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
-------
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
-------
>
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
-------
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:
-------
?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
-------
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
-------
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
-------
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,
-------
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
-------
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
-------
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
-------
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
-------
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
-------
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.
-------
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
-------
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
-------
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.
-------
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
-------
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
-------
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.
-------
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.
-------
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
-------
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.
-------
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
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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
-------
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.
-------
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
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DDT 106
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