DDT

   Chemistry, Metabolism, and
                  .by
               Ben H. Lira
   Division* ibf Enforcement .Proceedings
Office of Enforcement and General Counsel
     Environmental Protection Agency
        Watshington, 0. C.  '20460
                June 197:2

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                           DDT






     DDT, Anofex, Arkotine, Chiorophenothane, Dedelo,




Dichlorodiphenyltrichloroethane, Dicophane, DND, ENT 1506,




Genitox, Gesapon, Gesarex, Gesarol, Guesarol, Gyron, loxdex,




Kopsol, Neocid, Pentachlorin, p,p'-DDT, Rukseam, Zerdane, and




C(, o( - bis  (p-Chlorophenyl)-B/B/B-trichlorethane are alterna-




tive names  for a chlorinated hydrocarbon insecticide with the




official chemical name of l/l,l-Trichloro-2/2-bis  (p-Chloro-




phenyl) ethane.  The insecticide has a molecular weight of




'354.50.  Percentage wise, carbon is 47.43; hydrogen, 2.5.6;




and  chlorine, 50.01.  The compound has the following chemical




structure:
            Cl
     DDT consists of solid, white needle crystals with a




melting point of 108.5-109°C and has a vapor pressure of




1.5 X  10"7mm of Hg at 20°C and 3.0 X 10~7mm of Hg at 25°C.




Its solubility in water has been reported as low as 0.2 parts




per billion (ppb), but the best estimate appears to be about




1.2 ppb or 3.4 X 10~*  moles per 100 milliliters of water at




25°C.  The insecticide is, however, soluble in many of the




common organic solvents.  Table 1 shows solubilities of DDT




in some of the organic solvents at various temperatures.

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                                                                       2
Table 1

   Solubility of DDT in some Organic Solvents at 27°C

                                         Solubility
   Solvent                          (g/lOOg of solvent)

Acetone                                     74
Acetophenone                  •              65
Benzene                                     89
Carbon tetrachloride                        28
Chlorobenzene                               67
Cumene                                      43
Cyclohexqne                                 19
Cyclohexanone                              122
Cymene                                      34
o-Dichlorobenzene                           45
Dichloroethane                              47
Dimethylphthalate                           29
Dioxane                                     89
Dipentene                                   26
Ethyl alcohol (95% at 24°C)       '          2.2
Isoproryl alcohol                           14
Methylene chloride                          66
Methyl ethyl ketone                        100
Methylnaphthalenes (mono- and di-)          56
Monomethylnaphthalene                       51
Pinene                                      16
Tetrachloroethylene                         23
Tetralin                                    63
1,2,4-Trichlorobenzene                      28
Trichloroethylene                           38
m-Xylene                                    64
o-Xylene                                    66
     DDT was first described in 1874 by Othmar Zeidler in

Germany.  Its insecticidal effectiveness, however, was not

discovered until 1939 by Paul Muller at the Basal laboratories

of J. R. Geigy S.A. in Switzerland.   It was patented in 1942.

The insecticide was brought into the United States during the

same year for testing.  It was later "imported in quantity.

Because of the compound's insecticidal effectivesness, the

United States began producing DDT in large quantities for

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                                                                     3
military use by early 1944.

     The technical grade product is a complex mixture of

compounds in which the p, p1  or the 4,4'-isomer amounts to

75-76%.  In Table 2 the composition of three samples of

technical grade DDT is given. '

Table 2

            Composition of Technical Grade DDT

                                     Content (%) in samples
                                      with setting point
Compound
1 , 1 , l-Trichloro-2 , 2-bis
(p-chlorophenyl) ethane i/
1,1, l-Trichloro-2- (o-Chlorophenyl )-
2- (p-chlorophenyl) ethane ,2/
1 , 1 , l-Trichloro-2 , 2-bis
(o-Chlorophenyl) ethane ~J
1 , l-Dichloro-2 , 2-bis
(p-Chlorophenyl) ethane 4/
1, l-Dichloro-2- (o-Chlorophenyl )-
91.

72.

11.

0.



2- (p-Chlorophenyl) ethane 5/ 0.
p-Chlorophenyl trichloromethy lcarbinal.6/-
o-Chlorophenyltrichloromethylcarbinal
p-chlorobenzene sulfonate 2J
p, p1 -Dichlorodiphenyl sulfone §/
o-Chlorophenylchloroacetamide 2j
p-Chlorophenylchloroacetamide 1Q/
Ammonium p-Chlorobenzenesulfonate 1J-/
Sodium p-Chlorobenzenesulfonate i?_/
Inorganic Compounds
Unidentified Compounds and losses
I/
cl / \ ? /
~\ / Li~7V

0.
0.
_
, o.
0.
-
0.
14.


/
4UC

7

9

Oil



17

57
034

006
005

01
55

V-
f
91.

72.

19.

-



0.
0'

0.
0.
_
_
: -
0.
0.
5.



2UC

9

9





3
2

4
6



02
1
58



83.

70.

20.

-



4.

0.
0.
0.
0.
—
_
0.
2.



6°C

5

9





0

1
1
007
01


04
59



       1,1,l-Trichloro-2,2-bis (p-Chlorophenyl)ethane

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                                                                 4
                    Cl
                       H
                           \        '  "
                V,
     :ci
          1,1, l-Trichloro-2- ( c-Chlorophenyl ) -2- (p-Chloropheny 1 ) ethane

3/          _ f1     "v
                       f
                       c
          1,1,l-Trichloro-2,2-bis(o-Chlorophenyl)ethane
                       H
          /         \
       ci
                   / Ju\        r
         l,l-Dichloro-2,2-bis(p-Chlorophenyl)ethane
                    Cl
                       H
                       C -(v          />— Cl
/ITS        /
                      HCC12
         1,l-Dichloro-2-(o-Chlorophenyl)-2-(p-Chlorophenyl)ethane
                          H
         Cl—(v         ,)—  C  	' CC1-
            \	/    L
          p-Chlorophenyltrichloromethylcarbinol
Z/      .           Cl
                   \ CC13    0
                   \ '       T
                   r~°~l~°~-—" cl
                     OH      5
     o-Chlorophenyltrichloromethylcarbinol p-Chlorobenzenesulfonate
                         O
                         s
                         o
         p , p ' -Dichlorodipheny 1 sul f one

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                      >
        o-Chlorophenylchloroacetomide

10/
        p-Chlorophenylchloroacetamide
                               o
                               t
         Cl~\         /)— O — S — 0 — NH,
       Ammonium p-Chlorobenzenesulfonate

12/
                           O — S — O — Na
        Sodium p-Chlorobenzenesulfonate


     Of all the isomers of DDT only the p,p'-or the 4,4'-

isomer has valuable insecticidal properties.  The properties

of the DDT isomer are given in Table 3.

Table 3

          Insecticidal Properties of DDT Isomers
                       M.P.              Relative toxicity
Isomer	(°C)	    (house flies)

  4,4' 13/           108.5-109                  1
  2,2' 14/            92.5-93        '           0.011
  2,3' 15/             	                      0.015
  2,4' 16/            74-74.5                   0.018
  3,4' IT/             	                      °'9

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                                                                      6

         4,4' iomer:
14/      2,2" isomer:
                        ,C1    Cl
                                 V
                                 /2	*
                        lY-CH
               \5-   — v,   ,., ,
         2,3'  isomer:
                _ 71             cl
                £	§7    Cci3
16/      2,4'  isomer:
                         Cl
                                     .2-
                                    (1         f)—Cl
                              CC1.
iZ/      3,4'  isomer:
               V
                                CH
                                CC1
     Because technical grade DDT has a much lower melting

point than pure p,p'-DDT, it grinds poorly in ball mills,

thereby hindering the preparation of wettable powders with

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a high content of the active ingredient.  For preparing such




powders, DDT without oily impurities is required.  This is




achieved by producing DDT from specifically purified chloral.




     The principal method of producing DDT is the condensation



of.chlorobenzene with chloral:
               ci3c-






     chlorobenzene   chloral            p/p'-DDT





This reaction takes place in the presence of condensing agents




such as concentrated sulfuric acid, oleum, chlorosulfonic acid,




fluorosulfonic acid, hydrogen fluoride, anhydrous aluminum




chloride, and others.   In industry the most frequently used




method is condensation of chloral with chlorobenzene in the




presence of concentrated sulfuric acid or weals oleum at a




temperature not higher than 20°C, since at higher temperatures




the amount of p-Chlorobenzenesulfonic acid that is formed as




a by-product increases sharply.
                     H2S04-
                                 p-chlorobenzenesulfonic acid




Routes have now been developed for the use of p-Chlorobenzene-




sulfonic acid for .the production of acaricides, DDT synergists,




and other compounds.  It also is possible to regenerate

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chlorobenzene from p-Chlorobenzenesulfonic acid in 95 percent



yield by the action of dilute sulfuric acid at 200°-240°C.



p-Chlorobenzenesulfonic acid is produced in a minimal amount



when the condensation is carried out in the presence of



chlorosulfonic acid.  However, because of the high cost of



chlorosulfonic acid this method is almost never used in


industry.
                             O              /      —\  O
                             t             /       \ t
                  ,. + Cl — S — OH—?C1—/          V? — OH

                 /         4             \       /I
                     Chlorosulfonic acid




     Chloral is obtained by chlorination of ethyl alcohol or



acetaldehyde.  The chlorination of acetaldehyde proceeds


through an enol form and can be represented by the following


general equation:
     3C12 + H3C - C - H - - >   C13C - C - H + 3HC1



        acetaldehyde               chloral






     The mechanism of chlorination of ethyl alcohol is more



complex:



     1.  CH3CH2OH + C12 -
     2.  2H3CH2oci  + HC1 - * CH3C-H + HC1

            2                          ci P
     3.  CH3C-H + C12 + HOCH2CH3 - ^ H2C-C-0-CH2CH3 + H2O

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                                                                       9
                                    __. , . ,CH,
            p                        |     2  3
     4.  CH.,C-H+Clo+2HOCH9CH7	> H?C-C-H      + HC1 + HoO
                               Cl. .QCH2CH3
           Cl OCH2CH3            Cl OCH2CH3
      .  H2C—C—H    +C12	> H-C—C—H    +HC1
                 ioCrlo            Cl OCrloCrio
            Cl OCHoCHo
            I
6.  H— C— C— H    + H20
        l OCHoCHo
                                 H— C— C— H    + CHCHOH
                                                + HC1
                                          H2CH3
               OH
     8.  C13C—C—H    + H20 	> C13C—C—H
               OCH2CH3

     It con be seen from the equations presented above that the
chlorination of ethyl alcohol is best carried out in the
presence of a small amount of water.  It has been established,
experimentally, that 14 kilograms of water to 100 kilograms
of alcohol distillate is optimum.  The process can be carried
out as either a batch or a continuous process.  The chlorina-
tion is carried out in the first stage at 50°-60°C, and then
at 90°C.
     The product obtained by the chlorination of ethyl alcohol
contains chloral alcoholate and chloral hydrate, which on
treatment of the reaction mixture with concentrated sulfuric
acid go over to chloral:

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              ?H
     a.  -C13C-CH

              OH
              ?H
     b.  C13C-CH + H2S04-

              OCH2CH3
C13C
-C-OH +
H2S04'H20
C13C-C-OH+CH3CH20-S-OH+H20
     It is also possible to obtain DDT by the condensation

of chlorobenzene with pentachloroethane:
           pentachloroethane


However, the DDT produced by this method is strongly

contaminated with various by-products.

     An interesting method for the synthesis of DDT and

especially of its unsymmetrical analogs is the reaction of

chlorobenzene with p-Chlorophenyltrichloromethylcarbinol:
                                                                    10
                                                                 NC1+2HC1
          p-Chlorophenyl-
          trichloromethylcarbinol
This reaction proceeds readily in the- presence of sulfuric

acid or oleum.  The p-Chlorophenyltrichloromethylcarbinol

is prepared from chloroform and p-Chlorobenzaldehyde:
                                                                 C1+H2O

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                                                                      11
CHC13 + Cl -£         \  C-H	»CL/         Vc-OH
      For the synthesis of the radioactive C-14 labeled DDT

 the following laboratory reactions may be employed:

      1. *C + O2 	> *C02  (*C denotes C-14)
A                                    Modified
                       /> +*C02+HC1 Friedel-crofts  cl /         V*J-H + HC1
               ,	/     2      reaction        ^	^

                                  (dry AlCl3/Cu2Cl2)
                                                   ' p-Chlorobenzaldehyde
          3r%i_f          \   j./* TT •  /•"!   	v r«i /          \	*/* /^T  i U/"1!
          Cl-L         .f—*C-H + C12 	^ Cl ^.          /^-*C-C1  + HC1


                                           p-Chlorobenzoyl chloride
                          o                                o
      4.  Cl-(.         A-*C-Cl+HoN-C=N—>C1-^          \- *C-CH?C1  + No
                      f      c     .d     \	f
                ——'         Cyanamide              '
                         
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                                                                    12
7*
     The pure p,p'-isomer of DDT is  thermally  stable.  Its

decomposition starts above 195°C and proceeds  according  to the

equation :
Iron salt impurities sharply lower- the decomposition  temp-

erature of DDT.  For example,  in the presence  of  only 0.01

percent ferric chloride (FeCls), the decomposition  tempera-

ture is lowered to 120°C.

     When DDT decomposes under the influence of sunlight  in

alcohol solution, the following reactions  take place:
                     ;C1 C,.                      o
                  H-C-t=6-C-H        .    + 2 CH3?-H + 4HC1
                                           acetaldehyde

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2.  In the presence of the oxygen in air, the tetra,




p-chlorobenzene compound above undergoes the following




oxidation reaction:
                                                                     13
     Cl
              p,p'-dichlorobenzophenone




Apparently similar processes also occur "on the leaves of




plants.




     Pure DDT at room temperature does not affect most metals,




but technical preparations, especially those containing water

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and salt solutions, cause more or less corrosion.   Probably



this is associated with the evolution of HC1 as a result



of hydrolysis of the DDT by water as the following equation



indicates:
                           1+2H90
                               ft
                                     1+3HC1
                                                     - OH




                                     p,p'-dichlorodiphenylacetic acid



At room temperature, this reaction proceeds slowly, but when



an aqueous suspension of DDT is boiled, the process is



accelerated.  Caustic alkalies, lime, barium hydroxide, and



other alkaline agents increase the rate of hydrolysis of DDT.



