available  for the edible por-

tions of all four major groups of aquatic organisms consumed

in the United States.  Since data indicate that the BCF  for

lipid-soluble compounds is proportional to percent lipids,

BCF's can be adjusted  to  edible portions using data on per-

cent lipids and the amounts of various  species consumed  by

Americans.  A recent survey on fish and shellfish consumption

in the United States (Cordle, et al. 1978) found that the per

capita consumption  is  18.7 g/day.  From the data on the  19

major species identified  in the survey  and data on the fat

content of the edible  portion of these  species (Sidwell, et

al. 1974), the relative consumption of  the four major groups

and the weighted average  percent lipids for each group can be

calculated:

                          Consumption        Weighted Average
        Group              (Percent)          Percent Lipids

Freshwater fishes             12                    4.8

Saltwater fishes              61                    2.3

Saltwater molluscs             9                    1.2

Saltwater decapods            18                    1.2

Using the percentages  for consumption and lipids for each of

these groups, the weighted average percent lipids is 2.3 for

consumed fish and shellfish.

     No measured steady-state bioconcentration factor  (BCF)

is available for nitrobenzene, but the  equation "Log BCF =

0.76 Log P - 0.23"  can be used  (Veith,  et al. Manuscript) to

estimate the BCF for aquatic organisms  that contain about

eight percent lipids from the octanol-water partition coeffi-

cient (P).  Based on an octanol-water partition coefficient


                             C-6

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of 71, the steady-state bioconcentration factor  for  nitroben-



zene is estimated to be 15.  An adjustment factor of  2.3/8.0



= 0.2875 can be used to adjust the estimated BCF from the  8.0



percent lipids on which the equation is based  to the  2.3 per-



cent lipids that is the weighted average for consumed fish



and shellfish.  Thus, the weighted average bioconcentration



factor for nitrobenzene and the edible portion of all aquatic



organisms consumed by Americans is calculated  to be  15 x



0.2875 = 4.3.



Inhalation



     Nitrobenzene is readily absorbed through  the lungs with



retention of up to 80 percent  (Piotrowski, 1967).  There are



reports of nitrobenzene poisoning from inhalation of  an ex-



terminator spray for bedbugs which was sprayed on a  child's



mattress (Stevenson and Forbes, 1942; Nabarro, 1948).  Poi-



sonings have also resulted from inhaled nitrobenzene  used  as



a scent in perfume and soap (Dorigan and Hushon, 1976).



Chronic and acute poisoning from exposure to nitrobenzene



fumes in production plants are well documented  (Dorigan and



Hushon, 1976; Browning, 1950;  Zeligs, 1929; Hamilton, 1919),



but since nitrobenzene is also absorbed through  the  skin,  in-



dustrial poisoning cannot be attributed to inhalation alone.



A worker exposed to the TLV for nitrobenzene of  5 mg/m^



would absorb 18 mg/day through the lungs (Piotrowski, 1967).



Dermal



     Nitrobenzene is highly fat-soluble and can  be absorbed



through the skin at rates as high as 2 mg/cm2/hr  (Dorigan



and Hushon, 1976).  Medical literature contains  many  reports
                             C-7

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of poisonings from absorption of nitrobenzene  in  shoe  dyes



and laundry marking  ink.  These reports were common during



the 19th century and  the  first half of this century.



     Poisoning following  the wearing of newly-dyed wet  shoes



was reported in 1900  (Levin, 1927).  The poisoning can  result



from nitrobenzene or  aniline, both of which were  used  in  shoe



dyes and which cause  the  same toxic symptoms.   There have



been reports of cases of  shoe dye poisoning in  an army  camp



(Levin, 1927) , in children who were given  freshly dyed  shoes



(Zeitoun, 1959; Graves, 1928; Levin, 1927), and  in adults.



Generally the affected people are brought  to the  physician's



attention with symptoms of dizziness, bluish color of  lips



and nails (cyanosis), headache, and sometimes  coma.  All



these sypmptoms are  due to methemoglobin formation from the



absorbed nitrobenzene or  aniline.



     Cyanosis and poisoning of newborns who came  in contact



with diapers or pads  containing marking ink were  very  common.



Generally this occurred when the diapers or pads  were  freshly



stamped by the hospital laundry (Etteldorf, 1951; Ramsay  and



Harvey, 1959; MacMath and Apley, 1954; Zeligs,  1929; Rayner,



1886).  Often the imprint of the ink could be  seen on  the



infant's skin.  Removal of the diaper or pad and  thorough



washing of the skin  usually reduced toxic  symptoms, although



methylene blue and ascorbic acid have also been  used to re-



lieve cyanosis.  The  toxicity is often more severe in  prema-



ture infants who are  in an incubator and surrounded by  fumes



as well as the dye on the cloth (Etteldorf, 1951).  Washing
                              C-8

-------
of the marked diapers or pads before their use removes  the



hazard of absorption of nitrobenzene or aniline from  the



ink.



     In Egypt, "pure bitter almond oil" (a mixture  of two  to



ten percent nitrobenzene and 90 to 98 percent cottonseed



oil) has been rubbed on babies to remove crusts from  the skin



and to protect the children from other diseases.  Zeitoun



(1959) reports cases of nitrobenzene poisoning seen in  Alex-



andria hospitals as a result of this practice.



     Hamilton (1919) reported a case of chronic nitrobenzene



poisoning in a woman who used it as a cleaning fluid  for many



years.  The continuous dermal absorption caused her to  exper-



ience symptoms of multiple neuritis, extreme indigestion and



hemorrhages of the larynx and pharynx.



     Dermal absorption of nitrobenzene is the cause of  many



of the chronic and acute toxic effects seen  in nitrobenzene



workers  (inhalation also accounts for industrial  toxicity  al-



though the routes of exposure often cannot be distinguished).



The amount of cutaneous absorption is a function  of the am-



bient concentration, the amount of clothing  worn, and the



relative humidity (high humidity increases absorption)  (Dori-



gan and Hushon, 1976).  A worker exposed to  the TLV of  5 mg/-



m^ could absorb up to 25 n*g per day; one-third of that  amount



would pass through the skin of a clothed man (Piotrowski,



1967).  Pacseri and Magos (1958) measured ambient nitroben-



zene in industrial plants and found levels of up  to eight



times the current TLV.
                             C-9

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     Hamilton  (1919) reported a  case of acute,  fatal,  nitro-



benzene poisoning that  resulted  from a soap  factory worker



spilling "oil  of mirbane" on his clothes.  Immediate  removal



of the contaminated clothing would probably  have prevented



his death.



     There are reports  of acute  and chronic  poisoning  due to



skin absorption of dinitrobenzene by workers  in munitions and



nitrobenzene plants.  Dinitrobenzene is believed to be  much



more toxic than nitrobenzene (Maiden, 1907).   Ishihara,  et



al. (1976) reported a case of poisoning where  a worker



handled a cleaning mixture containing 0.5 percent dinitroben-



zene.  The worker wore  gloves, but the dinitrobenzene  pene-



trated through the gloves to cause acute symptoms of methemo-



globinemia and hemolytic  jaundice.  Re^sek (1947) described



dinitrobenzene diffusion  through the skin of munitions



workers.  Some of these workers  who had chronic dinitroben-



zene poisoning experienced an acute crisis after exposure to



sun or drinking alcohol (beer).  Alcohol ingestion or  chronic



alcoholism can also lower the lethal or toxic  dose of  nitro-



benzene (Dorigan and Hushon, 1976).  This acute reaction



could occur as late as  six weeks after toxic symptoms  dis-



appeared .



     Although  there are many literature references dealing



with occupational exposure to nitrobenzene,  there are  few,  if



any, of nitrobenzene exposure resulting from water  intake.



Therefore, data derived from occupational exposure were



= ~,ployed  to develop information  for establishing the  water



<-  5_^_v criterion in tnis document.
                              C-10

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                       PHARMACOKINETICS



Absorption



     Nitrobenzene absorption can occur by all possible



routes, but it takes place mainly through the respiratory



tract and skin.  At the TLV of 5/mg/m3, a nitrobenzene



worker can absorb 18 mg/day through the lungs and 7 mg/day



through the skin (Piotrowski, 1967).  On the average, 80 per-



cent of the nitrobenzene vapors are retained in the human



respiratory tract (Piotrowski, 1977).



     Nitrobenzene,  as liquid and vapor, will pass directly



through the skin.  The rate of vapor absorption depends on



the air concentration, ranging from 1 mg/hr at 5 mg/m3 con-



centration to 9 mg/hr at 20 mg/m3.  Air temperature does



not affect the absorption rate, but an increase of relative



humidity from 33 to 67 percent will increase the absorption



rate by 40 percent.  Work clothes reduce cutaneous absorption



of nitrobenzene vapors by 20 percent (Piotrowski, 1977).



     Maximal cutaneous absorption of liquid nitrobenzene is



0.2 to 3 mg/cm^/hr depending on skin temperature.  Elevated



skin temperature will increase absorption.  Absorption will



decrease with duration of contact.  Cutaneous absorption can



be significant in industry, since contamination of skin and



clothes of dye manufacture workers may reach levels of 2 and



25 mg/cm2, respectively (Piotrowski, 1977).



     In view of this high level of absorption from lungs and



skin, individual protection of exposed workers is justified.



Distribution



     Upon entry into the body, nitrobenzene enters the blood
                             C-ll

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stream.  Here it reacts with the hemoglobin  to  form  its  oxi-
dation product, methemoglobin.  Methemoglobin has a  reduced
affinity for oxygen, and  the reduced oxygen  carrying capacity
of the blood is the cause of most of the toxic  effects of
nitrobenzene, including its lethality.  Methemoglobin levels
from nitrobenzene have ranged from 0.6 gm/100 ml  in  indus-
trial chronic exposure to 10 gm/100 ml in  acute poisoning
(Pacseri and Magos, 1958; Myslak, et al. 1971).   The normal
methemoglobin level is 0.5 gm/100 ml.  Under normal  condi-
tions methemoglobin will  slowly be reduced to oxyhemoglobin,
the normal form of blood  hemoglobin.
     Pacseri and Magos  (1958) have demonstrated that sulfhe-
moglobin is also formed in the blood after chronic exposure
to nitrobenzene.  They found average sulfhemoglobin  levels  in
nitrobenzene workers of 0.27 gm/100 ml  (compared  to  the  upper
limit of normal of 0.18 gm/100 ml).  Pacseri postulated  that
since blood sulfhemoglobin disappears more slowly than methe-
moglobin, it is a more sensitive indicator of nitrobenzene
exposure.  Sulfhemoglobin may be more specific  than  sensitive
because methemoglobin  is  normally found in the  blood but
sulfhemoglobin  is not.
     Uehleke (1964) measured the velocity  of methemoglobin
formation from  nitrobenzene  in cats.  He found  the rate  to  be
variable and not related  to  the blood concentration  of nitro-
benzene, although the  methemoglobin formation velocity was
maximal  in each animal  at the time of highest blood  concen-
tration of nitrobenzene.  He also found that metabolites of
nitrobenzene are able  to  oxidize hemoglobin. Methemoglobin
formation from  nitrobenzene  has also been  demonstrated
                              C-12

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in vitro (Dorigan and Hushon, 1976, cited from von Oettingen,
1941; Kusumoto and Nakajima, 1970).
     Further indications of the presence of nitrobenzene  in
the blood are the production of hemolytic anemia after acute
exposure (Harrison, 1977) and the alteration of the sodium
and potassium permeability of erythrocytes by derivatives of
nitrobenzene (Cooke, et al. 1968).
     Nitrobenzene is very lipid soluble, with an oil to water
partition coefficient of 800.  In a rat study the ratio of
concentration of nitrobenzene in adipose tissue versus blood
in internal organs and muscle was approxiraatley 10:1 one  hour
after an intravenous dose  (Piotrowski, 1977).  Rabbits intu-
bated with 0.25 ml of nitrobenzene had 50 percent of the  com-
pound accumulated unchanged in tissues within two days after
the intubation (Dorigan and Hushon, 1976).
     Dresbach and Chandler  (1918) have shown cerebellar dis-
turbance in dogs and birds  from nitrobenzene vapors, although
they found blood changes as described above to be the predom-
inant effects in other mammals they tested.  A histologic
study attributed these effects to changes in the Purkinje
cells of the cerebellum.  Reports of the effect of nitroben-
zene on the liver vary from description of liver damage from
accumulated nitrobenzene (Dorigan and Hushon, 1976) to the
statement that nitrobenzene does not cause severe renal nor
liver damage (Goldstein, 1975).  Goldwater (1947) has de-
scribed hyperplasia of the  erythropoietic centers of the  bone
marrow in workers chronically exposed to nitrobenzene, but he
                             C-13

-------
concluded that the hyperplasia  is a secondary result of  the
hemolytic effect of the compound.  Makotchenko and Akhmetov
(1972) observed secretory changes of the adrenal  cortex  of
guinea pigs given nitrobenzene  every other day at a dose of
0.2 gm/kg for six months.
Metabolism
     Available information on nitrobenzene metabolism  is
based on animal experiments and fragmentary human data.
There are two main metabolic pathways:  1) reduction to
aniline followed by hydroxylation to aminophenols, and 2)
direct hydroxylation of nitrobenzene to form nitrophenols.
Further reduction of nitrophenols to aminophenols may  also
occur (Piotrowski, 1977).  The  rate of nitrobenzene metabo-
lism is independent of the dose in later stages of acute or
chronic intoxication.  This can cause its accumulation in
highly lipid tissues (Dorigan and Hushon, 1976).
     The reduction of nitrobenzene to aniline occurs via the
unstable intermediates, nitrosobenzene and phenyl hydro-
xylamine, both of which are toxic and have pronounced  methe-
moglobinemic capacity.  The reactions occur in the cytoplas-
mic and microsomal fractions of liver cells by the nitro- re-
ductase enzyme system (Pouts and Brodie, 1957).   This  enzyme
system is active in mice, guinea pigs, and rabbits, and  is
less active in rats and dogs.   The aniline is then excreted
as an acetyl derivative or hydroxylated and excreted as  an
aminophenal.  Reddy, et al.  (1976) showed that the gut flora
                              C-14

-------
of rats was needed for the reduction of nitrobenzene and  sub-



sequent methemoglobin formation.



     The hydroxylation of nitrobenzene to nitrophenols  does



not occur in the microsomal fraction.  The reaction proceeds



via peroxidase in the presence of oxygen (Piotrowski, 1977).



     Robinson, et al. (1951) studied nitrobenzene metabolism



in the rabbit using ^C labeled material.  The main meta-



bolic product found was p-aminophenol  (35 percent) which  was



formed via phenylhydroxylamine.  Seven phenols and aniline



were detected as metabolite.s within 48 hours of a dose  of 150



to 200 mgAg body weight of nitrobenzene.  Nitrobenzene was



retained somewhat in the rabbits; its metabolites were  de-



tected in urine one week after dosing.  Little unchanged



nitrobenzene was excreted in the urine.  The major urinary



metabolites were p-aminophenol, nitrophenols, and nitro-



catechol.  These constituted 55 percent of the urinary



metabolites and were excreted conjugated with sulfuric  and



glucuronic acids.  About one percent of the dose was expired



as radiolabeled carbon dioxide.



     Yamada (1958) studied nitrobenzene metabolism in rabbits



in a three-month subcutaneous exposure study.  He found that



urinary excretion of detoxification products varied in  the



early stage of exposure, but did not in the later stages.



The reduction and hydroxylation pathways all became depressed



during the later stages of this chronic poisoning study.



     Parke (1956) reports metabolites of nitrobenzene iso-



lated four to five days after administering 0.25 mg/kg  orally



as a single dose in the rabbit  (see Table 2) .
                             C-15

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                                TABLE 2

Metabolic Fate of a Single Oral Dose (0.25 g/kg.) of  [14C] Nitrobenzene
         in the Rabbit During 4-5 Days After Dosing  (Parke,  1956)
Metabolite
        Percentage of Dose (average)
Respiratory CO2
Nitrobenzene
Aniline
o-N i trophenol
m-Nitrophenol
p-Nitrophenol
o-Aminophenol
m-Aminophenol
p-Aminophenol
4-Nitrocatechol
Nitroquinol
p-N i tropheny1
  Mercapturic acid
(Total urinary radio-
  activity)
Metabolized nitrobenzene
  in feces
Metabolized nitrobenzene
  in tissues
Total accounted for
 1   "I
 0.6*  h-2 in expired air
 0.4+_J            '
 0.1
 9
 9
 3
 4                 I	58 in
31                       urine
 0.7
 0.1

 0.3
                w^

(58)

 9

15-20
85-90%
60 total
        * 0.5%  in the  expired air and  <0.1%  in  the  urine.
        + 0.3%  in the  urine and <0.1%  in the  expired  air.
         6% of the dose was present in the  feces as  p-aminophenol.

     An investigation  of the metabolism of^"*  C  nitrobenzene

     in the cattle tick, Boophilus microplusf and spider,  Nephia

     plumipes,  was done by Holder and  Wilcox  (1973).   They found

     that the tick metabolized nitrobenzene  to  nitrophenol and

     aniline whereas no free phenols were  found as  metabolites  in

     the spider.  Aniline was the major metabolic product  in  both

     species.

          Nitrobenzene in water can be degraded by  some bacteria,

     such as Azobacter agilis, if present  in  sufficiently/small
                                   C-16

-------
amounts.  Nitrobenzene tends to inhibit its own degradation



at concentrations above 0.02 to 0.03 mg/1 (Dorigan and



Hushon, 1976; Lu and Metcalf, 1975).



     Lu and Metcalf (1975) studied nitrobenzene in a model



aquatic ecosystem to assess biodegradation and biomagnifi-



cation.  The ecosystem consisted of green filamentous algae,



Oedogonium cardiacium, snails, Physa, water fleas, Daphnia



magna, mosquito larvae, Culex quinquifasciatus, and mosquito



fish, Gambusia affinis, under controlled atmospheric condi-



tions.  0.005 to 0.5 mg/m3 (0.01 to 0.1 ppm) of 14C-labeled



nitrobenzene was added to the water and animals were removed



for analysis after 24 to 48 hours.  The radiolabeled metabo-



lites were extracted and separated by thin layer chroma-



tography.  The distribution of nitrobenzene and its degrada-



tion products is listed in Table 3.
                             C-17

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                                                   TABLE 3
     Distribution of Nitrobenzene and Degradation Products in Model Aquatic Ecosystem (Lu and Metcalf, 1975)
o
i
M
OQ
Nitrobenzene equivalents, ppm

Total 14C
Nitrobenzene
Aniline
Acetanilide
Aminophenolsk
Nitrophenolsk
Polar
Unextractable
Rfa

0.72
0.60
0.35
0.20
0.10
0.0

H20
0.53755
0.50681
0.01262
0.00180
0.00106
0.00466
0.00896
0.00164
Oedognoium
(alga)
0.0690
0.0162
0.0032
0.0160
0.0080
0.0016
0.0240

Daphnia
(daphnia)
0.1812
0.0709
0.0079
-
0.0315
0.0394
0.0315

Culex
(mosquito)
0.5860
0.3952
0.0272
0.0272
-
0.1226
0.0138
-
Physa
(snail)
0.6807
0.3886
0.0169
0.0169
-
0.2190
0.0393

Gambusia
(fish)
4.9541
4.0088
0.2963
0.3527
0.0986
0.0847
0.1130

   a  TLC  with  benzene:acetone:Skellysolve B (bp 60-68C):diethylamine=65:25:25:5 (v/v).

   b  The  isomers  could  not  be  separated reliably because  of small amounts and similar Rf values

-------
     Nitrobenzene was neither stored nor  ecologicaly magni-

fied.  It was reduced to aniline in all organisms,  acetylated

in fish and water extracts only, and hydroxylated  to nitro-

phenols by mosquito Larvae and snails.  The metabolites  of

nitrobenzene formed by the different organisms  are  illus-

trated in Figure 1.
                                     I
                                  n
                                  n
                                 .  '
                                 l_Li

                                   a. . !_!_
Figure 1:  Relative detoxication capacities  of  key organisms
of a model aquatic ecosystem following  treatment  with  radio-
active nitrobenzene (Lu and Metcalf, 1975).

Excretion
     In man the primary known excretion products  of nitroben-

zene are p-aminophenol and p-nitrophenol which  appear  in the

urine after chronic or acute exposure.  In experimental in-

halation exposure to nitrobenzene, p-nitrophenol  was formed

with the efficiency of 6 to 21 percent.  The efficiency of
                             C-19

-------
p-aminophenol formation  is estimated from observation  of



acute poisoning cases where the molar ratio of excreted



p-nitrophenol to p-aminophenol  is  two to one, since p-amino-



phenol is not formed at  a detectable level in short subacute



exposure (Piotrowski, 1977).