The first step of the reaction of DDT with alkalies is the



splitting out of HC1 and the formation of p,p'-dichloro-



diphenylethylene, which further reacts at a higher tempera-



ture to p,p'-dichlorodiphenylacetic acid:
1.  Cl-
Cl+KOH-
                                                          -C1+KC1+H2O
                                    p,p'-dichlorodiphenylethylene
2.  Cl
C1+2H 0-=-> Cl-
                                                             -C1+2HC1

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                                                                    15
This reaction is employed for the quantitative determination




of DDT and also of the p,p'-isomer of DDT in the technical




grade product.  Determination of p,p'-DDT is based on the




different rate of splitting out of HC1 by caustic alkalies




from the isomers of DDT.




     Extensive investigations have been carried out on




the synthesis and on the biological activity of homologs




and analogs of DDT.  When chlorine in the aromatic radical




of l,l,l-trichloro-2,2-bis (p-Chlorophenyl)  ethane is




replaced by hydrogen (Fig. 1), bromine (Fig. 2), iodine




(Fig. 3), hydroxyl (Fig. 4), a higher hydrocarbon radical




(Fig. 5), amino (Fig. 6), thiocyano (Fig. 7), carboxyl




(Fig. 8), nitro (Fig. 9), and cyano (Fig. 10) groups the




insecticidal activity of the compound is substantially




lowered.  A lowering of the insecticidal activity also




occurs when several methyl (Fig. 11) or alkoxy (Fig. 12)




groups are introduced into the aromatic radicals.
     Figure 1
     Figure 3
Figure 2
Figure 4
                                                      -Br

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CH3(CH2)4
Figure 5
                  H
                         (CH2)4CH3
     H2N
Figure  6
                         NH,
     NCS
Figure 7
                          SCN
                   CC1-
HO-
Figure 8
                   H
                  otlX
                             .-C-OH
                                                                 16
      02N





Figure 9
                   H
                   I
                  -C

                   CC1
                           NO,

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          NC
            \_/tc
   Figure 10
           HoC
                                                                    17
     Replacement of chlorine by fluorine (Fig. 13), methoyl




(Fig. 14), methyl (Fig. 15), or ethyl (Fig. 16), does not




substantially change the insecticidal activity of the




compound, but it lowers the toxicity for vertebrates and



man.

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                                                                   18
   Figure 13
   Figure 14
                    H  /^^X
                   - C -/     VCH2CH3
                       \\    A   '
                      4    /  '•
For examples,  1,1,l-Trichloro-2,2-bis (p-methoxyphenyl)

ethane (Fig.  14)  is one-fortieth as toxic as DDT for

mammals, while l/l/l-Trichloro-2,2-bis (p-ethoxyphenyl)

ethane (Fig.  17)  is two-thirds as toxic.

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                                                                    19
                                OCH2CH3
   Figure 17
     Trichlorodinaphthyl ethane (Fig. 18), trichloro-




dithienyl ethane (Fig. 19), and others are weak insecticides.




The unsymmetrical analogs also, as a rule, are considerably




less active than DDT.
   Figure 18
Figure 19
Removal of the trichloromethyl group from the aromatic




radicals leads to less active compounds, as does also the




splitting out of HC1 from compounds containing the trich-




loromethyl group.  Replacement of one chlorine (Fig. 20)




in the trichloromethyl group by hydrogen lowers the




insecticidal activity by two to four times, and the toxicity




for animals by 5 to 15 times.  When a second chlorine atom




(Fig. 21) is replaced by hydrogen, a fivefold decrease in




toxicity for animals is observed in comparison with DDT




and the insecticidal activity decreases 5 to 50 times.

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                                                                   20
Replacement of chlorine by fluorine (Fig.  23),  bromine




(Fig. 24), alkyl groups (Fig.  25),  and other similar  groups




in most cases leads to a substantial lowering of the




insecticidal properties.  p,p'-Dichlorodiphenylethane




(Fig. 22) has practically no toxic  effects toward insects,




but it is an acaricide.  The corresponding dichlorodiary-




lethylenes (Fig. 26) are also practically  nontoxic toward




insects.  The diaryltrichlorovinylmethanes (Fig. .27)  have  .




only weak insecticidal properties.
   Figure 20
Figure 21
   Figure 22
Figure 23
   Figure 24
                                               C(CH3CH2).
Figure 25

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   Figure 26
                                 Cl-
                                             ici3
                   Figure 27
     Of a large number of analogs and homologs of DDT that




have been studied, a few, which are described briefly below,



have found practical use.  These analogs are commonly known



as methoxychlor, DDD, Perthane, and DFDT.  The official



chemical name for methoxychlor is l,l,l-trichloro-2,2-bis



(p-methoxyphenyl) ethane.  It has the following chemical



structure:
                                                                  21
      CH30
                OCH3
Methoxychlor is obtained in good yields by the condensation




of chloral with anisole in the presence of sulfuric acid




as indicated in the equation below.
CH30-,
              OCH-:
                   -OCHo+H9O
                       O  £j
  anisole
chloral
methoxychlor

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                                                                  22
In contrast to the manufacture of DDT,  the use of oleum


(I^SoC^) is not recommended in making methoxychlor,  because


in this process a very large amount of  sulfonation products


is obtained.  The technical grade product contains not less


than 88 percent of the p,p'-isomer and  a small amount of


the o,p'-isomer.  Methoxychlor has limited use because not


only does it cost more than DDT,  but it also has little


effectiveness toward a number of insects.  It often  is


contained in various preparations based on DDT and lindane


as a third component.  Methoxychlor formulations are


completely similar to those of DDT.


     In addition to methoxychlor, DDD has found wide use


in agriculture.  DDD is also known as TDK (tetrachloro-


dipheny1ethane).  Its official chemical name is 1,1-


Dichloro-2,2-bis (p-Chlorophenyl) ethane and has the


following chemical structure:
DDD consists of white crystals with a melting point of 112°C.



The technical grade preparation has a setting point of about


86°C and contains as the main impurity the o,p'-isomer; i.e.

                         /Cl

                             H
                                /      \
                                         -Cl

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The o/p|-isomer has indicated promising results  in  the




medical treatment of malignant tumors of the adrenal




glands.  DDD is one of the metabolites of DDT.   It  is




produced by the condensation of chlorobenzene with




dichloroacetaldehyde, which must be very pure and not




contain chloral as an impurity.  The reaction goes  as




follows:
                                                                  23
  Chlorobenzene    Dichloroacetaldehyde






DDD differs somewhat from DDT in its chemical  properties.




Where DDT splits out one molecule of HC1 when  treated




with ferric chloride, DDD splits out two molecules  of




HC1.  The respective equations are as follows:




a.  DDT:
                                                                C1+H2O
                                                            -C1+HC1
b.  DDD:
              H
  Cl
/     Y.4
                            Fed-
c=c-
                                                           C1+2HC1
                                     Dichlorotolan

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     Perthane is a selective insecticide to control pests




of stone fruits, lettuce, and spinach and also to control




flies in animal husbandry.  Ferthdne is a white crystalline




substance with a melting point of 56°-57°C.  It is insoluble




in water, highly soluble in organic solvents.  It is produced




by the condensation of dichloroacetaldehyde with ethylbenzene




in the presence of sulfuric acid:




a.
 r*u Friedel-Crafts
,oH"lo

*  °Anhydrons
                                           >\

                                            V
                                                    CH2CH3
                                         ethylbenzene
                                                                   24
2CH3CH2~~>
                          2:
                        H-C-
                                              Perthane
                        H-i
                                     CH3CH2-
        Cl,
Perthane is one-fifth to one-tenth as active as DDT.




     DFDT is another analog of DDT.  It has a melting point




of 45°C.  It is almost insoluble in water,  but more soluble




than DDT. in organic solvents.  DFDT is produced by the
                                     /



condensation of chloral with fluorobenzene  in the presence




of sulfuric acid.




   a.
                    +  Br,
                           Fe

                                             :H2CH3

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                                                                  25
   b.
                   Br + AgF

—F + AgBr
   c.
                                                     DFDT


The official chemical name of the insecticide  is  1,1,1-


Trichloro-2,2-bis(p-fluorophenyl)ethane.   DFDT is similar


to DDT in chemical properties.  Its toxicity for  insects


is close to that of DDT.   The analog is less persistent than


DDT, which in some cases  is a great advantage. It is, however,


more expensive than DDT because of the relatively high cost


of fluorobenzene.


Metabolism of DDT:


     There are five principal routes of DDT metabolism in


various organisms:


          1.  Oxidation to DDA, common name for
                                    f

              bis(chlorophenyl)acetic acid.


          2.  Oxidation to Kelthane, common name  for


              1,1-bis(p-chlorophenyl)-!,!,1-trichloro-


              ethanol


          3.  Oxidation to dichlorobenzophenone.

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                                                                  26
       .   4.  Dehydrochlorination to DDE,  common name




              for dichlorodiphenyldichloroethylent.




          5.  Reductive dechlorination to  DDD, common




              name for l,l-Dichloro-2,2-bis(p-chloro-




              phenyl)ethane.




Figure 28 gives the routes and the chemical structures




of the metabolites of DDT.
                                                                Cl
                                                 Kelthane
                       3   oxidation  ^  ci-
                                          Dichlorobenzophenone
                                                   DDD
   Figure 28.  Routes of DDT Metabolism

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                                                                    27
     In vertebrates,  it has been known for almost 20 years

that DBA, a common name for bis(chlorophenyl)  acetic acid,

is a major metabolite in feces and urine.   Early reports

claimed substantial amounts of unchanged DDT in urine and

feces, but more recent findings have cast  doubt on these

reports, although there is no doubt that modest amounts of

DDT occur in feces.  Furthermore, the best study has shown

that fecal DDA occurs as some derivatives  whose nature is

unknown; because DDA is produced from the  derivative on

acid, but not on alkaline, hydrolysis, it  was suggested

that the derivative might be an amide but  was certainly

not the usual glycoside.  The pathway for  DDA production

in rats has recently been reported to involve alternate

reduction and dehydrochlorination, followed by hydration

and oxidation.  This pathway is given in Figure 29.
  C1~
                                    Figure 29.  A possible  route  for
                                    DDA synthesis in  the  rat.
        DDA

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The evidence for this sequence is that the feeding of each

compound to rats gave the subsequent intermediate as the

major metabolite in liver.  An exception is the last step,

which is presumptive, for the metabolism of "DDOH" was not

examined.

     The simplifying view that DDA is the product of DDT

metabolism in vertebrates is complicated by species varia-

tion.  In man, DDE, a common name for dichlorodiphenyl-

dichloroethylene, is the principal storage form of ingested

DDT, and since DDE is not on the above route to DDA, that

route is probably inoperative in man.  By contrast, rats

convert only small amounts of DDT to DDE, and monkeys none

at all.

     Only recently has it been realized that even in mammals

another route of DDT metabolism is common; namely, reductive

dechlorination to DDD, a common name for l,l-Dichloro-2,2-

bis(p-chlorophenyl)ethane.  The reason for the delayed re-

alization is that the standard Schechter-Haller method -i/

does not distinguish DDT from DDD; consequently, chrqmato-

graphic techniques are required to separate them.  Because

DDD is insecticidal, this metabolism is not a detoxification.

In 1963, it was reported that DDD was widely found in samples

of water, soil, plants, and animal tissues obtained from
T7The Schechter-Haller Method:  ^/Schechter, M.S., Soloway,
    S.B., Hayes, R.A., and Haller, H.L. (1945).  Ind. Eng.
    Chem., Anal. Ed., Vol. 17 (11), p. 7QA-JJ  DDT is nitrated
    to the tetranitro derivative which produces a colored
    solution in benzene with a maximum absorption at 596 nyu
    when reacted with methanolic "sodium methylate.  However,
    any aromatic compound that may be nitrated is a potential
   • source of interference.  Homologs and analogs of DDT also
    respond to this analytical method.

-------
                                                                   29
areas where DDT, but not DDD,  has been sprayed.   In the




same year, the reaction was demonstrated in yeast;  and in




1965 in rats, mice, and rumen fluid.   In the case of rats,




it was found that DDD was present in  the liver,  but none




was in body fat.  In 1965, it was found that even lake




water can convert DDT to DDD,  and so  can the two porphyrins,




hematin and hemoglobin, in the reduced form.  Since porphyrins




are astonishingly stable with a half-life of billions of years




in many media -, it may be that the conversion in  lake water




utilizes porphyrins; and thus, the conversion reaction from




DDT to DDD may not be enzymatically induced.




     Isomerization in vivo is an unusual reaction for exotic




compounds, but it has recently been shown that feeding




o,p'-DDT leads to substantial residues of p,p'-DDT in the •




liver of rats.
               °'P'-DDT
                                           p,p'-DDT
Although there are other metabolities,  the p,p'-DDT isomer




was a major one.  The parts per million (ppm)  of DDD,  p,p'-DDT,




o,p'-DDT, and DDE were, respectively,  o.64, 0.47, 0.12 and




0.10 after feeding 50 ppm of o,p'-DDT.   The reverse isomeriza-




tion of p,p'-DDT to o,p'-DDT is of very small, perhaps zero,

-------
                                                                   30
importance.   In seven experiments in which p,p'-DDT was




fed at 50 ppm, the o,p'-DDT was found only in two cases




and at low levels: 0.09 and 0.03 ppm.




     In insects, the best known metabolite of DDT is DDE.




The ability of the housefly to dehydrochlorinate DDT to DDE




may be a major cause of the exposed groups resistance to the




DDT.  The enzyme responsibel for this reaction is DDT-dehy-




drochlorinase.  The enzyme has been greatly purified and




its properties extensively studied.  It exists, in very




small concentrations, in susceptibel as well as in resis-




tant houseflies.  The enzyme is of moderate specificity, in




that it also dehydrochlorinates DDD, but it is ineffective




against o,p'-DDT.  The Mexican bean beetle, which is normally




tolerant to DDT and related compounds such as DDD and meth-




oxychlor, has a relatively high titer of DDT-dehydrochlorinase.




The level of this enzyme, measured by DDD dehydrochlorination,




fluctuates during the development of the beetle, and this




fluctuation is mirrored by parallel fluctuations in DDD




tolerance.




     Work with homogenates of houseflies, rather than with




purified enzyme, has suggested that there may be more than one




dehydrohalogenase enzyme.  This is indicated by the fact




that susceptible houseflies with little ability to dehydro-




chlorinate DDT can dehydrobrominate the CBr3 analog excellently.