     Ikeda and Kita  (1964) measured the urinary excretion  of



p-nitrophenol and p-aminophenol in a patient admitted  to a



hospital with toxic  symptoms resulting from a 17-month



chronic industrial exposure to  nitrobenzene.  The  results  of



their study are shown in Figure 2, which demonstrates  that



the rate of excretion of the two metabolites parallels the



level of methemoglobin  in blood.   The authors exposed  five



adult rats to a nitrobenzene vapor of 125 mg/m^ (25 ppm)



for eight hours and  measured the subsequent excretion  of



p-aminophenol and p-nitrophenol.   The results are  shown  in



Figure 3.  The urinary  excretion ratio of p-aminophenol  and



p-nitrophenol corresponded to  their findings in the human



case.



     Studies of nitrobenzene concentrations in the blood



of an acutely exposed person indicate that the compound  re-



mains in the human body for a  prolonged period of  time.



Similar observations have been  made from excretion of  the  two



urinary metabolites  in  patients treated for acute  or  subacute



poisoning.  The excretion coefficient of urinary p-nitro-



phenol, followed  for three weeks,  is about 0.008 per  hour.



Metabolic transformation and excretion of nitrobenzene in  man



is slower by an order of magnitude than in rats or rabbits



(Piotrowski, 1977).
                              C-20

-------
O  9 ?
J 5< o
J J HOSPITAL Oars j
I S iO '5 JO 3O 35 4O



z
I
o
1  soo-
4 
I G
& ~
 3
 I 400-
O-r
^ 
-J 5
O 
 ?
0 ~
a
= 2OO-
z
"
IOO-

.a-//....
1056 ' "
I







1





a
/
B 	 "
/
/
t

v'






\
Vv
v\
\\

"" \v
\C-
Vi5-^--X>. ^ /, /,
"'*
o

-.2 0
O
n z
-'O 0 "
. O

O"
-8 ' C
ff ,4
5 I
4 ^
_ A ^
* 0
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- * z
n
M
- 2
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IS 2O 2S 3O 1 S IS JO 25
AUGUST SEPrM8q
Figure 2.  Changes in the levels of total hemoglobin and methae-
  moglobin in blood and of p-nitrophenol and p-aminophenol  in
  urine.   The usual daily volume of urine was about 1 litre.
Figure 3.  Excretion of p-nitrophenol and p-aminophenol  in  the
  urine of rats exposed to nitrobenzene.
                             C-21

-------
     Because of  the  slow  rate  of  nitrobenzene  metabolism in



man, the concentration  of p-nitrophenol  in  the urine  in-



creases for about  four  days  during  exposure and the  concen-



tration on the first  day  is  only  about 40 percent  of  the peak



value.  An estimate  of  the mean daily dose  of  nitrobenzene  in



chronic industrial exposure  can be  obtained by the measure-



ment of urinary  p-nitrophenol  in  specimens  taken on  each of



the last three days  of  the work week.  The  level of.  nitroben-



zene exposure can  be  approximated using  the formula  y =



O.lSz, where y is  the daily  excretion of urinary p-nitro-



phenol in mg/day and  z  is the  mean  daily dose  of absorbed



nitrobenzene in  mg  (Piotrowski, 1967).   The extended  systemic



retention and slow excretion of metabolites of nitrobenzene



in man is determined  by the  low rate of metabolic  transforma-



tion (reduction  and  hydroxylation)  of the nitrobenzene it-



self.  The conjugation  and excretion of  the metabolites,



p-nitrophenol and  p-aminophenol,  is rapid  (Piotrowski,



1977).



     The urinary metabolites in man account for only 20 to  30



percent of the nitrobenzene  dose; the fate  of  the  rest of the



metabolites is not known  (Piotrowski, 1977).  Parke  (1956)



studied 1*C - nitrobenzene metabolism in rabbits and was able



to account for 85  to 90 percent of  the dose which  was admin-



istered by intubation.  One  percent of the  nitrobenzene was



exhaled as CC>2 in  air,  and 0.6 percent was  exhaled as un-



changed nitrobenzene.  Fifty-eight  percent  of  the  dose ap-



peared as urinary  metabolites, p-aminophenol,  nitrophenols,



aminophenols, nitrocatechols,  and aniline.   Thirty percent  of
                              C-22

-------
of 71, the steady-state bioconcentration factor for  nitroben-



zene is estimated to be 15.  An adjustment factor of  2.3/8.0



= 0.2875 can be used to adjust the estimated BCF from the  8.0



percent lipids on which the equation is based to the  2.3 per-



cent lipids that is the weighted average for consumed fish



and shellfish.  Thus, the weighted average bioconcentration



factor for nitrobenzene and the edible portion of all aquatic



organisms consumed by Americans is calculated to be  15 x



0.2875 = 4.3.



Inhalation



     Nitrobenzene is readily absorbed through the lungs with



retention of up to 80 percent (Piotrowski, 1967).  There are



reports of nitrobenzene poisoning from inhalation of  an ex-



terminator spray for bedbugs which was sprayed on a  child's



mattress (Stevenson and Forbes, 1942; Nabarro, 1948).  Poi-



sonings have also resulted from inhaled nitrobenzene  used  as



a scent in perfume and soap (Dorigan and Hushon, 1976).



Chronic and acute poisoning from exposure to nitrobenzene



fumes in production plants are well documented  (Dorigan and



Hushon, 1976; Browning, 1950; Zeligs, 1929; Hamilton, 1919),



but since nitrobenzene is also absorbed through the  skin,  in-



dustrial poisoning cannot be attributed to inhalation alone.



A worker exposed to the TLV for nitrobenzene of 5 mg/m3



would absorb 18 mg/day through the lungs (Piotrowski, 1967).



Dermal



     Nitrobenzene is highly fat-soluble and can be absorbed



through the skin at rates as high as 2 mg/cm2/hr  (Dorigan



and Hushon, 1976).  Medical literature contains many  reports
                             C-7

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of poisonings from absorption of nitrobenzene  in  shoe  dyes



and laundry marking  ink.  These reports were common during



the 19th century and  the  first half of this century.



     Poisoning following  the wearing of newly-dyed wet snoes



was reported  in 1900  (Levin, 1927).  The poisoning can result



from nitrobenzene or  aniline, both of which were  used  in  shoe



dyes and which cause  the  same toxic symptoms.  There have



been reports of cases of  shoe dye poisoning in an army camp



(Levin, 1927), in children who were given  freshly dyed shoes



(Zeitoun, 1959; Graves, 1928; Levin, 1927), and  in adults,



Generally the affected people are brought  to the  physician's



attention with symptoms of dizziness, bluish color of  lips



and nails (cyanosis), headache, and sometimes  coma.  All



these sypmptoms are  due to methemoglobin formation from the



absorbed nitrobenzene or  aniline.



     Cyanosis and poisoning of newborns who came  in contact



with diapers  or pads  containing marking ink were  very  common.



Generally this occurred when the diapers or pads were  freshly



stamped by the hospital laundry (Etteldorf, 1951; Ramsay  and



Harvey, 1959; MacMath and Apley, 1954; Zeligs, 1929; Rayner,



1886).  Often the imprint of the ink could be  seen on  the



infant's skin.  Removal of the diaper or pad and  thorough



washing of the skin  usually reduced toxic  symptoms, although



methylene blue and ascorbic acid have also been  used to re-



lieve cyanosis.  The  toxicity is often more severe  in  prema-



ture infants  who are  in an incubator and surrounded by fumes



as well as the dye on the cloth (Etteldorf, 1951).  Washing
                              C-8

-------
of the marked diapers or pads before their use removes  the



hazard of absorption of nitrobenzene or aniline from  the



ink.



     In Egypt, "pure bitter almond oil" (a mixture of  two  to



ten percent nitrobenzene and 90 to 98 percent cottonseed



oil) has been rubbed on babies to remove crusts from  the skin



and to protect the children from other diseases.  Zeitoun



(1959) reports cases of nitrobenzene poisoning seen in  Alex-



andria hospitals as a result of this practice.



     Hamilton (1919) reported a case of chronic nitrobenzene



poisoning in a woman who used it as a cleaning fluid  for many



years.  The continuous dermal absorption caused her to  exper-



ience symptoms of multiple neuritis, extreme  indigestion and



hemorrhages of the larynx and pharynx.



     Dermal absorption of nitrobenzene is the cause of  many



of the chronic and acute toxic effects seen in nitrobenzene



workers  (inhalation also accounts for industrial  toxicity  al-



though the routes of exposure often cannot be distinguished).



The amount of cutaneous absorption is a function  of the am-



bient concentration, the amount of clothing worn, and  the



relative humidity (high humidity increases absorption)  (Dori-



gan and Hushon, 1976).  A worker exposed to the TLV of  5 mg/-



m-* could absorb up to 25 irg per day; one-third of that  amount



would pass through the skin of a clothed man  (Piotrowski,



1967).  Pacseri and Magos (1958) measured ambient nitroben-



zene in industrial plants and found levels of up  to eight



times the current TLV.
                             C-9

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     Hamilton  (1919)  reported  a  case of  acute,  fatal,  nitro-



benzene poisoning that  resulted  from a soap  factory worker



spilling "oil  of mirbane" on his  clothes.   Immediate  removal



of the contaminated clothing would probably  have  prevented



his death.



     There are reports  of acute  and chronic  poisoning  due to



skin absorption of dinitrobenzene by workers  in munitions and



nitrobenzene plants.  Dinitrobenzene is  believed  to be much



more toxic than nitrobenzene (Maiden, 1907).   Ishihara,  et



al. (1976) reported a case  of  poisoning  where  a worker



handled a cleaning mixture  containing 0.5  percent dinitroben-



zene.  The worker wore  gloves, but the dinitrobenzene  pene-



trated through the gloves to cause acute symptoms of methemo-



globinemia and hemolytic jaundice.  Rejsek (1947)  described



dinitrobenzene diffusion through  the skin  of munitions



workers.  Some of these workers  who had  chronic dinitroben-



zene poisoning experienced  an  acute crisis after  exposure to



sun or drinking alcohol (beer).   Alcohol ingestion or  chronic



alcoholism can also lower the  lethal or  toxic  dose of  nitro-



benzene (Dorigan and  Hushon, 1976).  This  acute reaction



could occur as late as  six  weeks  after toxic  symptoms  dis-



appeared.



     Although  there are many literature  references dealing



with occupational exposure  to  nitrobenzene,  there are  few,  if



any, of nitrobenzene  exposure  resulting  from water intake.



Therefore, data derived from occupational  exposure were



^"iployed to develop  information  for establishing  the  water



 .2_i_v criterion in  tnis document.
                              C-10

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                       PHARMACOKINETICS


Absorption


     Nitrobenzene absorption can occur by all possible

routes, but it takes place mainly through the respiratory


tract and skin.  At the TLV of S/mg/m3f a nitrobenzene


worker can absorb 18 mg/day through the lungs and 7 ing/day

through the skin (Piotrowski, 1967).  On the average, 80 per-


cent of the nitrobenzene vapors are retained in the human
                                       
respiratory tract (Piotrowski, 1977).

     Nitrobenzene, as liquid and vapor, will pass directly


through the skin.  The rate of vapor absorption depends on

the air concentration, ranging from 1 mg/hr at 5 mg/m3 con-


centration to 9 mg/hr at 20 mg/m3.  Air temperature does


not affect the absorption rate, but an increase of relative

humidity from 33 to 67 percent will increase the absorption


rate by 40 percent.  Work clothes reduce cutaneous absorption


of nitrobenzene vapors by 20 percent (Piotrowski, 1977).

     Maximal cutaneous absorption of liquid nitrobenzene is


0.2 to 3 mg/cm^/hr depending on skin temperature.  Elevated


skin temperature will increase absorption.  Absorption will

decrease with duration of contact.  Cutaneous absorption can

be significant in industry, since contamination of skin and


clothes of dye manufacture workers may reach levels of 2 and


25 mg/cm2, respectively (Piotrowski, 1977).

     In view of this high level of absorption from lungs and


skin, individual protection of exposed workers is justified.


Distribution

     Upon entry into the body, nitrobenzene enters the blood
                             C-ll

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stream.  Here it reacts with the hemoglobin to form  its oxi-
dation product, methemoglobin.  Methemoglobin has a  reduced
affinity for oxygen, and  the reduced oxygen carrying capacity
of the blood is the cause of most of the toxic effects of
nitrobenzene, including its lethality.  Methemoglobin levels
from nitrobenzene have ranged from 0.6 gm/100 ml in  indus-
trial chronic exposure to 10 gm/100 ml in acute poisoning
(Pacseri and Magos, 1958; Myslak, et al. 1971).  The normal
methemoglobin level is 0.5 gm/100 ml.  Under normal  condi-
tions methemoglobin will  slowly be reduced to oxyhemoglobin,
the normal form of blood  hemoglobin.
     Pacseri and Magos  (1958) have demonstrated that sulfhe-
moglobin is also formed in the blood after chronic exposure
to nitrobenzene.  They found average sulfhemoglobin  levels in
nitrobenzene workers of 0.27 gm/100 ml (compared to  the upper
limit of normal of 0.18 gm/100 ml).  Pacseri postulated that
since blood sulfhemoglobin disappears more slowly than methe-
moglobin, it is a more sensitive indicator of nitrobenzene
exposure.  Sulfhemoglobin may be more specific than  sensitive
because methemoglobin  is  normally found in the blood but
sulfhemoglobin  is not.
     Uehleke (1964) measured the velocity of methemoglobin
formation from  nitrobenzene in cats.  He found the rate to be
variable and not related  to the blood concentration  of nitro-
benzene, although the  methemoglobin formation velocity was
maximal  in each animal at the time of highest blood  concen-
tration of nitrobenzene.  He also found that metabolites of
nitrobenzene are able  to  oxidize hemoglobin.  Methemoglobin
formation from  nitrobenzene has also been demonstrated
                             C-12

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in vitro (Dorigan and Hushon, 1976, cited from von Oettingen,
1941; Kusumoto and Nakajima, 1970).
     Further indications of the presence of nitrobenzene  in
the blood are the production of hemolytic anemia after acute
exposure (Harrison, 1977) and the alteration of the  sodium
and potassium permeability of erythrbcytes by derivatives of
nitrobenzene (Cooke, et al. 1968).
     Nitrobenzene is very lipid soluble, with an oil  to water
partition coefficient of 800.  In a rat study the ratio of
concentration of nitrobenzene in adipose tissue versus blood
in internal organs and muscle was approximatley 10:1  one  hour
after an intravenous dose (Piotrowski, 1977).  Rabbits intu-
bated with 0*25 ml of nitrobenzene had 50 percent of  the  com-
pound accumulated unchanged in tissues within two days after
the intubation (Dorigan and Hushon, 1976).
     Dresbach and Chandler  (1918) have shown cerebellar dis-
turbance in dogs and birds  from nitrobenzene vapors,  although
they found blood changes as described above to be the predom-
inant effects in other mammals they tested.  A histologic
study attributed these effects to changes in the Purkinje
cells of the cerebellum.  Reports of the effect of nitroben-
zene on the liver vary from description of liver damage from
accumulated nitrobenzene (Dorigan and Hushon, 1976)  to the
statement that nitrobenzene does not cause severe renal nor
liver damage (Goldstein, 1975).  Goldwater (1947) has de-
scribed hyperplasia of the  erythropoietic centers of  the  bone
marrow in workers chronically exposed to nitrobenzene, but he
                             C-13

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concluded that the hyperplasia  is a secondary  result  of  the
hemolytic effect of the  compound.  Makotchenko and Akhmetov
(1972) observed secretory changes of  the adrenal  cortex  of
guinea pigs given nitrobenzene  every  other day at a dose of
0.2 gin/kg for six months.
Metabolism
     Available information on nitrobenzene metabolism is
based on animal experiments  and  fragmentary  human data.
There are two main metabolic pathways:  1) reduction  to
aniline followed by hydroxylation to  aminophenols, and 2)
direct hydroxylation  of  nitrobenzene  to form nitrophenols.
Further reduction of  nitrophenols to  aminophenols may also
occur (Piotrowski, 1977).  The  rate of nitrobenzene metabo-
lism is independent of the dose  in later stages of acute or
chronic intoxication.  This  can cause its accumulation in
highly lipid tissues  (Dorigan and Hushon, 1976).
     The reduction of nitrobenzene to aniline  occurs  via the
unstable intermediates,  nitrosobenzene and phenyl hydro-
xylamine, both of which  are  toxic and have pronounced methe-
moglobinemic capacity.   The  reactions occur  in the cytoplas-
mic and microsomal fractions of liver cells  by the nitro- re-
ductase enzyme system (Fouts and Brodie, 1957).  This enzyme
system is active  in mice, guinea pigs, and rabbits, and  is
less active  in rats and  dogs.   The aniline is  then excreted
as an acetyl derivative  or hydroxylated and  excreted  as  an
aminophenal.  Reddy,  et  al.  (1976) showed that the gut flora
                              C-14

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of rats was needed for the reduction of nitrobenzene  and  sub-



sequent methemoglobin formation.



     The hydroxylation of nitrobenzene to nitrophenols  does



not occur in the microsomal fraction.  The reaction proceeds



via peroxidase in the presence of oxygen  (Piotrowski, 1977).



     Robinson, et al. (1951) studied nitrobenzene metabolism



in the rabbit using l^c labeled material.  The main meta-



bolic product found was p-aminophenol  (35 percent) which  was



formed via phenylhydroxylamine.  Seven phenols and aniline



were detected as metabolites within 48 hours of a dose  of 150



to 200 mg/kg body weight of nitrobenzene.  Nitrobenzene was



retained somewhat in the rabbits; its metabolites were  de-



tected in urine one week after dosing.  Little unchanged



nitrobenzene was excreted in the urine.  The major urinary



metabolites were p-aminophenol, nitrophenols, and nitro-



catechol.  These constituted 55 percent of the urinary



metabolites and were excreted conjugated with sulfuric  and



glucuronic acids.  About one percent of the dose was  expired



as radiolabeled carbon dioxide.



     Yamada (1958) studied nitrobenzene metabolism in rabbits



in a three-month subcutaneous exposure study.  He found that



urinary excretion of detoxification products varied in  the



early stage of exposure, but did not in the later stages.



The reduction and hydroxylation pathways all became depressed



during the later stages of this chronic poisoning study.



     Parke (1956) reports metabolites of nitrobenzene iso-



lated four to five days after administering 0.25 mg/kg  orally



as a single dose in the rabbit  (see Table 2).
                             C-15

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                                TABLE 2

Metabolic Fate of a Single Oral Dose (0.25 g/kg.) of  [14C] Nitrobenzene
         in the Rabbit During 4-5 Days After Dosing  (Parke,  1956)
Metabolite
        Percentage of Dose (average)
Respiratory CO2
Nitrobenzene
Aniline
o-Nitrophenol
m-Nitrophenol
p-Nitrophenol
o-Aminophenol
m-Aminophenol
p-Aminophenol
4-Nitrocatechol
Nitroquinol
p-Nifcrophenyl
  Mercapturic acid
(Total urinary radio-
  activity)
Metabolized nitrobenzene
  in feces
Metabolized nitrobenzene
  in tissues
Total accounted for
 1   ~j
 0.6*  j-2 in expired
air
 0.4+.J
 0.1
 9
 9
 3
 4
31
 0.7
 0.1

 0.3

(58)

 9

15-20
85-90%
           60 total
  58 in
    urine
        * 0.5%  in  the expired air and  <0.1%  in  the urine.
        + 0.3%  in  the urine and  <0.1%  in the expired  air.
         6% of  the dose was present in the  feces as  p-aminophenol.

     An investigation of the metabolism ofl^ c  nitrobenzene

     in the cattle tick, Boophilus microplus, and spider,  Nephia

     plumipes,  was done by Holder and  Wilcox  (1973).   They found

     that the tick metabolized nitrobenzene  to  nitrophenol and

     aniline whereas no free phenols were  found as metabolites  in

     the spider.   Aniline was the major metabolic product  in  both

     species.

          Nitrobenzene  in water  can be degraded by some  bacteria,

     such as Azobacter  agilis, if present  in sufficiently/small
                                  C-16

-------
amounts.  Nitrobenzene tends to inhibit its own degradation



at concentrations above 0.02 to 0.03 mg/1 (Dorigan and



Hushon, 1976; Lu and Metcalf, 1975).