-------
                                                                    31
                                                           Cl  + HBr
Studies on homogenates  of resistant houseflies show that




the CBr3 analog is dehydrobrominated four times faster




than DDT; the CHBr2 or  CHClBr,  15  times faster; and the




CHC12 analog at one-thirtieth the  rate..




 1.
2.
                                    -> Cl
.Cl +HC1
                                          Relative  rate = 1
                                                               Cl +HBr
                                         Relative rate = 4
4.
                                         Relative rate = 15
                                                   HCC1




                                         Relative rate = 15
                                                               Cl+HBr

-------
                                                                   32
5.
                                                              C1+HC1
                                        Relative rate = 1/30









In view of the above suggestion of the existence of more




than one dehydrohalogenase, these findings may not describe




the specificity of a single enzyme, but rather suggest that,




in resistant insects too, more than one such enzyme exists.




     Degradation to DDE is also a major pathway in some




insects other than houseflies, including Mexican bean beetle,




pink bollworm, and Aedes aegypti mosquito.




     It seems that in resistant pink bollworms and in Aedes,




as in the housefly, the resistance is primarily due to a




far greater titer in resistant than in susceptible strains.




However, the DDT dehydrochlorinases may differ in different




species.  In the resistant housefly, the enzyme is specific




enough that o,p-DDT is not dehydrochlorinated, so that .-




resistant insects can be killed by this compound.




     An inhibitor for DDT-dehydrochlorinase has been




developed; i.e., "WARF antiresistant."  (WARF are the




initials of the Wisconsin Alumni Research Foundation).




Its chemical name is N,N-Dibutyl-4-chlorobenzenesulfonamide.




It has the following chemical structure:
I
                        ^CH2CH2CH2CH3

-------
                                                                    33
This compound has the ability to synergize the toxicity of




DDT to resistant houseflies and Aedes,  and is therefore




considered to operate by blocking DDT-dehydrochlorinase.




     It was found that DDA, originally  thought to be




primarily a metabolite in vertebrates,  is also important




in at least one insect; body house.  In the homogenates




of this insect, DDT is degraded to DDA, dichlorobenzo-




phenone, and DDE in the ratio 2:2:1. The enzyme (or




enzymes) involved showed astonishing heat stability, for




it could be boiled for an hour withour  loss of activity!




Fractionation suggested that at least two enzymes were




involved.




     In the domestic fruit fly Drosophila melanogaster,




quite different metabolic routes exist.  In 1959, an




investigator showed that there was quite extensive




matabolism to Kelthane.  Later studies  made with C  -DDT




showed that dichlorobenzophenone is the other major




metabolite in the larvae.  In adult fruit flies, two other




principal metabolites were demonstrated without being




identified.  In this study, a remarkable strain variation




was found; Kelthane was the major metabolite in strain




Oregon Re, but was not a metabolite in strain Oregon R.




An enzyme system for converting DDT to Kelthane, or a




Kethane-like material, has been shown in microsomers from




German cockroaches.

-------
                                                                    34
Mechanism of Action

     It is painful to have to admit that,  after decades

of intensive research, there is still much to be desired

in the full explanation of the mechanism of action of DDT.

It is, however, generally agreed now that the insecticide's

primary effects are virtually all upon the nervous system,

both in vertebrates and invertebrates.  The evidence for

this view is as follows:

     (1)  The symptoms of poisoning suggest nervous

impairment.  In the American cockroach, for example, there

is tremor throughout the body and appendages.  These

symptoms are commonly called "DDT jitters".  Additionally,

the treated insect showed hyperexcitability, followed very

slowly by loss of motion or ataxia and total paralysis

within 24 hours.  In more sensitive insects, such as house-

flies, fruit flies, and bees, the symptoms are similar but

appear more rapidly, with paralysis in a few hours.

Similarly, in mammals there is hyperexcitability and tremor,

which is particularly evident in the face, and later there

are convulsions, which may be both tonic,  (i.e., the animal

is rigid) with opisthotonus (the animalls head is arched
                                    /
back) and clonic (the animal is frenzied with uncoordinated

movements).  Finally, there is weakness and prostration.


     (2)  When DDT is applied to isolated tissues and

enzymes, only nervous tissue is sensitive to very low

concentrations.  This statement is true only if one

-------
                                                                    35
excludes those effects given equally by DDT and nontoxic




analogs, such as DDE.




     (3)  In the DDT-treated rat,  an excellent correlation




has been found between the level of DDT in the central




nervous system and the intensity of symptoms.




     On the above evidence it seems safe to conclude that




DDT is a neurotoxicant.  Two questions are begged from




this conclusion; namely, 1. Where does the disruption of




the nervous system occur?  2.  What is the explanation




for the disruption?




     In 1945, a group of investigators showed the now




familiar physiological symptoms of DDT poisoning of the




nerve.  According to these investigations, the phenomenon




was a multiple effect; i.e., single nerve impulses arriving




at a DDT-treated region of the nerve give rise to prolonged




volleys of impulses.  These volleys may account for the




"DDT-jitters" symptoms.  This was demonstrated in crayfish,




crabs, lobsters, and cockroaches.   Cockroach nerve, however,




was at least ten times less sensitive than the crustacean,




a fact that paralleled the 40-fold lower toxicity of DDT




to whole cockroaches than to whole crayfish.  The multiple




effect could also be demonstrated on isolated nerve trunk.




Therefore, it may be reasonable to assume that DDT acts




on axonic rather than on synoptic transmission, a property




shared by very few drugs, the best known of which is




veratrine.

-------
     The question of the relative sensitivity of various

nerves to DDT has provoked different answers.  Some investiga-

tors claimed that the motor nerves were more sensitive than

the sensory ones.  Others claimed the opposite to be true.

It appears that the latter may be the case from investigations

designed specifically to settle this moot point.  In 1946,

one investigation showed definitely that although 1,000 ppm

of DDT in solution could affect motor nerves and even muscle

fibers in the American cockroach, low concentrations in the

order of 0.01 ppm have no effect on these, or on the central

nervous system, but only upon sensory nerves.  These findings

were later confirmed by another investigation.  It therefore

seems extremely likely that DDT is lethal because of its

effects on sensory nerves.

     This conclusion, in turn, begs the following question:

What physiological mechanism gives rise to this effect?  An

early speculation was that calcium ion permeability of the

nerve was reduced, because the investigators' work in

crayfish showed that lowering the calcium ion concentration

antagonized the DDT effect.  However, these calcium ion

effects are not seen in insect nerve; and, therefore,
                                    {
probably have no connection with the poisoning of insects.

     The most revealing studies on the mechanism of DDT

excitation were begun in 1957 by a group of Japanese

investigators, working with the crural nerve of the American

cockroach, and using intracellular electrodes, which show

-------
                                                                    37
the response of a single neuron rather than responses of




bundles of nerves.  The results of the investigations




indicated that DDT affected the action potential in a




specific way; namely, it increased the negative after-potential




(NAP).  Because the NAP is associated with potassium ion




efflux in cockroaches as well as vertabrates, the Japanese




investigators suggested that DDT specifically inhibits




this efflux.




     For reasons of clarity, it is well, perhaps, to digress




briefly and discuss the neurobiological aspects of this




particular phenomenon of potassium ion efflux and impulse




transmission in nerves.




     There are two quite different modes of nerve impulse




transmission in the nervous system:  axonic transmission




which conveys an impulse from its arrival point, then along




the axon to the meeting place with another cell, which may




be another neuron or may be a muscle, gland, or sensory




receptor cell.  Across the junction between cells, synoptic




transmission occurs.  The term synapse was formerly used




for junctions between two neurons, but is now generally




used for junctions of neurons with other cells, even for




the junction between neuron and muscle which has the specific




name of neuromuscular junction.




     Neurophysiologists used to be either "sparks" men or




"soup" men: the sparks men believed all transmission was




electrical; the soup men argued that it was chemical.  In

-------
                                                                   38
fact, it is now firmly established that virtually all




axonic transmission is "electrical",  in a manner of




speaking, and virtually all synoptic  transmission is




chemical.




     The modern understanding of the  basis of axonic




transmission is due largely to A.L. Hodgin and A.F. Huxley.




If one pokes an electrode into a resting axon, and measures




the internal potential of the axon with respect to some




outside point, the inside is found to be more negative




than the outside; i.e., the axon is polarized.  The




resting potential difference is the "membrane potential."




When a nerve impulse goes by, the inside suddenly becomes




more negative than the outside, but recovers as the




impulse passes on.  In fact, it is this propagated reversal




of polarity that constitutes the impulse.  This moving




deploarization is called an action potential.




     The resting potential is believed to be caused by




the existence of a higher potassium ion level inside the




nerve than outside.  On the other hand, the sodium ion




level is higher on the outside portion of a nerve's




membrane.  The first or rising part of the action potential




is caused by the sudden development of leakiness to sodium




ions in the axon's outer membrane, so that sodium rushes




in and the potential rapidly drops to zero and even goes




positive.  These potentials are commonly called after-potentials,

-------
either negative or positive.  Then, microseconds later,




the membrane becomes leaky to potassium ions that, because




their concentration is higher inside than out, rush out and




restore the equilibrium.  It is further believed that a




system, picturesquely called the sodium pump, is always




busy pumping sodium ions out of the nerve to maintain its




low internal sodium ion concentration, and hence the nerve's




ability to be fired.  The causal relationships to explain




the sudden sodium ion leakiness in the membrane is un-




answered.




     To return to the experiments done in Japan, the




investigators showed that high potassium concentrations




reduced the effects of DDT on cockroach nerve, and low




potassium concentrations enormously enhanced them, so




that the negative after-potential (NAP) in a potassium-




free environment appeared as a plateau rather than as a




shoulder as given in Figure 30.
                     •
                       N'A?
                     A  "f
                          "I'-rnt.
   Figure 30.  Action potentials recorded intracellularly.

-------
     A.  A schematic diagram (S=spike, NAP=negative




         after-potential, PAP=positive after-potential).




     B.  Drawing based on normal cockroach giant axon




         data.




     C.  100 minutes after 10~4 M DDT.




     D.  Same in potassium-free solution.




     In addition to the "potassium-sodium" ions exchange




explanation above, there is also a molecular basis for




the physiological disruption in impulse transmission




caused by DDT.




     For a clearer understanding of this concept, a few




words concerning the chemistry of impulse transmission at




the synoptic juncture may need to be said at this point.




When an impulse, propagated along an axon, reaches a




synapse, the impulse itself dies out.  However, it causes




release, from the end of the axon, of a little burst or




quantum or cloud of a chemical, the transmitter substance,




which diffuses across the synapse and triggers off another




action potential if the synapse is between neurons, or an




appropriate response if the synapse is between a neuron




and some effector, such as muscle or gland.  There are two




known kinds of transmitter substance; namely, acetylcholine




and norepinephrine.  The chemical structures of these




substances are given below.

-------
                                                              41
                                     CH-CH2-NH2
                                           OH




            *    CH3
   P        + X C
CH3C-OCH2CH2N — CH3


                 CH3                 HO


                                           OH


     acetylcholine                   norepinephrine


Synapses which utilize acetylcholine are called cholinergic,


those that utilize norepinephrine are called adrenergic.


     The above explanation assumes that the transmitter


substances stimulate some component on the far side of


the synapse, or the post-synaptic side.  This component is


called the receptor.  One explanation is that the transmitter


substance combines with the receptor to produce a configura-


tional change which alters the sodium ion permeability at


that point, thus triggering an action potential or appropriate


response.  A diagram of a synapse is given in Figure 31.
             ; • «

            ', ( Vr. ^V^. ..^-"""VJ--'

                                          I ,
                             f.'
                                       o > I *•*' ?^


                                     
-------
                                                                   42
     In order to restore the sensitivity of the synapse,
the transmitter substances must be eliminated so that the
receptor can return to its resting condition.  At cholinergic
junctions, acetylcholine is very promptly removed by
cholinesterase, which hydrolyzes it to inactive components;
i.e., acetic acid and choline.   The reaction is as follows:
                 OH
                                              H
The cholinesterase is commonly on the presynaptic side of
the synapse rather than immediately adjacent to the recptor,
but since the synoptic cleft, the zone between the presynaptic
and postsynaptic endings, is only about 500 angstroms (A) wide
(1A=10   cm.), and each acetylcholine molecule is about 9 A
long, it is not difficult to imagine that presynaptic
cholinesterase could rapidly eliminate acetylcholine through-
out the synoptic cleft.  In adrenergic junctions, the
corresponding degrading enzyme is monoamine oxidase, but its
precise localization is not known, and the current view is
that diffusion away from the site is the major mode of loss,
followed by oxidative removal at a relatively slow pace.
     The enzymatic hydrolysis of acetylcholine is strongly
inhibited by the alkaloids physostigmine, neostigmine, and
atropine at levels as low as one part per million with
respect to the inhibitor.  The esterases are irreversibly

-------
                                                                   43
inhibited by diisoprpylfluorophosphate.  Chemical structures
for these compounds are given below.
                                        N(CH3)
                     CH-
        Physostigmine
                                            N(CH3).
                                Neostigmine
   H2C
    -CH-
-CH-
           •CH-
            CH
   •*    yn
   :-o-LcH
   H2C
   —CH—

Atropine
-CHr
                                O-CH

                              F-P	

                                O-CH
                                    CH3

                                    CH3

                                   .CH
                       Diisopropylfluorophosphate


     Returning to the discussion.of  the molecular basis

for the physiological disruption  in  nerve  caused  by DDT,

there are several theories, which overlap  a good  deal, that

are based upon the formation  of some sort  of  complex

between DDT and "the nerve membrane." By  this, it is

generally meant that the axonic membrane is involved  in

the action potential.

-------
                                                                   44
     The- results of one investigation demonstrated the




strong affinity of DDT for cholesterol,  which is  common




in nerve tissue, and speculated that combination  with




some important nerve-cell lipoids might  cause "a  state of




excitability."  Two attempts have been made to put this




concept on a more specific footing.   One attempt  was made




by L.J. Mullins in 1955.  Mullins theorized that  the target




for lindane and DDT was a hypothetical lattice in the nerve




membrane.  This is a steric conception,  comparable to that




part of theorectical organic chemistry in attempting reaction




mechanisms.




     Mullins1 hypothesis stemmed from his observations that




certain isomers of 1,2,3,4,5,6-Hexachlorocyclohexane (HCH),




popularly and chemically incorrect-called !'benzene hexa-




chloride" or "BHC", had little or no insecticidal effects




on the target organism, while others were quite potent.