     Lu and Metcalf (1975) studied nitrobenzene in a model



aquatic ecosystem to assess biodegradation and biomagnifi-



cation.  The ecosystem consisted of green filamentous algae,



Oedogonium cardiacium, snails, Physa, water fleas, Daphnia



magna, mosquito larvae, Culex quinquifasciatus, and mosquito



fish, Gambusia affinis, under controlled atmospheric condi-



tions.  0.005 to 0.5 mg/m3 (0.01 to 0.1 ppm) of 14C-labeled



nitrobenzene was added to the water and animals were removed



for analysis after 24 to 48 hours.  The radiolabeled metabo-



lites were extracted and separated by thin layer chroma-



tography.  The distribution of nitrobenzene and its degrada-



tion products is listed in Table 3.
                             C-17

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                                                   TABLE 3
     Distribution of Nitrobenzene and Degradation Products in Model Aquatic Ecosystem (Lu and Metcalf, 1975)
n
i
M
00
Nitrobenzene equivalents, ppm

Total 14C
Nitrobenzene
Aniline
Acetanilide
Aminophenolsk
Nitrophenols*3
Polar
Unextractable
Rfa

0.72
0.60
0.35
0.20
0.10
0.0

H20
0.53755
0.50681
0.01262
0.00180
0.00106
0.00466
0.00896
0.00164
Oedognoium
(alga)
0.0690
0.0162
0.0032
0.0160
0.0080
0.0016
0.0240

Daphnia
(daphnia)
0.1812
0.0709
0.0079
-
0.0315
0.0394
0.0315

Culex
(mosquito)
0.5860
0.3952
0.0272
0.0272
-
0.1226
0.0138
-
Physa
(snail)
0.6807
0.3886
0.0169
0.0169
-
0.2190
0.0393

Gambusia
(fish)
4.9541
4.0088
0.2963
0.3527
0.0986
0.0847
0.1130
-
   a  TLC  with  benzene:acetone:Skellysolve B (bp 60-68C):diethylamine=65:25:25:5 (v/v).

   b  The  isomers  could  not  be  separated reliably because  of small amounts  and similar Rf values

-------
     Nitrobenzene was neither stored nor ecologicaly magni-

fied.  It was reduced to aniline in all organisms, acetylated

in fish and water extracts only, and hydroxylated to nitro-

phenols by mosquito Larvae and snails.  The metabolites of

nitrobenzene formed by the different organisms are illus-

trated in Figure 1.
                   LadL
                                       n
                                  .a.,
                                            -*S	
Figure 1:  Relative detoxication capacities of key organisms
of a model aquatic ecosystem following treatment with radio-
active nitrobenzene (Lu and Metcalf, 1975).

Excretion
     In man the primary known excretion products of nitroben-

zene are p-aminophenol and p-nitrophenol which appear in the

urine after chronic or acute exposure.  In experimental in-

halation exposure to nitrobenzene, p-nitrophenol was formed

with the efficiency of 6 to 21 percent.  The efficiency of
                             C-19

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p-aminophenol formation  is estimated  from observation  of



acute poisoning cases where the molar ratio of excreted



p-nitrophenol to p-aminophenol  is two to one, since  p-amino-



phenol is not formed at  a detectable  level in short  subacute



exposure (Piotrowski, 1977).



     Ikeda and Kita  (1964) measured the urinary  excretion  of



p-nitrophenol and p-aminophenol in a  patient admitted  to a



hospital with toxic  symptoms  resulting from a 17-month



chronic industrial exposure to  nitrobenzene.  The  results  of



their study are shown in Figure 2, which demonstrates  that



the rate of excretion of the  two metabolites parallels the



level of methemoglobin  in blood.  The authors exposed  five



adult rats to a nitrobenzene  vapor of 125 mg/m^  (25  ppm)



for eight hours and  measured  the subsequent excretion  of



p-aminophenol and p-nitrophenol.  The results are  shown  in



Figure 3.  The urinary  excretion ratio of p-aminophenol  and



p-nitrophenol corresponded to their findings in  the  human



case.



     Studies of nitrobenzene  concentrations in the blood



of an acutely exposed person  indicate that the compound  re-



mains in the human body for a prolonged period of  time.



Similar observations have been  made from excretion of  the  two



urinary metabolites  in  patients treated for acute  or subacute



poisoning.  The excretion coefficient of urinary p-nitro-



phenol, followed  for three weeks, is  about 0.008 per hour.



Metabolic transformation and  excretion of nitrobenzene in  man



is slower by an order of magnitude than in rats  or rabbits



(Piotrowski, 1977).
                              C-20

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                                    s    is ao
                                    SEPTEMBER
                                                    O

                                                    |
                                                  ^ Z
                                                  t. a
                                                  g:
                                                   P
Figure 2.  Changes in the levels of  total  hemoglobin and methae-
  moglobin in blood and of p-nitrophenol and  p-aminophenol in
  urine.  The usual daily volume of  urine  was about 1 litre.
                  3000,
Figure 3.  Excretion of p-nitrophenol  and  p-aminophenol in the
  urine of rats exposed to nitrobenzene.
                             C-21

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     Because of the  slow  rate  of  nitrobenzene  metabolism  in



man, the concentration  of p-nitrophenol  in  the urine  in-



creases for about  four  days  during  exposure and the  concen-



tration on the first day  is  only  about 40 percent  of  the  peak



value.  An estimate  of  the mean daily dose  of  nitrobenzene  in



chronic industrial exposure  can be  obtained by the measure-



ment of urinary p-nitrophenol  in  specimens  taken on  each  of



the last three days  of  the work week.  The  level of  nitroben-



zene exposure can  be approximated using  the formula  y =



0.18z, where y is  the daily  excretion of urinary p-nitro-



phenol in mg/day and z  is the  mean  daily dose  of absorbed



nitrobenzene in mg  (Piotrowski, 1967).   The extended  systemic



retention and slow excretion of metabolites of nitrobenzene



in man is determined by the  low rate of metabolic  transforma-



tion (reduction and  hydroxylation)  of the nitrobenzene it-



self.  The conjugation  and excretion of  the metabolites,



p-nitrophenol and  p-aminophenol,  is rapid  (Piotrowski,



1977).



     The urinary metabolites in man account for only 20 to  30



percent of the nitrobenzene  dose; the fate  of  the  rest of the



metabolites is not known  (Piotrowski, 1977).  Parke  (1956)



studied l^c _ nitrobenzene metabolism in rabbits and  was  able



to account for 85  to 90 percent of  the dose which  was admin-



istered by intubation.  One  percent of the  nitrobenzene was



exhaled as CC>2 m  air,  and 0.6 percent was  exhaled as un-



changed nitrobenzene.   Fifty-eight  percent  of  the  dose ap-



peared as urinary  metabolites, p-aminophenol,  nitrophenols,



aminophenols, nitrocatechols,  and aniline.  "Thirty percent  of
                              C-22

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the nitrobenzene was still in the rabbit tissue  four  to  five



days after dosing, and nine percent of the nitrobenzene



metabolizes was in the feces.



     Urinary p-nitrophenol in man is determined  after hydrol-



ysis of the conjugated metabolites.  Analytical  methodology



(of which there are several methods) involves  removal of in-



terfering color substances, hydrolysis, extraction  of



p-nitrophenol, re-extraction into an aqueous system,  reduc-



tion to a p-aminophenol, and reaction to indophenol,  which  is



a blue colored product.  The sensitivity is 5  ug per  sample



(Piotrowski, 1977).



                           EFFECTS



Acute, Sub-acute, and Chronic Toxicity



     Acute exposure to nitrobenzene can occur  from  accidental



or suicidal ingestion of the liquid nitrobenzene or ingestion



as false bitter almond oil in food or medicine.   Cutaneous



absorption causing acute toxic reactions can result from



wearing wet, freshly dyed shoes  (Levin, 1927), marking ink



used on diapers or protective pads  (Etteldorf, 1951), use of



soap or skin oil containing nitrobenzene (Zeitoun,  1959), or



an untreated spill of nitrobenzene on the skin in an  indus-



trial plant (Hamilton, 1919).  The  fatal dose  of nitrobenzene



in humans varies widely; values  from less than 1 ml to over



400 ml have been reported  (Wirtschafter and Wolpaw, 1944)



Chronic toxic effects in man generally result  from  industrial



exposure to vapors that are absorbed through the lungs or  the



skin.  One case of chronic toxicity was reported in a woman



who used nitrobenzene as a cleaning solution for many years



(Hamilton, 1919).



                             C-23

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     Symptoms of chronic occupational nitrobenzene absorption
are cyanosis, methemoglobinemia, jaundice, anemia, sulfhemo-
globinemia, presence of Heinz bodies in the erythrocytes,
dark colored urine, and the presence of nitrobenzene metabo-
lites (e.g. nitrophenol) in the urine (Pacseri and Magos,
1958; Hamilton, 1919; Wuertz, et al. 1964; Browning, 1950;
Maiden, 1907; Piotrowski, 1967).
     The symptoms of dinitrobenzene poisoning  include  those
found in nitrobenzene toxicity as well as abdominal pain,
weakness, enlarged liver, and basophilic granulations  of red
corpuscles  (Beritic, 1956; Maiden, 1907).  Dinitrobenzene
poisoning also causes unequal responses in different exposed
workers.
     The outstanding symptom of acute nitrobenzene poisoning
is cyanosis as a result of methemoglobin formation (up to  80
percent) (Piotrowski, 1967).  If the cyanosis  is  severe  or
prolonged the patient will go into coma and may die.   Often
anemia is seen a week or two after acute poisoning as  a  re-
sult of the hemolytic effect of nitrobenzene  (Stevenson  and
Forbes, 1942).  Suicidal ingestion of nitrobenzene has been
reported (Nabarro, 1948; Leinoff, 1936; Myslak, et al. 1971),
and  it has  also been used unsuccessfully to induce abortion
(Nabarro, 1948; Dorigan and Hushon, 1976).  Harrison  (1977)
reported a  case of poisoning from an aniline-nitrobenzene
mixture which was accidentally ingested from  a pipette by  a
chemistry student.  The mortality due to ingested nitroben-
zene in the above cases was variable, depending on the health
                              C-24

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of the patients and the treatments they received.   Common
treatments include gavage, transfusions, oxygen  therapy,
methylene blue, ascorbic acid, and toluidine blue.   Treatment
is usually directed to reduce the methemoglobinemia which  is
the immediate effect, and often the cause of death  in  nitro-
benzene poisoning.  Death has resulted from  intake  of  less
than 1 ml of nitrobenzene (Wirtschafter and Wolpaw, 1944).
     Some of the reported toxicity values are  summarized  in
Table 4 (Fairchild, 1977).  The term LDLo designates the
lowest reported lethal dose and TDLo is the  lowest  published
toxic dose.
                             C-25

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                           TABLE 4



            Acute Toxicity Values (Fairchild, 1977)