He attempted to explain the marked variation in neural




activity caused by the various HCH isomers on the basis




of varying abilities to "fit" into a hypothetical lattice




in the axonic membrane.  He suggested that one might consider




the lattice to resemble the spaces seen  when one  looks, end-




on, at a pile of cylinders packed in the tightest possible




way, which would be a hexagonal array (see Figure 32).

-------
                                                                    45
     Figure 32.  Mullins1  model of on insecticide
     molecule in the axonic membrane lattice.   The
     space is formed from a gap between three
     cycliders of 40A diameter, separated from
     each other by 2A.
The spaces are the hypothetical pores in the membrane

lattice, and Mullins suggests that compounds are excitatory

if they fit tightly into these pores.  Compounds that are

too large should have no effect.  Compounds that can fit

snugly might well distort the membrane structure and

produce excitability.  Compounds that are small enough

to enter, but can only bind at one or two places of

contact, would block the pores but not distort the

membrane, and thus lessen excitability..  Mullins

hypothesized that if one considered a lattice made

up of axons with diameters of 40 angstroms (A) and with a

separation gap of 28, the resultant spaces or pores made

-------
                                                                    46
up of adjacent cylindrical axons were such that in the




plane orientation - that is, pushing the HCH isomer.in




with its flat face flushed with the plane of the pore -,




the gamma HCH isomer fitted neatly.  On the other hand,




other known isomers of HCH; namely/ those that are alpha,




beta, delta, and epsilon, were found to be too large.  The




gamma HCH isomer is commercially known as lindane and shows




very strong excitation in the treated organism.  Mullins




further hypothesized that all of the HCH isomers could




be pushed into the lattice-space, in an end-on orientation




of the molecule, but then none fitted tightly enough to




the walls of the adjacent axons.  Mullins showed that DDT,




if pushed into the lattice space in an end-on position,




fitted rather tightly, and was therefore prepared to




extend his steric concept to DDT activity.  To augment his




hypothesis, Mullins noted that 2-chloro-DDT cannot be




correctly oriented; and thus, explains its activity.




     Although Mullins1 hypothesis has never been disproved,




it remains still entirely speculative and awaits confirming




experimental evidence.  It would-appear that the key




experimental results would be those equivocally showing




lindane or another active agent actually binding or




adhering to some vital surface of the axon membrane and •




thereby altering the axon's or its membrane's properties.




     Mullins1 steric hypothesis may be contrasted with




the almost purely electronic explanation by F.A. Gunther

-------
                                                                   47
and his co-workers.  They examined the role of bonding

energies, which they speculated were entirely of the

van der Waals type.  Gunther and his colleagues examined

30 DDT analogs, and treated the data by considering the

substituted phenyls together as a "head" and the sub-

stituted alkane portion as the "tail."  The DDT molecule

may be diagrammatical ly represented as follows:
                                R4
                                R.5

          "Head"             "Tail"

The various R's could be Cl, H,  or CH3,  so that for any

tail, for instance, one could have six different heads;

namely, 1. Cl and Cl;  2. H and H;  3. CH3 and CH3;

4. Cl and H;  5. Cl and CH3; and 6. H and CH3.  Of the

many tails that could be made, they chose five; namely,

1. H, H, and H;  2. H, H, and CH3;  3. H, Cl,  and Cl;

4. H, H, and Cl; and 5. Cl, Cl,  and Cl.   They  reported

that if one end of DDT or one of its analog, a head or a

tail, were held constant, then the toxicity for mosquitoes
                                    f
(Culex quinquefasciatus) increased linearly with each

logarithmic increment of the total van der Waals forces

of the chlorines plus the methyls plus the hydrogens.

The slopes of the eleven graphs  prepared in this way were

remarkably constant in eight cases, varying only between

0.5 and 0.7.  Unfortunately, neither correlation coefficients

-------
                                                                  48
nor the graphs themselves were reported.   However,  if one


were to assume that the graphical fit was good,  then the


implication is that by increasing nonspecific bonding forces


one can improve affinity for a hypothetical target  whose


shape is complementary to DDT, and hence increase potency.


Just why it is the logarithms of the van der Waals  forces,


rather than the force values themselves,  is not  clear.  It


may imply that it becomes progressively more difficult to


improve the affinity, so that the logarithm of the  van der  Waals


forces via successive additions of DDT or its analogs will


achieve only linear toxicity increments.   In other  words, it


is a simple case of diminishing marginal return  for each


successive input.  The underlying assumption is  that the


fit to the target is comparable in all the compounds, and


indeed chlorine and methyl groups have fairly similar

                                   o
van der Waals radii, 1.80 and 2.27 A, respectively.  Now

                                         o
hydrogen has a very different radius, 0.3A, so one  might


imagine the H-substituted analogs to fit badly,  and it is


perhaps this that causes the slopes for an all-hydrogen


head or all-hydrogen tail abnormal; i.e., 1.6 and 0.1,


respectively.  Yet in four out of the eight cases in which
                                    /

the slopes were so-called normal, there was at least one


hydrogen in the head or tail of the whole group, and one


would have thought that in these cases a different  relation-


ship would hold.  The conclusions in Gunther's study was as


follows:  "These data are consistent and therefore  in

-------
agreement'with our postulate that these particular




insecticides are reacting with a protein-like substance,




presumably an enzyme."  This conclusion may overstate




the experimental evidences because there was really




nothing in the paper which would indicate an enzymatic




mechanism.  Further, there was no indication in the ex-




perimental evidences whether the target is proteinaceous




or not.




     At this point of the narrative it may be appropriate




to mention some of the very extensive structure-activity




studies on DDT analogs, for they relate to the structural




view of DDT action which is particularly appropriate to




hypotheses that demand a close fit into a lattic.e or onto




a membrane component.  E.F. Rogers and his colleagues




suggested in 1953 that the important property of the CC13




group was its bulk.  If one considers the diphenylmethylene




nucleus:
then, if substituents on the methylene group are small,




the phenyl groups rotate freely around their bond to the




methylene.   But if a bulky substituent is inserted on the




methyline, such a rotation is inhibited, especially if one




imagines the bulky substituent "demanding" room to rotate




freely, and the phenyls then take up the well-known

-------
                                               .PLEASE RETURN TOi
                                            KCIC/OTS CHEMICAL LIB
                                             401 M ST., S.W., TS-793
                                             WASHINGTON,,D.C. '0460
"butterfly" configuration seen in DDT, in which the phenyls

are as coplanar as they can be.  Rogers concluded that

coplanarity was necessary in the DDT molecule  to  have

insecticidal properties.  He apparently based  the conclusion

on the fact that bulky substituents other than CC13 were

also effective insecticides.  Thus, the unchlorinated

compound dianisyl neopentane had good insecticidal activity.

Rogers1 hypothesis also helps to explain the toxic properties

of Prolan /2-Nitro-l,l-bis(p-chlorophenyl)propane7 ano^

Bulan J/T/l-Bis(p-chlorophenyl)-2-nitrobutane7. On the other

hand, it turns out that if one replaces the CC13  group of

DDT by CH(NO2)C1, or C(CH3)2C1, or C(Np2)Cl2, these compounds

have no insecticidal properties.
   CHoO
OCH
     Dianisyl  neopentane (active)
                                     50
           Prolan (active)
       Bulan (active)
               not active

-------
                                                                    51
          not active
                             Cl    Cl
                   not active
     Another hypothesis attempting to explain the toxic

mechanism of DDT seems at first almost the opposite of

Regers1 "bulk" explanation.  R. Riemschneider and H.D. Otto

argued that some ability of the phenyls to rotate was a

requirement for activity.  However, in harmony with Rogers'

view, these investigators considered that free rotation

was required to permit the taking up of the "almost planar"

configuration described above.  This explains, these

investigators reasoned, the 0,0'-DDT isomer's lack of

insecticidal properties because the two chlorines in the

ortho positions in the phenyl groups restrict their

rotation around the 1 carbon atom of the ethane molecule.
                                         Cl
                  2'
3'
                               Cl
               CC13
         p,p'-DDT
 (free rotation of phenyl
  groups; an active agent)
               0,0'-DDT
            (restricted rotation of
             phenyl  groups;  an inactive,
             agent)

-------
                                                                    52
Similarly, one could account for the properties of analogs with

two methyl substituents per ring; when these were in the 2 and

4 positions, or the 2 and 5, rotation was impossible and the

insecticidal activity lacking, but when they were in the 3

and 4 positions, rotation was possible and activity was

found.  The "partial rotation" hypothesis has one major flaw.

The o, p'-DDT isomer does not have such rotability; yet it is

a perfectly good insecticide.

                        Cl
                     o,p'-DDT
               (restricted rotation of phenyl
                groups; an active agent)
     The very extensive studies on structure-activity

relationships in DDT analogs, mostly carried out before

1950, have been admirably reviewed by A.W.A. Brown in his

book, Insect Control by Chemicals.  Investigations centered

on other compounds built on the 1,1-diphenylethane framework.

More specifically, the points at which the DDT molecule were

modified to produce new compounds, and the trends in synthesis

during these early investigations of new analogs were as

follows:

     1.  Alteration of the positions of the chlorine

         atoms on the benzene rings to produce

         structural isomers of DDT.

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                                                                   53
     2.  Replacement of the chlorine  atoms with




         other halogens.




     3.  Substitution of other radicals  on the




         benzene rings.




     4.  Subtraction, or addition,  of chlorine




         atoms on the trichloroethane location




         in the molecule.




Compounds developed along these lines of synthesis are given




in Tables 4 and 5, in which their relative insecticidal




activity is indicated.

-------
Table 4.   Toxicity of DDT Analogs:  Substitution of Other
          Halogens, and Structural  Isomers

(The number indicates the relative  degree of toxicity)
                                                                  54

-------
Table 5.   Toxicity of DDT Analogs:   The Effect of Removing
          Chlorine from the Ethane  Nucleus and Substituting
          Other Groups on the Benzene Rings
55
                                                               OC4H9

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                                                                    56
     Additionally, it was observed by several  early




investigators that the most toxic of the diphenyl  sulfo




esters, sulfides, sulfoxides,  and sulfones,  diphenyl




ethers, and phenyl benzyl ethers were those  in which  the




benzene rings were halogenated in the p,p' position.




These early investigations, although primarily concerned




with stomach poisons for the clothes moth, led to  the




discovery of DDT, a contact poison rather than a stomach




one.  Chemical structures for some of the above diphenyl




sulfur compounds are as follows:
  a diphenyl  sulfo  ester
                            1     Cl
                                a diphenyl sulfide
           :H2-S-CH2







a dibenzyl sulfide
                               1 Cl
                                 a diphenyl sulfoxide
  Cl —
  a diphenyl  sulfone
                          Cl    Cl-
                                a diphenyl disulfide

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                                                                   57
  cr
  a diphenyl disulfoxide
a diphenyl ether
               0-CH
   a phenyl benzyl ether
a diphenyl methane
     Substitution .of the sulfoxide group by a trichloroethyl




group, i.e, ^H-lp-CCljy /  which is also strongly electro-




negative, confers lipoid solubility on the resulting




molecule of DDT.  Thus,  the symmetrical apolar molecule of




DDT is capable of showing toxic action by mere contact of




DDT with the lipoid epicuticle of insects.  An early




hypothesis suggested that the high contact toxicity of




DDT may be related to its molecular structure with a




lipoid—soluble narcotic.  This explanation has some




merit on the grounds that the DDT molecule may be regarded




as methane substituted with two chlorobenzene groups and




a chloroform group.

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                                 chlorobenzene groups
                   DDT
                                 methane nucleus




                                 chloroform group
Chlorobenzene is toxic to insects, and addition of a second




chlorine in the para position produces the well-known highly




toxic fumigant, p-dichlorobenzene.




          Cl                           Cl
      chlorobenzene
p-dichlorobenzene
Chloroform is a strong narcotic and is soluble not only in




the lipoids of nerve sheaths but also in the waxy epicuticle




of insects.  Combination of two moles of chlorobenzene with




one mole of other narcotics; such as, bromoform




chloromethane /H^CC'Lj, nitromethane ^-^CNO^, ethylene




                   ether ^C2ti5oc2ti^7', and divinyl ether




               /  also produced excellent contact insecticides,




and condensation with the anaesthetic cyclopropane resulted




in a contact insecticide of outstanding effectiveness.

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                                                                   59
           cyclop
                      p / p ' -Dichlofopheny 1 cyclopropane
     On the other hand,  it has been suggested that it is the




chlorobenzene group which confers lipoid solubility and the




remainder of the molecule is  responsible for the toxicity.




In methoxychlor, it is  the methoxybenzene portion of the




molecule which is lipoid soluble, whereas methoxybenzene is




not toxic to insects.
                                                    OCH-
                              Methoxychlor

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     It was further suggested that the toxicity of DDT is

related to the release of toxic HC1 in the insect tissues,

since the insecticide is highly susceptible to dehydro-

halogenation by mild alkali.  The reaction is as follows.
          H

     Cl-fC-fd -sS^jU Cl-cU-fci H- HC1

          CC13                 CC12

     Careful examination was made by early investigators of

the relation between dehydrohalogenation and toxicity in

DDT and a large number of its analogs.  Some general

correlation was found with DDT analogs classified in three

categories, that are:

     1.  Compounds split off HC1 readily in

         the presence of alcoholic KOH.  Some of

         the compounds in this category include

         DDT, DFDT (1,1,l-Trichloro-2,2-bis

         (p-fluorophenyl) ethane), and DDD

         (1,l-Dichloro-2,2-bis(p-chlorophenyl)

         ethane).

     2.  Compounds split off HC1 slowly in

         the presence of alcoholic KOH.  Some of

       •  the compounds in this category include

         methoxychlor (1,l,l-Trichloro-2,2-bis

         (p-methoxyphenyl) ethane), methyl-DDT

         (l,l,l-Trichloro-2,2-bis (p-methylphenyl)
         <.
         ethane),  and methyl-DDD (1,1-Dichloro-

         2,2-bis(p-methylphenyl)ethane).

     3.   Compounds that split off HC1  with comparative

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                                                                     61
         resistance in the presence of alcoholic




         KOH.  None of the compounds in this




         category show any more than the sligh-




         test toxicity.  Dichlorodiphenyltetra-




         chloroethane (1,1,l-Trichloro-2-chloro-




         2,2 bis(p-chlorophenyl) ethane),
         is an example.