Animal              Route              Toxic Dose



woman               oral               TDLo:  200 mg/kg



human               oral               LDLo:  5 mg/kg



rat                 oral               LD50:  640 mg/kg



rat                 skin               LD50:  2100 mg/kg



rat                 intraper.          LD50:  640 mg/kg



rat                 subcutan.          LDLo:  800 mg/kg



mouse               subcutan.          LDLo:  286 mg/kg



dog                 oral               LDLo:  750 mg/kg



dog                 intraven.          LDLo:  150 mg/kg



cat                 oral               LDLo:  2000 mg/kg



cat                 skin               LDLo:  25 mg/kg



rabbit              oral               LDLo:  700 mg/kg



rabbit              skin               LDLo:  600 mg/kg



guinea pig          intraper.          LDLo:  500 mg/kg
Aquatic  toxicity  at  96  hours  -  10-100 mg/1  (ppm).
                              C-26

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     Levin (1927) demonstrated in vivo production of methemo-



globin by nitrobenzene in dogs, cats, and rats, but not  in



guinea pigs or rabbits.  Dresbach and Chandler  (1918)  found



that nitrobenzene fumes caused cerebellar disturbances  in



dogs and birds, while blood changes were the principal  toxic



effects in other mammals they studied.  Reddy,  et al.  (1976)



reported a delay in methemoglobin formation  in  germ free rats



by nitrobenzene and postulated that the gut  flora of rats was



responsible for the reduction (in vivo) and  methemoglobin



forming capacity of nitrobenzene.  Shimkin  (1939) measured



the toxicity of nitrobenzene in mice when absorbed through



the skin.  He found the minimum lethal dose  to  be 0.0004 ml/



gm body weight by a subcutaneous route of administration.



The nitrobenzene caused respiratory failure, reduction  of the



white blood cell count, and liver pathology  in  the mice.



     Yamada (1958) did a chronic toxicity study in rabbits



that received a subcutaneous dose of 840 mg/kg  body weight



per day for three months.  He found a decrease  in erythrocyte



number and hemoglobin content early in the  exposure.   These



values increased during the three months but did not return



to normal levels.  Urinary excretion of detoxification  pro-



ducts was variable in the early stages of the exposure,  but



then all the detoxification reactions (reduction, hydroxyla-



tion, and acetylation) were depressed.  As  a result of  these



observations, Yamada divided this response  in the rabbit into



three stages: initial response, resistance,  and exhaustion.
                             C-27

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     The effects of subacute nitrobenzene exposure  in  rats



were studied by Kulinskaya  (1974).  Vasilenko and Zvezdai



(1972) measured blood changes and found suIfhemoglobin



formation to be the most regular and persistant change noted.



Increased methemoglobin levels with Heinz body formation and



anemia were also seen.



     The cytotoxicity of nitrobenzene to cultured Erlich-



Landschutz diploid  (ELD) cells was measured by Holmberg and



Malmfors (1974).  They found no significant increase  in cell



injury after five hours incubation with nitrobenzene.  How-



ever, a 3M nitrobenzene solution reduced cell proliferation



by 50 percent  in cultured hamster cells (Raleigh, et  al.



1973).  Nitrobenzene  increases oxygen consumption by  cultured



cells (Biaglow and  Jacobson, 1977), and its derivatives are



used to sensitize malignant cells in vitro for radiation



(Chapman, et al. 1974).  They suggest that this effect is due



to radical oxidation  and increased cellular damage.



     Nitrobenzene derivatives have a wide variety of  toxic



effects.  1-Chloro  -  2, 4 dinitrobenzene (DNCB) is  a  well



known skin sensitizer in guinea pigs, mice, and man  (Hama-



guchi, et al.  1972; Jansen  and Bleumink, 1970; Maurer, et al.



1975; Weigand  and Gaylor, 1974; Noonan and Halliday,  1978).



Cooke, et al.  (1968)  showed that nitrobenzene derivatives



react with cell membranes to alter sodium-potassium con-



ductance, and  sometimes affect action potentials of nerve



cells.
                              C-28

-------
     M-dinitrobenzene is a potent methemoglobin former, and



is more toxic than nitrobenzene (Ishihara, et al. 1976;



Pankow, et al.  1975).  Pentachloronitrobenzene (PCNB) is a



common fungicide with varying toxic effects in different



mammalian species (Courtney, et al. 1976).



     Some of the toxic effects of nitrobenzene are summarized



in Table 5 and  Apendix A (Dorigan and Hushon, 1976).
                             C-29

-------
                                     TABLE 5

                        Toxicological Effects of Nitrobenzene
Organism Route
Human Inhalation
Inhalation
Inhalation
Oral
Oral
Rabbit Cutaneous
absorption
Oral
Oral
Exposure
0.2-0.5 mg/1
(40-100 ppm)
0.129 mg/m3
6-30 ug/1
333 ml
0.4 ml
0.7 gm/kg
700 mg/kg
600 mg
Exposure
Time
ca. 6 hrs.
-
6 hrs.
Single
Single
Single (?)
Single
Single
Oral
50 mgAg
Single
                                                     Response

                                          Slight effects, e.g. headache,
                                           fatigue.

                                          Threshold level for electroen-
                                           cephalograph distrubance.

                                          Retained 80% of vapor in lungs.
                                           Urinary excretion of p-nitro-
                                           phenol  (max. in 2 hrs. still
                                           detected after 100 hrs.).

                                          Max. dose with recovery report-
                                           ed (folowing severe symptoms)

                                          Mm. lethal dose reported.

                                          After 52 hrs.: lethal
Lethal dose

Dizziness, loss of reflexes,
 methemoglobinemia, congestion
 of brain tissue-12 hrs.-death.

Tissue degeneration, especially
 heart, liver, kidney.
                                                                    Source

                                                              von Oettingen,
                                                               1941.

                                                              Andreeshcheva,
                                                                1964.

                                                              Salmowa, et al.
                                                               1963.
                                                              von Oettingen,
                                                               1941.

                                                              von Oettingen, 1941

                                                              von Oettingen,
                                                               1941.

                                                              Stecher, 1968

                                                              Chandler, 1919.
Papageorgiou &
 Argoudelis, 1973.

-------
                                            TABI,E 5 (Continued)
  Organism

  Guinea
   pig
  Rat
o
i
u>
  Cat
  Dog
  Route

Inhalation


Oral



Oral
               Exposure
Exposure
  Time
             Saturated air  2-5 hrs.
             (0.04 vol. %)
                          ca.  3 gm
                          ca 1.2 gm
Inhalation   5 mg/m3
                            Single
                            Single
                            8 hrs.
Inhalation   ca. 0.03mg/    Daily up to
             m3             98 days
             Inhalation   0.06-0.1 mg/   70-82 days
                          m3
Oral

Inhalation


Oral


Oral
                          0.6 gm/kg
                            Single
             Saturated air  2-5 hrs.
             (0.04 vol. %)
                          2.4 gm
             2.4 gm
                            Single
Single
           Response
      Source
              Death following tremors, paraly- Chandler, 1919,
               sis of hind legs.
              0.5 hrs.: tremors, faint heart-
               beats, labored respiration
               2 hrs.: death.

              Immediately motionless, then
               complete recovery.

              Metabolites excreted in 3 days.
                                          Increased ability  to  form  sulf-
                                           hemoglobin  in preference  to
                                           methemoglobin.

                                          Cerebellar disturbances, in-
                                           flamed  internal organs.
             Inhalation   0.008 mg/m3    73 days       No effect.
              LD50

              Death following tremors, pa-
               ralysis of hind legs.

              Death in 12-24 hrs.
1 hr.:  vomiting, then sleepy
 continuing for 6 hrs.
 104 hrs.: death.
                                 Chandler, 1919.
                                 Chandler, 1919.
                                 Ikeda and Kita,
                                  1964

                                 Andreeshcheva,
                                  1970.
                                 Khanin, 1969.


                                 Andreeshcheva.
                                  1970.

                                 Smyth, et al. 1969

                                 Chandler, 1919.
von Oettingen, 1941;
 Chandler, 1919.

Chandler, 1919.

-------
Synergism and/or Antagonism
     Alcohol has a synergistic effect on nitrobenzene poison-
ing.  Ingestion of an alcoholic beverage has induced immedi-
ate acute toxic symptoms, including coma, in a worker recov-
ered from the effects of chronic nitrobenzene exposure.  Al-
cohol ingestion or chronic alcoholism can lower  the lethal or
toxic dose of nitrobenzene (Dorigan and Hushon,  1976).   In
subchronic dinitrobenzene poisoning, drinking of one beer or
exposure to sun can bring on an acute crisis as  late as  six
weeks after the disappearance of other symptoms  (Rejsek,
1947).  Therefore, workers exposed to subacute levels of
nitrobenzene or dinitrobenzene should be aware of  the pos-
sible deleterious synergistic effects of alcoholic beverages
and sunlight.
     Smyth, et al. (1969) studied the synergistic  action be-
tween nitrobenzene and  27 other industrial chemicals by  intu-
bation in rats.  Most of the compounds tested did  not alter
the LD50.  In another study, ingestion of 2 to 20  ml of
ethanol increased the severity of reaction to a  0.1 ml  intra-
venous dose of nitrobenzene in rabbits.  This observation
agrees with the clinical data on the synergism of  ethanol and
nitrobenzene (Dorigan and Hushon, 1976).
     Kaplan, et al.  (1974) studied the effect of caffeine,
an  inducer of microsomal enzymes, on methemoglobin formation
by  nitrobenzene in rats.  Methemoglobin was formed and  then
decreased in induced animals.  The increased microsomal  en-
zyme level increased the rate of metabolism and  excretion of
nitrobenzene and thus caused a rapid decline of  methemoglobin
levels.
                             C-32

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Teratogenecity



     There is a paucity of information on the teratogenic



effects of nitrobenzene.  In one study (Kazanina, 1968b),  125



mg/kg was administered subcutaneously to pregnant rats during



preimplantation and placentation periods.  Delay of embryo-



genesis, alteration of normal placentation, and abnormalities



in the fetuses were observed.  Gross morphogenic defects were



seen in four of 30 fetuses examined.



     Changes in the tissues of the chorion and placenta  of



pregnant women who worked in the production of a rubber



catalyst that used nitrobenzene were observed.  No mention



was made of the effects on fetal development or viability



(Dorigan and Hushon, 1976).  Menstrual disturbances after



chronic nitrobenzene exposure have been reported.