     The susceptibility of the isomers of DDT to dehydro-




halogenation follows their order of toxicity as- follows:




p,p'J-DDt, o.99%; m,p'-DDT, 0.87%; o,p'-DDT, 0.10%; and




6,0'-DDT, 0.0%.  A similar correlation is evident in the




halogenated analogs; i.e., fluoro  chloro  bromo  iodo,




in both the DDT and DDD series.  All the dichloroethylene




derivatives are less toxic than the parent trichloroethane




analogs.  This fact could be interpreted to indicate that




they have no toxic HC1 to liberate in the tissues by de-




hydrohalogenation of the ethane nucleus.




     When the question of lipoid solubility is examined, it




is found to bear no relationship to contact toxicity.




Although DDT is appreciably liposoluble, most of its analogs,




including the non-toxic ones, are considerably more so.

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     It may be concluded that although there is gegeneiral




relationship of toxicity with: 1. the p-chlorophenyl




group, 2. the lipoid-soluble or narcotic nucleus of




trichloroethane, 3. solubility in lipoids, and 4. sus-




ceptibility to dehydrohalogenation, the following important




exceptions can be found in each of the early hypotheses




attempting to explain the toxic mechanism of DDT:




     a.  Methyl-DDT and methoxychlor are highly




         toxic.




     b.  There are analogs lacking the tri-




         chloroethane nucleus which are no less




         toxic than DDT and are more lipoid-




         soluble than DDT.




     c.  The relation of toxicity with lipoid




         solubility is erratic, and more inverse




         than direct.




     d.  Methoxychlor, methyl-DDT, and methyl-DDD




         are comparatively resistant to dehydro-




         halogenation.




     Before ending the discussion on the mechanism of DDT




action, two other recent hypotheses, the "toxin" and




cholinesterase concepts, should be mentioned.  The toxin




hypothesis was introduced by J. Sternburg and C. W. Kearns




in 1952.  These workers observed that, cockroaches which




had been poisoned with DDT contained in their blood a




factor which could kill flies and cause DDT-like effects;




i.e., DDT "jitters", when applied to untreated cockroach

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                                                                    63
     nerve cords.  This factor was not DDT itself;  for,



unlike DDT, it was not ether extractable.   Since this



observation, others have shown that a variety of stressful



treatments cause cockroaches to produce such factors in



their blood.  For instance, pinning cockroaches down so



that they struggle for days or putting them on a kind of



treadmill that forces them to walk continuously for many



hours, alters their blood composition so that transfer



of it into untreated cockroaches causes paralysis or



death.  Other chemicals than DDT, specifically tetraethyl



pyrophosphate (TEPP) and dieldrin, have also induced a



blood-borne paralysis factor in cockroaches.



            CoHcOL     0     O    vOCoHc
             2 5^SV  -r     +•   -s^ 2 5


                   ^ P.—o—P>


            CoHca
             /. O                    tA ~l



                       TEPP



            tetraethyl pyrophosphate




     The observations suggest a refined version of an



early view that death from DDT was "due to exhaustion,"


caused perhaps by excessive activity induced in sensory



nerves. " The refinement would consist in having a chemical



factor, induced by DDT or other stressful  treatments, as



the immediate cause of death.  It seemed that the story



was complete when it was shown in 1958 and 1959 that



certain heart cells (corpora cardiaca) of cockroaches



have a sort of "neuroactive principle" which reduces the

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                                                                    64
spontaneous activity of an isolated nerve cord,  and this
         «

principle was depleted from these heart cells by stimulating


the cockroaches electrically.  It was also shown that both


respiration rates and acetylcholine levels of cockroach


nerve, and that cockroach strains tolerant of DDT were


also tolerant of mechanical stress.


     However, findings of later researches made this


apparently attractive stress "toxin" hypothesis more


complex.  It turns out that there are three separate


blood factors: (a) A heart accelerating factor released


by mechanical stress in a particular type of heart cell


(corpus cardiacum).  The factor is also released in the


nerve cord, brain, blood, and other organs.  This factor


has no effect on nervous action.  (b) A neuroactive factor


present only in the heart cells.  The factor whose depletion


by electrical stimulation is described above; and (c) the


neuroactive factor which appears in the blood of DDT-


poisoned cockroaches.  Factors (b) and (c) have no effects


on heart rate.  These two factors are also readily separable


chromatographically.  However, factor (c) chromatographs the


same way as the neuroactive factors found in the blood of


mechanically stressed cockroaches, which contained no factor


(b).  In summary, it does seem that DDT-and mechanical stress


produce in blood, a neuroactive principle which plays an


important role in causing deaths.


     Later studies on this principle have yielded only


moderate information concerning the DDT-induced factor,

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                                                                    65
but have no led to an elucidation of the factor's chemical




structure.  What is known about the factor, other than its




existence, is that it is dialyzable and it losses activity




on standing in the presence of cockroach blood.  The




factor can be chromatographed'on paper.  Spot color tests




on chromatograms suggested that the neuroactive factor (b)




was an aromatic amine containing an ester group.




     The "toxin" theory could, in principle, be compatible




with the theories on interference with the nerve membrane.




One might argue that DDT causes toxin production, which




then causes membrane disruption. . However, the toxin is




detectable only after fairly prolonged DDT treatment; and




furthermore, the underlying notion is that hyperexcitability




in the sensory nervous system is the equivalent of mechanical




or electrical excitation, and gives rise to toxin production.




Therefore, the "toxin" hypothesis does not offer an explana-




tion for the observed primary lesion in the nerve membrane




after it is treated by DDT.  It is the lesion which is




presumed to be membrane destabilization.




     A hypothesis which runs like an undercurrent through




the literature on DDT is that the compound has an effect




upon cholinesterase, an enzyme which catalyzes the hydro-




lysis of acetylcholine to acetic acid and choline.  There




were early reports that DDT was a cholinesterase inhibitor




in vitro, but these findings have been either unconfirmed




or specifically denied.  J. M. Tobias and colleagues




reported in 1946 that poisoning of the American cockroach

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                                                                     66
or housefly by DDT led to a two-fold elevation in the


acetylcholine level in the nervous system at the late


stages of poisoning; cholinesterase was unaffected.   Some


controversey surrounded these experimental evidences; i.e.


whether the compound found was' acetylcholine or trichloracetic


acid or something else.  A thorough-investigation"byFE.5-H.


Colhoun in 1959 confirmed the delayed 27-fold rise in


acetylcholine in the nerve cord of DDT-poisoned cockroaches.


However, this occurred in 24 hours after treatment.   The


acetylcholine level remained at this high level for about


another 36 hours.  After that period, the level declined


rapidly and dropped below the normal level during the last


stages of poisoning when the cord becomes or is already


necrotic.  From these observations, Colhoun and colleagues


suggested that acetylcholine synthesis took place at an


elevated rate during the initial stages of poisoning, but


in a form unavailable for hydrolysis by cholinesterase.  He


supports his suggestion by the fact that free acetylcholine


was found in the nerve cord along with cholinesterase


which, judged by subsequent homogenization and assay, was


not inhibited.  Colhoun and others concluded that the ace-
                                    /

tylcholine effect was only a secondary effect of DDT poisoning.


However, in 1962, J. Sternburg and P. Hewitt pointed out that


cholinesterases assays made subsequent to homogenization


could give erroneous results, if the cholinesterase was


inhibited reversibly in vivo.  Such an inhibitor would be


diluted away upon homogenization and thusly, they reasoned,

-------
inhibition of cholinesterase to hydrolyze acetylcholine . -



would disappear.  To make their point, they made ingenious



use of an organophosphate; i.e., tetramethyl pyrophosphate



(TMPP).

                        O     O
                        T


                        P—O—
                         TMPP

              Tetramethyl pyrophosphate
They selected the compound as an "irreversible" anti-



cholinesterase, which hydrolyzes spontaneously and



rapidly, so that it eliminates itself within a few hours.



     When the control and DDT-treated groups of cockroaches



were treated with TMPP, Sternburg and Hewitt observed that



the resultant cholinesterase inhibition was far less in



the DDT-treated insects.  They reasoned that the cholinesterase



of the DDT-treated cockroaches was protected from inhibition



by TMPP.  The protection increased as DDT poisoning deepened.



No such protection was afforded when cockroaches were



treated with lindane.  It was also observed that when



temperature was manipulated during TMPP induction, there



was a reversal of DDT-poisoning symptoms;, thus, temperature



removes the protective effect.  In other words, at lower



temperatures DDT jitters in the treated insects were more



severe than at higher temperatures.  The temperature



effect will be explored further in ensuing paragraphs of



this discussion.  One can argue that there may be other



factors other then a protective inhibition factor in

-------
DDT-treated insects against the anticholinesterase




property of TMPP.  The following alternative possibilities




to explain the protective factor were experimentally dis-




proved by Sternburg and Hewitt:




     1.  Poor circulation existed in the DDT-




         treated insects.




     2.  TMPP did not penetrate the DDT-treated




         insects' nerve cords.




     At least two mechanisms for the protective effect




can be speculated.  1) DDT, or a derivative of it, or a




compound whose production or release is stimulated by




it, might be a reversible inhibitor of cholinesterase,




thus protecting it from phosphorylation.  Putting this




speculated mechanism more simply, there may be something




in the DDT-treated nerve cord, and not present in the




untreated ones, that protects the cholinesterase




enzyme from being attacked by TMPP; namely, phosphory-




lation.  It is unlikely that DDT itself affords this




protection to cholinesterase.  If it did, an effect




would have been found at high concentrations in vitro.




2)  The well-established increased levels .of acetylcholine




might be protective; i.e. a case of protection by substrate




rather than protection by a reversible inhibitor.  There




is, however, evidence against acetylcholine protection,




for J. B. Waller and S. E. Lewis have shown that lindane,




aldrin, and pyrethrin cause similar increases in acetyl-




choline, but, as mentioned above, lindane does not protect

-------
the cholinesterase against phosphorylation action of TMPP.




     A determination of which of these alternatives is




correct is of major importance:.if DDT provokes reversible




inhibition of cholinesterase, the increases in acetylcholine




might be a result, and the mechanism could be important in




explaining central effects of DDT.  But if it is the excess




acetylcholine that is protective, then the TMPP findings




tell us nothing more than that acetylcholine is elevated,




a fact well established by more direct evidence.




     The temperature effect will now be briefly discussed




in the following paragraphs.  A well-documented fact is




that DDT shows a negative temperature coefficient of




activity, i.e., it is more active at low than at high




temperatures.  Insects treated with the appropriate dose




can be cooled to 15°C and thrown into violent symptoms,




then warmed to 35°C and appear entirely normal.  This




temperature effect is observed in various insects




that have been treated with DDT.  The effect is peculiar




with DDT because it is the opposite to that of other




types of insecticides; such as, prganophosphates, pryethrins,




and several other chlorinated hydrocarbons.  This negative




temperature effect of DDT seems to suggest that a chemical




complex, formed by DDT and some other component in the




nervous system, may be the basis of the toxic mechanism




of DDT.  A complex formation is suggested because virtually




any type of complex formation indicate just such a negative

-------
                                                                    70
temperature dependence,  presumably because thermal




agitation is disadvantageous for complexes.  By contrast,




chemical reactions usually show a positive temperature




coefficient.  In 1964, J. L. Eaton and J.  Sternburg




described an interesting analysis of DDT's temperature




effect.  They showed that the destabilization of sensory




nerves by DDT gave a positive temperature coefficient,




but that in the central nervous system showed a negative




one.  The overall a. response to DDT poisoning also showed




a negative coefficient.   These findings suggest that




central nervous system phenomena are the more crucial




in the DDT poisoning process. . This position, however,




conflicts sharply with the generally held view that




sensory nerves are of primary importance.




     In summary, relatively coherent hypotheses of the




mode of DDT's action have been sketched in this discussion.




Many other bits and pieces of information exist pertaining




to the subject matter, that seemingly do not fit into any




of the hypotheses so far.  For example, it has been




experimentally demonstrated that -DDT causes a sharp and




substantial increase in oxygen consumption in all the




insects studied so far.   One explanation for this phenomenon




is that increased uptake of oxygen results from excessive




muscular activity, which in turn was caused by excessive




nervous activity resulting from DDT.  It has also been




experimentally demonstrated that amino acid metabolism




is disturbed by DDT.  In 1963, J. J. Corrigan and C. W. Kearns

-------
showed that treatment of cockroaches with DDT  sufficient  to

give symptoms at 15°C led to alteration in blood  amino  acid

levels.  For example, tyrosine, proline, and o^-Ketoglutaric

acid levels fell 71, 61, and 50 percents, respectively.  On

the other hand, phenylalanine level rose 131 percent.
    NHoCH C-OH
                        -H2C-OH

  /                e^-Ketoglutaric acid
  proline             -i\clu^"l_i-tcirr^.v. cc.Ld.
(pyrrolidine-2-carboxylic acid)
    tyrosine
    (of-amino-B-^p-hydroxyphenyl7propionic acid)
                 H2N-C—C—OH
                     H  o
                 phenylalanine («f-amino-B-phenylpropionic acid)


Corrigan and Kearns explored the proline effect further.

When the temperature was raised to 34°C, the effect dis-

appeared and the proline level returned to normal.  When

radioactively-tagged proline/ namely/ C  -proline, was

injected into cockroaches, it was observed that the DDT-

treated insects respired two to three times more of the

tagged proline as C  02 than did the untreated ones.

Tht C  -proline was not only respired more, but three

times more of it was converted to glutamine than was

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                                                                     72
converted in controls.  Corrigon and Kearns suggested that

this extra proline ulitization was simply a reflection of

a "demand for oxidizable carbon."  The conversion of proline

to glutamine probably follows the equations as given below:
     9H2-
     CH2    .CH C-OH

      \  /   °
        NN/
                       - 2 H
          H
     proline
      CH
H2O + CH2

  H2N-C-H

      ;-OH
glutamine
                         -C-H
                            °H
                    H2N
                   glutamic acid
                                     ftj     f**U

                                     CH    ,CH C-OH
                                      V   s
                                     pyrroline carboxylic

                                            acid
C-OH (
CH7 <
1 i- -i
+NH-, 1 t L°J
f r*M £ ~"^ c
>
»
:-i
:H
'•H
H2N-C-
                                               |-OH
                                              glutamic acid
                                              semialdehyde
     The discussion above is an attempt  to  present  briefly

the most cogent hypotheses to explain DDT's toxic mechanism.

The full and unequivocal elucidation of  the insecticide's

mode of action still remains to be discovered.

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                                                                   73
Toxicoloqical Effects;




A.  Fish and Other Aquatic Biota




     The toxicity of DDT to fishes has been subjected to




considerable study.  Among the variables that have been




cited are the type of water course and bottom, depth,




vegetation, silt, turbidity, hardness, temperature, dis-




solved oxygen, organic content, species and age of fish,




various commercial product formulations, volume and flow




of water, and size and shape of receptacles.  The effect




of differing DDT formulations on rainbow trout have been




cited as: 1. DDT in acetone solution, not lethal at 30




mg/1; 2. DDT in fuel oil, not lethal at 20 mg/1; 3. DDT




in xylene, toxic at 5 mg/1; 4. DDT in emulsion, toxic at




3 mg/1; 5. DDT in kerosene, toxic at 0.3 mg/1.




     Owing to these many variables, it is not surprising




to find that there is a variation in concentrations of




DDT lethal to fish as reported in the literature.  The




effects of various concentrations of DDT on different




fishes are given in Table 6.

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Table 6.  Toxicity of DDT to Various Species of Fish
                                                                   74
Fish Species
                      Concentration (mg/1)
                           Results
Common sucker
Speckled trout

Common sucker
Speckled trout

Creek chub
Bluegill
Dace
Gambusia
Guppies
Bluegill fingerlings
Bass fingerlings

Bass fry
Rainbow trout
Bluegills
Salmon, young
Bass fingerlings

Goldfish
Goldfish
Bluegill adults

Bluegills
Darters
Sculpins
Trout

Bluegill fingerlings
Bluegills &. Croppies
Goldfish
Fatheads
Golden shiners
Goldfish
                      0.001 (alcohol suspension)  Killed
                      0.001 (alcohol suspension)  Killed

                      0.005                      Killed
                      0.005 (alcohol suspension)  Killed
0.01  (alcohol suspension)
0.01
0.01
0.01
0.01
0.01
0.01

0.025-0.04
0.0237-0.074
0.04
0.047
0.05

0.1
0.1
0.1

0.14  (in fuel oil)
0.14  (powder)
0.14  (powder)
0.14  (powder)

0.15
.0.18
0.20
0.40
0.50
1.60
                                                 Killed
                                                 Toxicity threshold
                                                 Killed
                                                 LD50
                                                 Toxicity threshold
                                                 Killed
                                                 Killed
                                                 Killed
                                                 96-hour
                                                 Killed
                                                 24-hour
                                                 Killed
                                                 Toxic limit

                                                 Killed

                                                 Killed
                                                 Killed
                                                 Killed
                                                 Killed

                                                 Killed
                                                 Killed
                                                 Killed
                                                 Killed .
                                                 Killed
                                                 Killed
     Table 7 gives the median lethal concentration

of DDT for various species of fish.

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                                                                    75
Table 7.  The LCcn for Various  Fish to  DDT
Fish Species
Brook trout
Land locked salmon
Mosquito fish
Largemouth bass
Brown trout
Coho salmon
Redear sunfish
Black bullhead
Rainbow trout
Bluegill
Yellow perch
Carp
Channel Catfish
Fathead minnow
Goldfish
Goldfish
Exposure
Time(hr)
36
36
. 36
96
96
96
96
96
96
96
96
96
96
96
96
96'
LC50 I/
(mq/1)
0.0323
0.08
0.32
0.002
0.002
0.004
0.005
0.005
0.007
0.008
0.009
0.010
0.016
0.019
0.021
0.027
     With increasing time and declining temperature the LC5Q

to DDT for rainbow trout decreased from 0.012  mg/1  to  0.0041.

This fact indicates that DDT is negatively temperature de-

pendent and positively time dependent.   Data is  given  in Table 8,
Table 8.  Effects of Time and Temperature on the Toxicity
          of DDT to Rainbow Trout averaging approximately
          one gram
Temperature, °F
45 -
55
65

24 hrs.
7.5
8.2
12.0
LC5n (mcr/1)
48 hrs.
4.7
5.2
7.3

96hrs.
4.1
5.0
6.0
I/ LC5Q, median lethal dose,  is  the milligrams  of  toxicant
   per kilogram of body weight lethal' to  50  percent  of  the
   test animals to which it is administered  under  the
   conditions of the experiment.

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                                                                    76
     Resistance to DDT may be induced up to a certain




concentration.  For example, guppies which had been exposed




to sublethal doses of DDT for 14 days and then placed in a




toxic concentration of DDT (0.032 mg/1) demonstrated that




they had increased their tolerance to the toxicant by this




procedure.




     Cutthroat trout were exposed in the laboratory for




30 minutes once a month for 1.5 years to the following




quantities of DDT in water baths: 0.01 mg/1, 0.03 mg/1,




0.1 mg/1, 0.3 mg/1, and 1.0 mg/1.  By the end of the




experimental period from about 50 to 75 percent of the 636




fish in each group were dead at the three highest quantities




of DDT.  The number and volume of eggs produced by the trout




were not reduced by these levels of DDT, but mortality among




sac fry was higher at the 0.3 and 1.0 mg/1 levels of DDT.




     Mosquito fish collected from waters near cotton fields




heavily treated with chlorinated insecticides exhibited




significant levels of resistance to DDT, compared with fish




from unexposed areas.  A concentration of 0.05 mg/1 of DDT




caused only about a 20 percent mortality in the resistant




fish, whereas this same concentration caused about a 90




percent mortality in the susceptible or unexposed fish.  In




another study about five percent of mosquito fish surviving




after exposure to DDT at concentrations above the threshold




toxicity aborted their young.




     Some species of fish are extremely sensitive to DDT.




For example, the extrapolated LDc  dosage for young chinook

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                                                                     77
and coho salmon was 0.0275 and 0.064 mg/kg per dayf  respectively.




The chinook salmon appeared to be two to three times more




sensitive to DDT than were coho salmon.




     Atlantic croakers were fed 2.57 micrograms of DDT per




gram weight of fish for 67 days.  The accumulation of DDT




resulted in mortality starting on the 14th day and continuing




until all fish were dead by the 67th day.  DDT is not only




toxic to fish but may also alter the normal behavior of fish.




For example, it was found that New Brunswick salmon  from a




DDT-sprayed region were unusually sensitive to low temperatures




and selected water of higher temperature than usual.  If this




response occurred in nature, salmon might place their eggs




in regions where the young fry could not survive. It was




also found that mosquito fish exposed to low levels  of DDT




in the range of 0.1 to 20 parts per billion (ppb) for 24 hours




tended to prefer waters with a higher level of salinity than




unexposed fish.  The amount of DDT taken up by pinfish




reached a maximum level about two weeks  after exposure  to




dosages of 0.1 ppb and 1.0 ppb.  At this time, the pinfish




had residues ranging from 3.8 ppiu to 11.5 ppm.  DDT  residues




in coho salmon eggs from Lake Michigan measured during 1968




ranged from 1.1 to 2.8 ppm.  Mortalities in the salmon fry




after hatching ranged from 15 to 73 percent.   The higher




residues of DDT in the eggs of these salmon were generally




correlated with higher mortalities in the fry.




     In Idaho and Wyoming, treatment of  forests with DDT




at 1, 2.5, 5, and 7.5 pounds per acre influnced some fish

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                                                                    78
populations.  At the one pound per acre dosage in Idaho,




some cottids (Cottus beldingii), mountain suckers, and black




bullheads were killed by the DDT, but speckled dace,  redside




shiners (Richardsonius balteatus hydrophlox),  rainbow trout,




eastern brook trout, and cutthroat trout apparently were  not




affected.  A few cutthroat trout were killed by the 2.5




pound per acre application of DDT.  The most striking in-




fluence of DDT was on the diet of fish.  Before treatment,




there was no crayfish in the diet, but immediately after




the treatment the percentage increased to 99 percent, as




in the case of the brook trout sampled.  No measure of the




long term effects of the change in food organisms was made




in the investigation.




     A spray calculated to give a DDT content of 0.09 ppm .




in water was used to treat a stream.  Eight miles downstream




from the treated area hundreds of fish were reported dying,




and the concentration of DDT at a point ten miles downstream




was 0.017 ppm.  In 1955 when the fish hatchery on Lake George




lost all of nearly 350,000 eggs removed from lake trout,  DDT




was suspected as the cause.  For'several years, about 10,000




pounds of DDT had been distributed yearly for control of




gypsy moth and biting flies in the watershed associated with




Lake George.  Careful studies revealed that DDT stopped




reproduction of lake trout in Lake George and several other




heavily contaminated lakes in the adjacent Adirondack region.




Although the trout eggs contained from 3 to 355 ppm of DDT,




little or no mortality occurred in the egg stage.  The fry,

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                                                                     79
however,.were highly sensitive to these dosages and were




killed at the time of final absorption of the yolk sac,




just when they were ready to feed.  For example, at levels




of DDT in eggs that would produce 3 ppm in fry, few fry




survived; and at 5 ppm DDT, none survived.




     The spraying of New Brunswick forests with DDT between




1953 and 1958 was reported to be responsible for the severe




reduction in salmon fishing success in the province.  The




severest reduction occurred in 1959 and 1962.  In a succeeding




investigation, the mortality of young Atlantic salmon and




eastern brook trout was observed in cages and free-living




streams in forested areas of New Brunswick sprayed with DDT




for spruce budworm control.  There were no short-term effects




on salmon parr with DDT at one-quarter pound per acre, but




many yearlings were killed.  Two applications of one-quarter




pound per acre ten days apart were as harmful as a single




application of one-half pound per acre.  DDT at one-half




pound per acre caused a loss between 50 to 98 percent of




underyearling and parr salmon.  In a similar investigation,




applications of DDT to control nuisance insects appeared to




be associated with the decline of the salmon fishery at




Sebago Lake, Maine.  Average DDT residues in salmon collected




in 1962, 1963, and 1964 were, respectively, 1.1, 3.2, and 1.8




ppm by total weight.  Salmon in the three year age group had




1.2 ppm; in the four year age group, 8.0 ppm; and in the five




year age group, 8.8 ppm of DDT.

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                                                                    80
     It appears that there is a link between the feeding

habits of Atlantic Salmon and DDT.   For example, the

application of DDT at one-half pound per acre to a forest

watershed of the Northwest Miramichi River,  New Brunswick,

changed the kinds of food found in  stomachs  of young

Atlantic salmon.  Salmon under one  year typically consume

immature aquatic insects, whereas salmon over one year

consume all sizes of aquatic insects.  After the DDT

application, the surviving young salmon fed on worms,

snails, and fish which previously had been unimportant

in their diet.  With the resurgence of aquatic insects, the

young salmon went back to its pre-spray feeding habits.

     Observations in the field confirm laboratory findings

that DDT is highly toxic to some fish and especially to

fry.  For example, one investigation reported that when

levels of DDT and its metabolites were above 400 parts

per billion in the eggs of hatchery trout, the "mortality

in the resulting fry ranged from 30 to 90 percent in the

60-day period following the swim-up stage."   In a similar

investigation, it was reported that DDT residues in c'oho

salmon eggs from Lake Michigan measured during 1968 ranged
                                    f
from 1.1 to 2.8 ppm.  Mortalities in the fry after hatching

ranged from 15 to 73 percent.  The  higher residues of DDT

in the eggs of these salmon were generally correlated with

higher mortalities in the fry.  DDT's high toxicity to

salmon fry, parr, and underyearlings would seemingly account

for the relatively low commercial salmon catches during and

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in immediately succeeding years in which salmon spawning




waters were heavily contaminated with DDT.




     Fish also accumulates DDT.  In one investigation




reporting the effects of an application of one pound per




acre of DDT over a 72,000-acre area in the Yellowstone




River drainage in 1957 for spruce budworm control, DDT




was found up to 0.03 ppm in the water.  Samples of mountain




whitefish, rainbow trout, and brown trout contained DDT up




to 14.00 ppm and DDE (Dichlorodiphenyldichloroethylene), a




degradation product of DDT, up to 6.53 ppm.  The author




further reported that "DDT was found in trout 85 miles below




the spray area, and fish taken more than two years after the




spraying still contained DDT."  In another report, DDT was




applied at 0.2 pound per acre to a tidal marsh in Florida.  .




Total kills of caged striped mullet, sheepshead, longnose




killifish, rainwater killifish, and tidewater silverside




occurred in 1 to 24 days.  Fish accumulated up to 90 ppm of




DDT within five weeks after treatment.




     In addition to fish, DDT is toxic to amphibians,




reptiles, arthropods, aunelids, arid other aquatic insects.




One investigation reported the impact of an application of




one pound per acre of DDT for the control of tent




catapillars in Hubbard County, Minnesota.  Before spraying,




111 small frogs (Rana sylvatica) were counted around two pools.




The frogs seemed well a day after spraying, but the water had




oil film and was covered with dead poisoned caterpillars.  Two




and a half days later, 35 dead frogs were found, and after a

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few more days, no living ones remained.  All but two of the




34 frog stomachs contained tent caterpillars, among other




insects.  Whether frogs were killed directly or indirectly




by eating poisoned insects, the local population was




drastically reduced.  Later investigations showed that DDT




is toxic toward frogs, especially the young.  The 24-hour




median lethal concentration of DDT for Fowler's toad




tadpoles and chorus frog tadpoles was, respectively, 2.4




and 1.4 ppm.




     Investigations showed that DDT is harmful for the p




propagation of molluscs and that they accumulate the




insecticide.  For example, an investigation reported that




seawater with a DDT level of 0.1 ppm halted the growth of




eastern oysters, and dosages as low as 0.0001 ppm signifi-




cantly reduced oyster growth.  Eastern oysters containing




about 151 ppm of DDT requires approximately three months




in clean water to lose 95 percent of their load of DDT.




Their growth returned to normal after only ten days of




flushing in clean water.  Several other mollusc species




lost about 75 percent of accumulated DDT after 15 days




of flushing in clean water.  Table 9 gives the accumulation




and retention of DDT by molluscs.     '

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Table 9.  Accumulation and Retention of DDT by Molluscs
          Exposed for Seven Days to 1.0/ig/l in Flowing
          Seawater and Then Placed in Clean Water


Mollusc
Hooked mussel
Eastern oyster
Pacific oyster
European oyster
Crested oyster
Northern quahog

After 7 Days
Exposure
24
26
20
15
23
6
Residue
After 15 Days
Exposure

2.5
16.0
8.0
5.0
0.5

After 30 Days
Exposure

1.0
	 •
4.0
	
	
     Fiddler crabs fed natural organic plant detritus for

11 days in estuaries containing DDT exhibited grossly

modified behavior.  Within five days on the DDT containing

detritus, the crabs became uncoordinated.  When threatened,

they did not scurry away, but moved a short distance, lost •

coordination and equilibrium and rolled over.