     Garg, et al.  (1976) tested substituted nitrobenzene



derivatives for their ability to inhibit pregnancy in albino



rats.  Two of the compounds tested (p-methoxy and p-ethoxy



derivatives) inhibited implantation and pregnancy 100 percent



when administered on days one through seven after impregna-



tion.



     The available data, although sketchy, indicate that



women who are or wish to become pregnant should avoid expo-



sure to nitrobenzene.  Further studies of nitrobenzene tera-



togenicity in mammals are needed.



Mutagenicity



     Chiu, et al.  (1978) tested nitrobenzene and 53 com-



mercially available ion, heterocyclic + aliphatic nitro
                             C-33

-------
compounds for mutagenicity using the Ames Salmonella

typhimurium TA 98 and TA 100.  They reported that 34 of the

53 compounds tested were mutagenic.  Nitrobenzene was not

found to be mutagenic.

     Trinitrobenzene was mutagenic in two in vitro assays,

the Ames Salmonella microsome assay, and the mitotic recom-

bination assay in yeast (Simmon, 1977).  Other nitrobenzene

derivatives have demonstrated mutagenicity  in in vitro

assays, so that the mutagenicity of nitrobenzene is still in

question and additional work is needed in this area.

Carcinogenicity

     The available literature does not demonstrate the car-

cinogenicity of nitrobenzene, although it is suspect (Dorigan

and Hushon, 1976).  This is another aspect  of nitrobenzene

effects that needs more research.

     Some nitrobenzene derivatives have demonstrated carcino-

genic capacities.  Pentachloronitrobenzene  (PCNB) induced

hepatomas and papillomas in mice (Courtney, et al. 1976).
                                                   4.
     1 - Fluoro -2,4- dinitrobenzene (DNFB) was demon-

strated by Bock, et al. (1969) to be a promoter of skin tu-

mors in mice, although it does not induce them when admin-

istered alone.
                             C-34

-------
                    CRITERION FORMULATION



Existing Guidelines and Standards



     The maximum allowable concentration of nitrobenzene  in



air in industrial plants is 5 mg/m^.  This value was  set  by



the joint ILO/WHO Committee on Occupational Health  in 1975



(Goldstein, 1975).  The OSHA (Occupational Safety and Health



Administration) standard for nitrobenzene in air is 5 mg/m^



(1 ppm) set in 1977 (Am. Conf. Gov. Ind. Hyg., 1977).  This



is also the threshold limit value  (TLV) in Germany  and Sweden



while the TLV in the USSR is 3 mg/m^ (Dorigan and Hushon,



1976).



     There are no standards for nitrobenzene levels in water.



Nitrobenzene was not listed among  the substances for which a



maximum concentration has been set.



Current Levels of Exposure



     A worker exposed to the current occupational standard of



5 mg/m^ (1 ppm) nitrooenzene for an eight-hour work day



would absorb approximately 24 mg by inhalation and  9 mg



cutaneously.  The maximum eight-hour uptake would be  33 mg,



which is less than the "reasonable safe" level of 35 mg/day



(Dorigan and Hushon, 1976).  Doses of up to 70 mg/day have



been reported for factory workers  and up to 80 mg/day have



been reported in a dye stuff factory in England  (Dorigan  and



Hushon, 1976, citing Piotrowski, 1967).



     Nitrobenzene can be a contaminant  in industrial waste



water, and companies utilizing or  making nitrobenzene are re-



quired to monitor its level in their effluent waste (Pierce,



1979).  The minimum detectable level of nitrobenzene  in
                             C-35

-------
drinking water by gas chromatography is 0.7 ng  (Austern, et



al. 1975).



     Nitrobenzene may be vented to the atmosphere.  The vents



are usually equipped with absorbers or scrubbers, but some



nitrobenzene vapors can escape.  Atmospheric nitrobenzene



levels outside a plant are not monitored by industry.  Since



inner plant levels are below the TLV of 5 mg/m^  (i ppm) and



nitrobenzene vapors accumulate at the floor level due to



their high density, the external air nitrobenzene concentra-



tions are expected to be very low (Dorigan and Hushon, 1976).



Special Group at Risk



     Workers in plants producing or using nitrobenzene have



the greatest risk of toxic exposure.  At the current TLV



level of 5 mg/m^ (1 ppm) a worker could absorb as much as



33 mg/day.  This is enough to produce symptoms of chronic



toxicity in some susceptible individuals (Dorigan and Hushon,



1976).  The amount of nitrobenzene absorbed by a worker via



inhalation and cutaneous absorption can be estimated from the



level of total (free and conjugated) p-nitrophenol in urine



as described by Piotrowski (1977).



     Due to the current widespread use of disposable diapers



and underpads in hospitals, nitrobenzene poisoning in infants



from laundry marking dyes is no longer a problem.



     Pregnant women may be especially at risk with respect to



nitrobenzene as with many other chemical compounds, due to



transplacental passage of the agent.  Individuals with



glucose-6- phosphate dehydrogenase deficiency may also be
                             C-36

-------
special risk groups (Calabrese, et al. 1977; Djerassi, et  al.



1975).  Additionally,  because alcohol ingestion or chronic



alcoholism can lower the lethal or toxic dose of nitrobenzene



(Rejsek, 1947; von Oettingen, 1941), individuals consuming



alcoholic beverages may be at risk.



Basis and Derivation of Criterion







        Because there  are little or no data available on the



toxicity of nitrobenzene ingested in drinking water, or on



the teratogenic, mutagenic, or carcinogenic effects of nitro-



benzene in general, experimental testing is necessary before



an oral ingestion based criterion can be derived.  It is



recommended that testing in these areas of  toxicity be imple-



mented so that the effects of nitrobenzene  on mammals may  be



better understood.



     Using the methodology of Stokinger and Woodward  (1958),



a water quality criteria (WQC) is derived using the organo-



leptic level and the TLV.



     Organoleptic Level: minimum detectable odor level in



water is 0.03 mg/1 = 30 ug/1.



     Assuming a daily intake of 2 liters of water, the total



intake of nitrobenzene based on this criteria would be 60



micrograms/day.  Recommended WQC = 30 ug/1.



     A calculation of the percentage of exposure attributable



to fish and shellfish products is not applicable to a cri-



terion based upon organoleptic effects.  Since an organolep-



tic effect is not based on a toxicological  assessment, it
                             C-37

-------
would be inappropriate  to apportion a percentage  of  exposure

to the consumption of toxicologically contaminated fish.

     TLV:  TLV = 5 mg/m3; air  intake = 10 m3/day; assume

80 percent absorption:

           (5 mg/m3) x  (10 m3/day) x  (0.8)  =  40 mg/day

average over seven days:

           40 mg/day x  5/7 = 29 mg/day

     Assuming 100 percent gastrointestinal  absorption of

nitrobenzene and consuming 2 liters of water  daily and  18.7

grams of contaminated fish having a bioconcentration factor

of 4.3, would result in a maximum permissible  concentration

of 13.9 mg/1 for the ingested  water:

         	29 mg/day	 =         ,
         2 liters +  (4.3 x 0.0187) x 1.0    *'*   g/

                  WQC using TLV = 13.9 mg/1

     Since the WQC using TLV is well above  the detectable

odor level of nitrobenzene, water containing  this concentra-

tion of nitrobenzene would not be esthetically acceptable  for

drinking.  Even though  the limitations of using organoleptic

data as a basis for  establishing a WQC are  recognized,  it  is

recommended that a WQC  of 30 ug/1 be established  at  the

present time.  This  level may  be altered as more  data are

developed upon which to calculate a WQC.

     The analysis and recommendations generated  in this docu-

ment are based on the literature available  to  date.  If

future reports indicate that nitrobenzene may be  carcino-

genic, mutagenic or  teratogenic, a reassessment of the  WQC

will be necessary.
                              C-38

-------
                                                 APPENDIX A

                                    Toxiological Effects of Nitrobenzene
  Organism

   Human
n
i
Ul
VD
   Route
Inhalation
  Exposure
              Inhalation  Poor ventila-
                           tion
Inhalation
0.2-0.5 mg/1
(40-100 ppm)
      Exposure
        Time

8 hrs./day for 17
 mos. factory worker

8 hrs./day for 1.5
 mos. factory worker
 paint firm

8 hrs./day for 4.5
 mos.
ca. 6 hrs,
              Inhalation  0.129 mg/m3
              Inhalation  "Large" amounts
                           poor ventila-
                           tion
              Inhalation  Acute
                Response

Cyanosis, headache, fatigue methemoglob-
 inemia (Ikeda and Kita, 1964).

Cyanosis, headache, fatigue, methemoglob-
 inemia, liver damage, hypotension (Ikeda
 and Kita, 1964).

Above plus: liver and spleen enlarged and
 tender, hyperalgesia in extremeties
 (Ikeda and Kita, 1964).

Slight effects, e.g. headache, fatigue
 (von Oettingen, 1941).

Threshold level for electroencephalograph
 disturbance (Andreeshcheva, 1964).

Hospitalized:

     2 - vertigo, coma, cyanosis
     3 - labored breathing, urine with
     7 - almond odor, methemoglobinemia
 recovery after 1 mo. (Ravault, et al.
 1946).

Burning throat, nausea, vomiting, gastro-
 intestinal disturbances, cold skin,
 livid face, cyanosis (von Oettingen,
 1941).

-------
                                          APPENDIX  A  (Continued)
Organism       Route      Exposure

 Human      Inhalation       -
n
i
            Inhalation  6-30 u.g/1




            Inhalation       -




            Inhalation       -




            Inhalation


            Inhalation  Acute
                                                Exposure
                                                  Time
                                         Nitrobenzene  factory
                                          worker
                                         6 hrs.
Factory worker (rub-
 ber accelerator)
                                         Factory worker
                                           (glass, porcelain)
                                          Industrial exposure
                                         Factory worker
                                           (filled containers
                                          with nitrobenzene)
                Response

Intermittent symptoms: cyanosis, pallor
 and jaundice, pharyngeal congestion,
 headache, changes in blood cell composi-
 tion (increased polynuclears and eosino-
 phils (von Oettingen, 1941).

Retained 80% of vapor in lungs, urinary
 excretion of p-nitrophenol (maximum in
 2 hrs., still detected after 100 hrs.)
 (Salmowa, et al. 1963).

Pregnant women: thickening of tissue in
 blood vessels, decreased placental ab-
 sorption, necrosis in placental tissue
 (Ferster, 1970).

Changes in bone marrow, increased lymphoid
 cell production, impairment of copper me-
 tabolism and certain iron-containing
 enzymes (Yordanova, et al. 1971).

Disturbance of motor impulses
 (Zenk, 1970).