     DDT caused reductions in numbers of natural insect

predators and parasites as well as the target insect species.

There is also ample field study evidence to show increases

of other pests that are DDT resistant once their natural

predators are reduced.  Some of the more salient evidences

are given in ensuing paragraphs.

     DDT was applied in apple orchards for the control of

apple pests to eliminate populations of certain highly

susceptible, predaceous ladybird beetles.  As these beetles

were the principal controlling agent for a red-mite pest,

the mite population subsequently reached outbreak levels,

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                                                                    84
causing severe damage to the apple trees.  This particular




mite is not susceptible to DDT and was therefore hardly




influenced by the chemical which killed the beetle.  Outbreaks




of the red-handed leaf roller occurred in apple orchards after




the use of DDT because the leaf roller's parasites and




predators were more susceptible than the leaf roller.  DDT




applications sharply reduced the parasitism of the apple




mealy bug, as DDT is highly toxic to its parasite.  In




another field study, it was found that DDT caused a reduction




in numbers of natural predators, followed by an increase in




numbers of European red mites and clover mites.  The study




showed the relationship between the concentration of DDT




used and the time applied and the frequency and magnitude




of population outbreaks attained by the mites.  The study




also showed the factors involved in reestablishing predator




populations and the time required for reattainment of




equilibrium of low populations of predators and mites. A




similar field study concerned an attempt to eliminate




.caterpillars, in particular Pieris rapae, on cole crops




with DDT.  The study reported that the survival of the




pest was better than expected because the insecticide




killed many of the caterpillars' natural enemies.  The




study indicated that it was impossible to predict the




changes in species populations with the application of any




one insecticide to a biotic community.  However, one common




trend was the reduction or elimination of natural enemies,

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                                                                   R5
frequently leading to outbreaks in the numbers of herbivores


or pest species on the cole crop under study.

                                                              ••*
     Aquatic insects are particularly susceptible to DDT.


An investigator studied the number and kind of aquatic insects


present in the forest-covered tributaries of the Miramichi


River of northern New Brunswick after aerial treatment with


0.5 pound per acre of DDT.  In the streams affected by DDT


fewer insect species emerged, and those species most severly


reduced were the larger ones, such as caddice flies.  The


treated streams generally had larger numbers of individuals,


but the weight of insect life was in some cases reduced by


half.  Furthermore, the insect fauna of the treated streams


was deficient in the species of insects on which salmon


mainly feed.  This aspect was mentioned in earlier paragraphs


of this discussion.  From two to three years were necessary


for the insect fauna to recover qualitatively for most groups;


however, for some recovery required four years.  In a similar


field study, DDT was applied at 1.0 pound per acre for control


.of the spruce budworm to the Swan Creek drainage area in


Montana.  Although the spraying aircraft did not treat within


one-quarter of a mile of the stream, 0.01 ppm of DDT was


measured in the water one-half hour after spraying.  Three


hours after the application samples of insects contained up


to 11 ppm of the insecticide.  Extreme mortalities occurred


in mayfly nymphs, caddice fly larvae, and stonefly larvae by


one hour after treatment.  These mortalities are not surprising

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                                                                  86
in view of the massive dose and the  susceptibility of these

aquatic insects.  Table 10 gives the median  lethal concentration

of DDT for some aquatic fauna.

Table 10.  The Median Lethal Concentration for Various
           Arthropods to DDT

Arthropod Species
Amphipod (Gammarus lacustris)
Amphipod (Gammarus lacustris)
Grass shrimp (Palaemonetes vulgaris)
Hermit crab (Pagurus longicarpus)
Sand shrimp (Crangon septemspinosa)
Stonefly (Pteronarcella badia)
Stonefly (Claassenia sabulosa)
Stonefly (Pteronarcys Californica)
Stonefly (Pteronarcys Californica)
Stonefly (Pteronarcys Californica)
Waterflea (Daphnia pulex)
Wat erf lea (Daphnia pulex)
Exposure
Time(hr)
24
48
24
24
24
24
24
24
48
48
48
48
LC5Q
(ppm)
0.0047
0.0021
0.012
0.007
0.003
0.012
0.016
0.041
0.019*
0.019*
0.00036
0.0004
* Different investigations
     In another investigation,  DDT was  applied at  0.5  pound

per acre for control of the elm spanworm.   In the  one  drainage

area where precautions were not taken to avoid the stream, a

90 percent kill of mayfly and stonefly  nymphs occurred.  A

similar incident occurred in Pennsylvania when 0.25 pound per

acre of DDT was applied directly to a stream.   Approximately

90 percent of the total stream insect population was exter-

minated and about 35 percent of the species eliminated.  Some

species did not repopulate the stream for two years or more.

After the treatment of 72,000 acres of  the  Yellowstone River

system with DDT at one pound per acre,  stream-bottom inverte-

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                                                                   87
brates were significantly reduced in number.  Total numbers




of invertebrates had recovered within a year, but the species




composition was still altered.  Both the folded-wing insects




(Plecoptera) and the very short-lived insects (Ephemeroptera)




were significantly reduced, but both the hairy-winged insects




(Trichoptera) and two-winged insects (Diptera) occurred at




higher numbers at the end of one year.




     DDT is also toxic toward earthworms and insect larvae.




In one field investigation, DDT applied at 25 pounds per acre




reduced earthworm activity by 80 percent.  Earthworm populations




were found to reflect the dosage of DDT in soil.  In soils




.containing 26.6 ppm, 4.1 ppm, and 3.6 ppm, the earthworms in




these soils averaged about 14 ppm, 7 ppm, and 3 ppm, respectively.




     DDT application at 0.1 ppm for control of black fly larva




in Bobby's Brook, Labrador, resulted in several faunal changes.




Caddice fly larval populations were reduced to zero or near




zero at all stations receiving the treatment, and the same was




true for stonefly and mayfly larvae.  The DDT also caused




mortalities in eastern brook trout by contamination of the




fish foods above maximum tolerance levels.




     Insects exposed to DDT have apparently become resistant




themselves or produced progenies that are resistant to the




insecticide.  For example, honeybees" at Riverside, California,




were found to be six times more resistant to DDT than honeybees




from unexposed areas.  One investigator reported that approxi-




mately 225 species of insects and mites have evolved resistance

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                                                                  88
to DDT.  Increasing number of insects have also evolved




resistance to, in addition to DDT,  cyclodiene (aldrin,




dieldrin, etc.) and organophosphorus (parathion,  malathion,




etc.) types of insecticides.   The 225 species were broken




down as follows:  121 crop pests, 97 man and animal pests,




6 stored-product pests,  and 1 forest pest.  These results




provide an idea of the amount of DDT and other insecticides




in the environment and intensity of selective pressure.




     Resistance to DDT may be induced by the accumulation




of the insecticide, especially if the dosages were sub-




lethal and small. Biological  concentration of DDT is




especially prevalent in molluscs and Crustacea.  Eastern




oysters placed in flowing seawater containing 1 part per




billion (ppb) of DDT for 40 days concentrated DDT some




70,000 times the level in the water.  Oysters exposed for




ten days to a mixture of eight pesticides in the water,




ranging from 0.001 to 0.05 ppm, increased the pesticide




concentrations in their bodies; DDT, for example, was




concentrated 15,000 times.




     Fish and birds also concentrate DDT and other pesticides.




Croakers, a saltwater fish, concentrated DDT 20,000 times the




level in water containing 0.001 ppm of the insecticide.  After




two weeks of exposure to 0.001 ppm of DDT in water, ten fish




concentrated the level of DDT in their bodies 12,000 times




the level of the water.   When ten fish were exposed to  a




lower concentration of DDT at 0.0001 ppm, they were found to

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                                                                   89
be able to concentrate the level in their own bodies 40,000




times that of the water.  DDT residues were found to reach




a level of more than 13 pounds per acre in a Long Island




saltmarsh.  In a sampling of a freshwater estuary and a




saltmarsh and the organisms therein, DDD in the water was




estimated at 0.05 ppb.  The organisms consisting mostly




plankton had about 40 ppb of DDT, a biological concentration




factor of 800.  The highest concentrations were detected in




the scavenging and carnivorous fish and birds; the birds




were reported to have 10 to 100 times more than the fish




species.




     In Lake Michigan sediments averaged 0.014 ppm of DDT,




DDE, and TDE.  From the same habitat the amphipod averaged




0.41 ppm for DDT and its related metabolites, or about 30




times the level found in the mud; various fish removed from




the lake had varying amounts of the insecticide's residues.




Alewives had 3.35 ppm; chub, 4.52 ppm; and whitefish, 5.60




ppm.  The breast muscle of gulls averaged 90.5 ppm of DDT,




approximately 27 times that found in alewives.  And body




fat of the gulls averaged 2,441 ppm of DDTI




     The chemical attributes of DDT make it susceptible to




biological concentration in algal living systems.  For




example, four species of algae concentrated DDT about 220-




fold when exposed to a concentration of DDT a 1 ppm in




water for seven days.  Daphnia, a zooplanktonic organism,




concentrated DDT 100,000-fold during a 14-day exposure to




water containing 0.5 ppb of DDT.  A fathead minnow concen-

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                                                                   90
trated DDT further in its tissues on being fed Daphnia


containing DDT.  Depending upon the DDT concentration in



the medium and in the food/ fish and Crustacea have been


observed to concentrate DDT from 50 to over 200 times that



of its medium's le'vel.  For example, it was observed in


ponds containing 0.02 ppm of DDT in water, rainbow trout,


black bullhead, and crayfish concentrated DDT to the levels


of 4.15 ppm or 208 folds, 3.11 ppm or 156 folds, and 1.47


ppm or 74 folds, respectively.


     In addition to aquatic biota and waterfowl, soil


organisms; such as earthworms and soil insects, also


accumulate DDT.  Animals and birds feeding on these insects


further contentrate DDT in their bodies.  It was observed


in one investigation that in some DDT-sprayed elm environments,


pesticide residues accumulated from 9.9 ppm in the soil to


141 ppm in earthworms; and, in turn, to 444 ppm in brains of


adult robins.  In another area where elm trees had been


sprayed with DDT for control of Dutch elm disease, the soils


had a residue to 19 ppm of DDT and earthworms from the same


soil contained 157 ppm.



     DDT is an extremely persistent insecticide.  Soil


residues in a Maine forest treated with DDT at one pound


per acre showed little decrease during the nine years after


application.  It was estimated that DDT residues may persist


over 30 years.  Supporting this estimation are results from



another investigation, which reported that the percentage



of DDT applied at a rate 100 ppm to sandy loam soil         k
                                          •


remaining after 17 years was still 39 percent.
                                                          y

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                                                                   91
Toxicological Effects:

B.  Higher Animals

     The median lethal concentration (LCso) of DDT and DDE

for various birds is given in Table 11.

Table 11.  The LCso of DDT and'DDE for Various Birds*
DDT(ppm)
Mallards
Pheasants
Bobwhites
Cotumix
850
300
600
400
to
to
to
to
1,200
700
1,000
600
DDE(ppm)
3,300
750
750
1,200
to
to
to
to
3,600
950
950
1,400
  *  Insecticides in diets of two-week-old birds when
     fed treated feed for five days followed by
     untreated feed for three days
Pheasants were maintained on diets containing different

dosages of DDT for an experimental period of 90 days.  In

the test, three out of ten females on 600 ppm of DDT died;

all the four males on 400 ppm of DDT died, whereas the 20

females on this dosage all survived; and one out of the

ten females on 200 ppm of DDT dies.

     DDT has been reported not only to be toxic to birds

but also to cause significant changes in the physiology of

some species of birds.  DDT fed daily to pheasant hens at

10, 100, and 500 ppm DDT in their food produced a normal

number of eggs which were fertile and hatched satisfactorily.

However, chick mortalities were reported to be highest from

parents who received 500 ppm of DDT.  In another investigation,

it was found that only 44 percent of the eggs laid by herring

gulls on the Lake Michigan side of the Door County peninsula

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                                                                    9
o
were  observed  to hatch, as compared with a 90 percent




level of hatching  found in the same species in Denmark.




This  reduction was reportedly due to the higher level of




DDT and its metabolites found in the Michigan gull  eggs.




      Bird  population  reduction since the advent of  DDT




and other  insecticides is also attributed to eggshell




thinning.  This is especially true of raptorial and upland




game  birds.  Bald  eagles fed controlled dosages of  DDT  in




the laboratory proved to be  susceptible to DDT.  The median




lethal dose for eagles is estimated to be 80 ppm of DDT.




•The investigators  pointed out that this level produced




chronic poisoning. The dosage also suppressed reproduction




and thinned eggshells.




      To see if there  was a correlation between;v*he:-thinning




of eggshells and DDT  and/or  its metabolites, historical




comparisons of eggshell weights and thicknesses were made.




In-one historical  survey, 614 peregrine falcon eggshells




were  both  weighed  and measured for thickness.  Eggs collected




•in California  from 1947 to 1952 had a significant decrease  in




both  thickness and weight of eggshells, compared with those




collected  in the same area from 1895 to 1939.  A regression




analysis was run between shell thickness and total  DDE




residues in herring-gull eggs collected in Maine, Michigan,




Minnesota, Rhode Island, and Wisconsin.  A high correlation




was found  between  the level  of DDE residue and the  thickness




of the eggshell; i.e., the more DDE residue, the thinner  the

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                                                                    Q
eggshell.  The weights of raptorial birds' eggshells in




museums and private collections were also measured to  '    •. .v-\




determine  if  there had been a change in the weights of these




eggshells  from the "pre-DDT" period of 1886 to 1939 to the




"post-DDT" period of  1947 to 1962.  In Brevard County,




Florida, bald eagle eggshells from the pre-DDT period




weighed 12.15 -±- 0.127 grams.  Eggshells from the post-DDT




period weighed 9.96 — 0.280 grams.  Hence, there was an




18-percent decrease in the weight of the eggshells.  Reports




were  also  received that  the bald eagle population was




declining  in  this area.  Similar results were reported




from  Osceola  County,  Florida.  From 1901 to 1944, the mean




weight for bald eagle eggshells was 12.32 -±- 0.240 grams.




From  1959  to  1962 the mean weight declined to 9.88 -±- 0.140




grams, a decline of 20 percent.  Bald eagle populations




were  reported to have declined also during the post-DDT




years. The mean weight  of 117 osprey eggshells  taken




between 1880  through  1938 was 7.08-±- 0.069 grams, whereas




•the mean weight of six osprey eggshells collected in 1957




was 5.30-±- 0.446 grams,  a decline of 25 percent.  It was




also  reported that osprey population in the area also




decreased  during the  post-DDT years.




      A Canadian investigation report indicated a 11 percent




drop  in the thickness of prairie falcon eggshells during




the post-DDT  years compared to those collected prior to




the advent of DDT and other halogenated hydrocarbon




insecticides. The report indicated a high correlation

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between the decline in the thickness of these eggshells




and DDE content in the eggs.  Associated with this decline




was a 34-percent decrease in the occupancy of territories




unknown to falcons during the previous ten years.  The




relatively high content of DDT and its metabolites found




in raptorial birds and in their eggs are not surprising




because these birds are at the end of a food chain.




     Laboratory experiments confirmed the eggshell thinning




phenomenon attributed to DDT or its metabolites.  In one




experiment, American sparrow hawks were fed for two years




a diet containing DDE, a. dosage equivalent to residue




levels commonly found in the foods of raptorial birds in




the field.  The investigators reported that there was no




difference in eggshell thickness between the treated and




non-treated birds during the first year.  However, they




noted an average ten percent decline in the thickness of




eggshells from treated hawks in the succeeding year.  In




another experiment, DDT and dieldrin were fed in combination




to the hawks.  Eggshells from the treated hawks were, on the




average, also thinner than the controls.  The investigators




also noted that there were also fewer eggs laid by the treated




hawks.




     The decrease in fertility was also observed in DDT-dosed




coturnix and ringdoves.  One investigation team reported that




coturnix fed p,p'-DDT in their feed at dosages of 2.5, 10,




and 25 ppm for 26 weeks produced overall 18 to 21 percent




fewer eggs than did the control group.  Downward production

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                                                                   95
trends continued for both the 10 ppm and 25 ppm dosages




with time.  Eggs produced by the three DDT-treated groups




of birds had, respectively 6.0, 6.4, and 7.3 percent thinner




eggshells than the untreated ones.  It was also observed that




hatching success declined significantly with time in all




groups except those fed 2.5 ppm doses.  In another investiga-




tion, mature coturnix were fed 0, 100, 200, and 400 ppm of




DDT in their feed for 60 days.  No effect on mortaliby, egg




hatchability, or fertility was observed in the 100 and 200 ppm




group.  However, the 400 ppm group suffered a 50 percent




mortality within 30 days after treatment began.  This group




also showed a marked decline in fertility.  There was some




decrease in hatchability of eggs from this group.  Young-..




chicks hatched from this group exhibited incoordination of




muscular action and spasms.




     Another team of investigators reported that coturnix,




fed relatively high dosages of DDT, produced eggs with




significantly less calcium.  The same team also reported




that the shell-forming glands of treated birds had 16 to




19 percent lower carbonic anhydrqse activity than the




untreated ones.  The eggshells from the treated group




were ten percent thinner than the control group.




Physiological changes brought on by DDT and its metabolites




were noted in experiments with ringdoves.  In one experiment,




a group of ringdoves fed 10 ppm of DDT in the feed showed a




significant decrease of estradiol in the blood.  Moreover,




this decrease occurred early in the breeding cycle.

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Consequently, egg-laying was delayed from a normal 16.5 —




1.6 days to 21.2 -±- 5.5 days.  The eggshells from the




treated group had approximately ten percent less weight




than those from untreated birds.




     A high correlation was found also between the amount




of DDE, a major matabolite of DDT, in eggs and eggshell




thickness of pelicans.  An important consequence of eggshell




thinning is the premature cracking of the eggs.  On Anacapa




Island off the coast of California, egg breakage resulted




in the complete reproductive failure of the brown pelican




on the island during 1969.  Shells, of a few intact eggs




measured shortly after egg-laying averaged only 0.38 milli-




meters (mm) compared to the average normal thickness of




0.57 mm, a decline of 34 percent.  Residues of DDT and its




metabolites were about 1,200 ppm, of which 85 percent was




DDE.  Residues in the fat of adult birds ranged between




738 and 2,603 ppm.  The investigators concluded that "these




findings, along with existing experimental evidence, clearly




implicate DDE as a cause of eggshell thinning, reproductive




failure, and population decline in brown pelicans."  These




findings.were confirmed by similar field studies done by




other investigators.  For example, when weights and thicknesses




of brown pelican's eggshells were compared between those




collected prior to 1947 and in 1969, both measurements




suffered decreases in the 1969 eggshells.  The investigator




further reported that the 16.2 percent decrease in eggshells

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                                                                    97
of South Carolina pelican eggs was about the same for the




eggshells of raptorial birds found in the area.  Other




investigators found that 15 pelican eggs taken in Texas




and Florida after 1949 were on the average 20 percent




below normal weight.  Shell thickness was found to have




decreased between 15 and 27 percent.  One investigator




advanced an explanation for the eggshell thinness




phenomenon, that it appears to be due to changes in the




storage and mobilization of calcium after ingestion,




rather than action at the initial step of this process.




     DDT's toxic effects toward birds and other non-target




species have been a subject of many field investigations.




Some of the more conclusive findings are given below.




     After the application of DDT at two pounds per acre




every year for four years, populations of American red-




starts, parula warblers, and red-eyed vireos in forested




areas declined 44, 40, and 28 percent, respectively, over




the four-year period compared with the non-sprayed area.




.Elm trees in a 430-acre area were sprayed with six-percent




DDT for control of disease.  The soil in the area contained




up to 18 ppm and the earthworms contained from 53 to 204 ppm.




The median DDT residue found in 21 dead robins was 3 mg.  If




this was taken as the lethal dose for robins, it would take




less than 100 worms from the area for a robin to accumulate




the lethal dose of 3 mg/kg of its body weight.  In Hanover,




New Hampshire elms were treated with 1.9 pounds per acre of




DDT.  The spraying resulted in the deaths of 151 birds

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                                                                    98
 compared to  only  ten in an untreated area in nearby Norwich,




 Vermont.   Moreover, the robin immigrant population in




 Hanover  by June 1, 1963, had declined to 70 percent below




 the original May  1 population level.  While at Norwich,




 there was no net  change.  Other birds affected included the




 myrtle warbler and the tree swallow.  DDT's disastrous




 effects  on birds  were also recorded in Michigan and Wisconsin.




 The insecticide was applied to elms on the Michigan State




 University campus for the control of Dutch elm disease.  The




 application  nearly killed all the robins as well as many




 other birds  on the campus.  Three habitats in Wisconsin




 received DDT for  control of Dutch elm disease, and three




 areas were unsprayed.  In the three DDT-treated areas, bird




 population averaged 31, 68, and 90 percent below those of the




 unsprayed areas.  Robin populations in the sprayed areas were




 69,70, and 98 percent below those of the unsprayed areas.




 Treatment of two  areas in Wisconsin with DDT to control




'.Dutch'elm disease with about two pounds of the insecticide




 per tree resulted in a robin mortality ranging from 86 to




 88 percent.




      The breeding success of New Brunswick woodcocks was




 closely  related to the amount of DDT used; i.e., an inverse




 relationship.  From 1961 to 1963, the level of residues of




 DDT in spring woodcock arrivals in New Brunswick increased




 significantly from an average of 2.0 to 5.4 ppm DDT.




      A survey reported that mortality among herring gulls




 found on the edge of Lake Michigan was attributed to DDT

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                                                                   99
present in the area.  Reproduction in these herring gulls


appeared to be reduced by the presence of DDT.   A sample


of nine eggs which appeared to be alive contained dosages of


202 -±- 34 ,ppm of DDE.  The ten dead eggs sampled had a higher


concentration of 919 -±- 117 ppni of DDE.  From 30 to 35 percent


of the eggs in 115 nests were dead, and this was felt to be


an exceptional egg mortality.  An investigation of a rice-


growing region in California where DDT-treated seed was used


for pest control, pheasants were found to have concentrations


of DDT averaging 740 ppm in their fat.  The survival rate of


young pheasants was lower than normal, prompting a restriction


against planting DDT-treated seed.  In an investigation of the


effect of temperature and DDT spraying on the ruffed grouse


population, a team of investigators reported an apparent


interaction between these two factors.  May and June


temperatures were related to the time of nest initiation,


to egg loss, and to other mortalities.  A synergistic


effect between DDT at levels of 0.25 and 0.5 pound per acre


and temperature was apparent in the loss of partially developed


eggs.  The DDT treatment was also correlated with a loss of


immatures and changes in fall age ratios.
                                     r

     DDT is acutely toxic to mammals on oral administration.


The oral toxicity averages about a tenth of the intravenous


route of administration.  Toxicity varies in the same species


according to absorption.  In general, solutions of the toxicant


are more toxic than the powder.  Reported median lethal doses


(LD50) ^or "^e same species as well as for different species

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                                                                 100
of mammals vary.  Presumably these dosage variations are due




to various factors such as temperature,  prior exposure to




the toxicant, purity of DDT, intrinsic factors in the




individual test mammal, and other conditions peculiar to




the individual laboratory experiment or field investigation.




Some of the reported median lethal doses via ingestion for




various mammals are:  rat, 420 to 800 mg/kg; mouse,  200




mg/kg; rabbit, 250 to 400 mg/kg; dog, 60 to 75 mg/kg; guinea




pig, 400 mg/kg.




     The symptoms of DDT poisoning in mammals start  with




twitching of the eyelids and general hyperexcitability.  The




twitching progresses to severe generalized tremors of long




duration.  This is then followed by alternate, irregular




contractions and relaxations of muscles characterizing a




clonic spasm.  The animal then undergoes continuous  muscular




tension or contraction characterizing a tonus convulsion.




Depression, paralysis, and death follow in rapid succession.




Animals that survive several weeks show extensive damage in




the liver, kidneys, and spleen.  There are generally no




significant lesions in the central nervous system.




Cumulative toxicity from repeated exposure to subtoxic




doses has been accused of producing digestive derangements




and various obsecure disturbances in man, but the connection




appears very dubious due to lack of experimental verification.




There are no definite data on continued DDT administration




in man.  It was estimated that two to five milligrams per




kilogram of body weight would probably cause mild illness.

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                                                                101
     It has been experimentally shown that prolonged




administration of the insecticide in mammals does not




increase their susceptibility to the acute effects,  but




rather decreases the initial symptoms.  For example,




continued feeding of dogs with 100 mg/kg daily results in




contractions of skeletal muscle.  When the dose is increased




to 150 and 250 mg/kg of body weight, severe but reversible




neurological disturbances appear.  These disturbances become




increasingly irreversible with larger and larger doses.




The neurological changes observed in the test dog resembled




those symptoms associated with the removal of certain portions




of the brain; i.e., cerebellum and cerebral cortex.   The




cerebellum of the test animal showed considerable damage




due to DDT poisoning, especially at high dosage levels.




Slow poisoning of rats with DDT begins with loss of appetite.




Also during the early stages of poisoning certain changes




occur in the fatty tissues of the rat's liver.  The significance




of these changes to the physiology of the test animal is a




moot point.  Moreover, these changes are reversible and




apparently only occur in rodents, especially in male rats.




Female rats, on the other hand, are more susceptible to




general toxicity.  When rats and mice are kept on a low fat




diet, the toxic effects seem to decrease.




     After administration of a large single dose, DDT is found




widely distributed in the tissues, but especially in the fat.




With continued ingestion, but without signs of toxicity, the




amount stored in the body may exceed several times the amount




that would be fatal with a single intravenous injection.

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                                                                  102
.Through slow accumulation  the amount of DDT on its metabolites




 in the fatty tissues may be up  to  ten  times that found in  the




 food.   Storage  in  the  fat  of rats  occurs  even with low




 insecticide dietary levels and  the concentration of DDT in




 these  tissues increases indefinitely with the duration of




 ingestion,  until the level reaches an  equilibrium plateau.




 This equilibrium point or  plateau  is characteristic of the




 dosage.  Smaller amounts of the insecticide or its metabolites




 are found in the lymph nodes, adrenals, heart, and thymus.




 Trace  amounts are  found in the  testes, liver, kidney, spleen,




 central nervous system, and lungs.




     When the administration of the toxicant is discontinued,




.to percent of the  stored DDT is still  present after a month




 and 25 percent  after three months. Up to 70 percent of the




-toxicant as bis(p-chlorophenyl)acetic  acid is eliminated in




 the urine of rabbits.  The remainder is excreted very slowly.




 DDT is also eliminated via milk, so that  this may produce




 typical toxic symptoms in  the sucklings of poisoned animals.




 For example, milk  cows fed on sprayed  alfalfa hay contained




 about  3.5 mg/1. The butterfat  may contain as much as 65 mg.




 Upon withdrawal of DDT contaminated feed,  the toxicant level




 in the body burden rapidly falls off within ten days.  However,




 small  amounts still persist after  six  months of withdrawal.




     The total  number  of reported  poisonings in man by DDT




 is very small relative to  the extent of its use; and




 clinical poisoning attributed to DDT is generally complicated




 by the possible toxic  effects of solvents and sometimes by

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                                                                103
other insecticides in mixtures.   No instance of chronic




poisoning in man has been 6onfirmed.  Experiments on volunteers




showed no effects after a single oral dose of 0.5 gram, and




only minor DDT symptoms after 1.5 gram.   Fatal poisoning in




a child indicates that 150 mg/kg, of body weight is lethal.




If this dose is taken, the calculated lethal dose for an




average adult weighing about 70 kg (approximately 150 pounds)




would be ten grams.  This dosage is similar to the median




lethal dose for many mammals.




     Clinical symptoms of acute DDT poisoning in man may




start in half an hour with large doses;  i.e., 300 to 500 mg




via ingestion.  Patients with mild cases have nausea,




vomiting, anxiety, a burning sensation in the lips and face.




In more severe cases, the patient may have, in addition,




tremors, convulsions, stiffness and pain in the jaws, and




soreness in the throat for several days.  No instance of




uncomplicated fatal poisoning by DDT is  on record.  However,




a man who had swallowed about 120 c.c. of a commercial




insecticide with five percent of DDT reported as the principle




toxic ingredient developed blood poisoning, suppression of




urinating functions, and involuntary spastic contractions of

         »
                                      f

the fingers and wrists.  The patient died in a deep coma in




six days after ingestion.  Autopsy revealed severe degeneration




in the patient's renal tubes and liver cells.  Although it is




not certain that DDT was alone responsible, the symptoms and




lesions are similar to those observed in animals.

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                                                                  104
                            DDT

                    General References
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                                                                 1  0 5
                            -2-
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                                 DDT                                    106

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