14 days: cyanosis, headache, backache,
 stomach ache, vomiting
 ca. 21 days: drank beer and fell uncon-
 scious, cyanosis, dilated pupils, re-
 tarded respiration, weak pulse
 1 yr.: intelligence dimmed
 2 yrs.: emaciated, atrophied muscles
 3 yrs.: memory failed
 6 yrs.: loss of perception of time and
 space (Korsakoff's syndrome)
 (Chandler, 1919).

-------
                                           APPENDIX A (Continued)
  Organism

   Human
o
i
  Rabbit
   Route

Cutaneous
 absorp-
 tion

Cutaneous
 absorp-
 tion

Cutaneous
 absorp-
 tion

Oral
              Oral
              Oral
  Exposure

Dye used in
 diaper stamps
                          Shoe dye
                          0.5% by
                           weight in
                           paper
Exposure
  Time
            333 ml
            0.4 ml
Subcuta-    0.8 mg/kg
 neous
 injection

Subcuta-    10-14 mg/kg
 neous
 injection

Cutaneous   0.7 gm/kg
 absorp-
 tion

Intraperi-  0.5gm/kg
 toneal
 injection
                ca. 7 hrs.
                (Handled carbon
                 paper)

                From human milk
                Single



                Single


                Daily



                Single



                Single



                Single
                Response

Babies: cyanosis, rapid pulse, shallow res-
 piration, vomiting, convulsions, recovery
 in 24 hrs. (von Oettingen, 1941).

Unconsciousness after consumption of alco-
 hol beverages, death (Chandler, 1919).
                                       Dermatitis (Calan and Connor, 1972).
                 Nurselings became cyanotic, recovery in 24
                  hrs. (mothers ate almond cake artifici-
                  ally flavored with nitrobenzene)
                  (Dollinger, 1949).

                 Maximum dose with recovery reported fol-
                  lowing severe symptoms (von Oettingen,
                  1941).

                 Minimum lethal dose reported (von
                  Oettingen, 1941).

                 Maximum dose not causing death (Yamada,
                  1958).
                                                                 Minimum dose producing observable effects;
                                                                  slow and lasting methemoglobinemia
                                                                  (von Oettingen, 1941).

                                                                 After 52 hrs.: lethal (von Oettingen,
                                                                  1941).
                                                                 Reduced blood pressure and myocardial
                                                                  glycogen level (Labunski, 1972).

-------
                                           APPENDIX A (Continued)
  Organism

   Rabbit
   Route
Exposure
n
i
  Guinea
    pig
Intraven-   0.1 gm
 ous
             Oral
             Oral
            1 mg/kg
            0.1 mg/kg
Inhalation  Saturated air
            (0.04 vol.  %)
              Inhalation

              Subcuta-
              neous
            0.2 gm/kg
      Exposure
        Time

Daily or every 5
 days
Oral
Oral
Oral
Oral
Oral
Oral
9 gm
4.8 gm
700 mg/kg
600 mg
300 mg
50 mg/kg
4 dose
15 ml
Single
Single
Single
Single
Single
              Single



              Single

              2-5 hrs.


              2-3 hrs.

              Every other day for
               6 mos.
                Response

Simultaneous doses of 2-20 ml ethanol in-
 creased severity of poisoning (Matsumara
 and Yoshida, 1959).

Convulsions, death (von Oettingen, 1941;
 Chandler, 1919).v

Lethal instantly  (von Oettingen, 1941;
 Chandler, 1919).

Lethal dose (Stecher, 1968).

Dizziness, loss of reflexes, methemo-
 globinemia, congestion of brain tissue -
 12 hrs. - death  (Chandler, 1919).

Fatigue for 1 week (Parke, 1956).

Tissue degeneration, especially heart,
 liver, kidney (Papageorgiou and
 Argoudelis, 1973).

Lowered hemoglobin, erthyrocytes and
 lymphocytes; increased leucocytes
 (Kazakova, 1956).

Threshold toxic dose (Kazakova, 1956).

Death following tremors, paralysis of
 hind legs (Chandler, 1919).

Death (Chandler, 1919).

Hemolytic anemia, loss of weight, de-
 creased motor activity, fluxes in
 urinary excretion of 17-hydroxy-cortico-
 steroids (Porter-SiIber chromogens)
 (Makotchenko and Akhmetov, 1972).

-------
                                           APPENDIX A (Continued)
  Organism       Route      Exposure

                          ca. 3 gm
                                             Exposure
                                               Time
Guinea     Oral
 pig


           Oral


           Oral


           Oral
o
i
   Rat
                          ca. 1.2 gm
                          50 mg/kg
                          1 mg/kg
           Oral        0.1 mg/kg

           Inhalation  5 mg/m3
Single



Single


1 year


Single



Single

8 hrs.
              Inhalation  ca. 0.03 mg/m3  Daily, up to 98
                                           days


              Inhalation  0.006-0.1 mg/   70-82 days
                          m3

              Inhalation  0.008 mg/m3     73 days

              Oral        0.6 gm/kg       Single

              Intraperi-  0.8gm/kg        Single
               toneal
               infection
                Response

0.5 hrs.: tremors, faint heartbeats,
  labored respiration
2 hrs.: death (Chandler, 1919).

Immediately motionless, then complete
 recovery (Chandler, 1919).

Tissue degeneration, especially heart,
 liver, kidney (Kazakova, 1956).

Lowered hemoglobin, erythrocytes,
 lymphocytes; increased leucocytes
 (Kazakova, 1956).

Threshold toxic dose (Kazakova, 1956).

Metabolites excreted in 3 days  (Ikeda
 and Kita, 1964).

Increased ability to form sulfhemoglobin
 in preference to methemoglobin
 (Andreeshcheva, 1970).

Cerebellar disturbances, inflamed inter-
 nal organs (Khanin, 1969).

No effect (Andreeshcheva, 1964).

LD50 (Smyth, et al. (1969).

Lethal (Magos and Sziza, 1958).

-------
                                          APPENDIX A  (Continued)
 Organism

  Rat
o
i
  Mouse
   Route

Subcuta-
 neous
 injection

Subcuta-
 neous
 injection

Subcuta-
 neous
 injection

Subcuta-
 neous
 injection
  Exposure

640 mgAg



300 mg/kg
                          200 mgAg
                             or
                          100 mgAg

                          125 mgAg
      Exposure
        Time

Single



Single



Single

Daily for 10 days

Single
             Subcuta-    100-200 mgAg   Single
              neous
              injection
Cutaneous   480
 absorption
             Intrap&ri-  1.23 gm/kg      Single
              jtoneal
              injection

             Intraperi-  1 gm/kg         Single
              toneal
                Response

Blood catalase activity decreased contin-
 uously over 96 hrs. (Goldstein and
 Popovici, 1959).

LD (14 days) - methemoglobinemia, anemia,
 sulfhemoglobinemia (Brown, et al. 1975).
                                       Methemoglobinemia, sulfhemoglobinemia,
                                        anemia (Zvezdai, 1972).
                                       Delayed embryogenesis, abnormal fetal
                                        development and embryo death, changes
                                        in polysaccharide composition of pla-
                                        centa (Kazanina, 1967, 1968a,c).

                                       Sulfhemoglobin (most regular and persis-
                                        tent form of hemoglobin) nitroxyhemo-
                                        globin, increased methemoglobin
                                        (Vasilenko and Zvezdai, 1972).

                                       30 min.: prostrate, motionless
                                       24 hrs.: death (von Oettingen, 1941).

                                       40 min.: 67% dead (Smith, et al. 1967).
                                                   10-15 min.: incoordination, comatose,
                                                    shallow respiration
                                                   Several hrs.: regained coordination
                                                   Immediately before death: lost coordina-
                                                    tion again, respiratory arrest
                                                   48 hrs.: death  (Smith, et al. 1967)

-------
                                           APPENDIX A (Continued)
  Organism

   Mouse
   Cat
n
i
fc.
Cn
   Dog
   Route      Exposure            	

Intraperi-  20 mgAg        Single
 toneal
 injection

Intraperi-  12.3 mgA9      Single
 toneal
 injection

Inhalation  Saturated air   2-5 hrs.
            (0.04 vol, %)

Inhalation        -         2-3 hrs.

Oral        24 gm          Single


Inhalation  "Thick vapor"   1.5 hrs.
              Intravenous 0015-0-25 gm/   Single
               injection   kg
                      Exposure
                        Time
              Oral
              Oral
2808 gm plus    2 doses, 0.5 hrs,
6 gm             apart
24 gm
Single
                                       Response

                       Lethal dose (Brown, et al. 1975).
                                                                 10 min.s 4.2% methemoglobin formed
                                                                  (Smith, et al. 1967).
Death following tremors, paralysis of
 hind legs (Chandler, 1919).

Death

Death in 12-24 hrs (von Oettingen, 1941;
 Chandler, 1919).

Complete anesthesia and sleep (Chandler,
 1919)o

Minimum lethal dose - lowered blood pres-
 sure, pulse rate increased then decreased
 respiration stimulated until paralyzed
 (von Oettingen, 1941).

Immediate; agitation, then motionless
1 hr.: convulsions, then motionless
4,5 hrs.; tremors, hind legs paralyzed
18 hrso: death (Chandler, 1919).

Few hrs.: "stupid"
12 hrs.; deep coma, slow respiration,
 lowered skin temperature, stomach
 strongly alkaline (Chandler, 1919).

-------
                                           APPENDIX  A  (Continued)
  Organism

   Dog
o
i
   Chicken
   Pigeon
Oral

Oral



Oral


Oral


Oral


Inhalation
              Exposure

            2.4 gm
1.2 gm
                          2.4 gm
                      Exposure
                        Time
                Single
                          0.75-1.0  gm/kg   Single

                          0.5-0.7 gm/kg    Single
Daily


Single


Single
                1 hr.
                2-3 hrs.
                Response

1 hr.: vomiting, then sleep continuing
 for 6 hrs.
6 hrs.: appeared normal
15-68 hrs.: rigid muscles
104 hrs.: death (Chandler, 1919).

Minimum lethal dose (von Oettingen, 1941).

Salivation, unrest, dizziness, tremors,
 increased pulse rate, sometimes con-
 vulsions (Chandler, 1919).

Formed methemoglobin continuously at
 "certain" concentration (Hashimoto, 1958)

Unsteady gait, recovery
 (Chandler, 1919).

Immediately unconscious
12 hrs.: death (Chandler, 1919).

No effects
Death (Chandler, 1919).

-------
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