United States
                                    Environmental Protection
                                   Health Effect Research
                                   Research Triangle Park NC 27711
                                    Research and Development
                                   EPA-600/S1 -82-011  Sept. 1983
Project  Summary
                                   Chemistry  and  Mode  of
                                   Action  of Insecticides:
                                   Phase II

                                   T. R. Fukuto, R. B. March, and T. A. Miller
                                     Results  gathered for the  period
                                   February 15, 1976-May 31, 1981 are
                                   presented.  Progress is reported  in
                                   areas dealing with the chemistry,
                                   metabolism, and mode of action  of
                                   organophosphorus,  carbamate, and
                                   organochlorine insecticides. Among
                                   the topics  included are:  insecticide
                                   metabolism and mode of action; insec-
                                   ticide selectivity; structure-activity
                                   relationships; biological and environ-
                                   mental  alteration of insecticides;
                                   effect of impurities on the potentiation
                                   of organophosphorus  insecticides;
                                   delayed toxicity of trialkyl phosphoro-
                                   thioates; delayed neurotoxicity  of
                                   organophosphorus esters; mode  of
                                   action of phosphoramidothioate esters;
                                   gas chromatography of insecticides;
                                   and neurophysiological studies on the
                                   mode of action of insecticides.
                                     This Project Summary was devel-
                                   oped by EPA's Health Effects Research
                                   Laboratory, Research  Triangle Park.
                                   NC, to announce key findings of the
                                   research project that  is fully docu-
                                   mented  in  a separate report of the
                                   same title (see Project Report ordering
                                   information at back).

                                   Insecticide Metabolism and
                                   Mode of  Action

                                   Studies on the Ox/dative
                                   Conversion of P=S to P=O
                                   Esters and the Effect of
                                   Chirality  on Toxicological
                                     The stereochemistry of P=S  to P=O
                                   conversion  effected by chemical model
                                   oxidation systems  and mouse  liver
                                   mixed-function oxidases (MFO), using
                                   the resolved isomers of fonofos (O-ethyl
                                   S-phenyl ethylphosphonodithioate) was
                                   examined. The stereochemical aspects
                                   of the chemistry, metabolism, and mode
                                   of action of the chiral isomers of fonofos
                                   are presented.
                                   The conversion of (5)-p- fonofos to (/?)p-
                                   fonofos oxon by  the action  of m-
                                   chJoroperoxybenzoic  acid occurred
                                   predominantly with retention of config-
                                   uration of the phosphorus atom. Oxida-
                                   tion to the rearrangement product, (/?)P-
                                   phenyl ethyl(ethoxy)phosphinyl disulfide
                                   took place with predominant  inversion
                                   of configuration. In the reaction leading
                                   to the oxon, the stereochemicai course
                                   was identical to that observed with
                                   mouse liver MFO.
                                    Toxicological data indicated a 2- to 4-
                                   fold higher  toxicity of (/?)p-fonofos to
                                   mice  and houseflies  relative to (S)p-
                                   fonofos.  Compared to fonofos, the
                                   difference  in toxicity between the
                                   fonofos oxon enantiomers was greater
                                   and (S)p-oxon was 2.6- (i.p.) and 12-fold
                                   more toxic to white mice and houseflies,
                                   respectively, than the (/?)P-oxon.
                                    The relative toxicities of (S)p-  and
                                   (/?)p-fonofos  oxon  to houseflies and
                                   white mice were qualitatively in agree-
                                   ment with their relative in vitro anti-
                                   cholinesterase activities toward fly-
                                   head, mouse-brain, and blood  ChE.
                                   Quantitatively the differences in anti-
                                   cholinesterase activities between the
                                   isomers were substantially greater (49-
                                   to  60-fold)  than  the  differences in
                                   toxicities to houseflies and white mice.
                                   At the same mtraperitoneal dosage of
                                   oxon isomers (0.6 mg/kg), the level of
                                   maximum inhibition of both brain and

blood ChE obtained from mice treated
with (S)p-oxon was approximately 3-fold
greater  than enzymes obtained  from
mice treated with the (/?)p-enantiomer.
Maximum  inhibition, however, oc-
curred at different time intervals after
treatment and the relationship was
further complicated by the differences
in recovery rates of the  inhibited
enzymes. Nevertheless, agreement be-
tween in vivo inhibition of mouse ChE
and intraperitoneal toxicity was good.
  The relationship between in vivo
inhibition of housefly-head acetylcho-
linesterase  (HFAChE)  and toxicity  of
houseflies treated with the two isomers
was less satisfactory. Maximum reduc-
tion  of HFAChE in flies treated with 5
fjg/g (/?)p-oxon was about 35% compared
to about 80% for flies treated with the
same amount of (S)p-oxon,  a slightly
greater  than 2-fold difference, a dif-
ference substantially smaller than the
12-fold difference observed for housefly
toxicity. Plots show that HFAChE inhi-
bited by (/?)p-oxon recovered to a signifi-
cantly greater extent (compare activities
at 12 h), and it is possible that ChE de-
pression over longer time periods
should be considered in relating in vivo
inhibition with toxicity.
  Studies on the in vitro metabolism of
the  two isomers  in the presence  of
mouse  liver MFO showed  that (R)p-
fonofos and (S)p-fonofos were convert-
ed to the respective (S)p-oxon and (R)p-
oxon with  70-80% stereospecificity.
Since (S)p-oxon  is  a  stronger  anti-
cholinesterase and is more toxic than
(/?)P-oxon,  the order of toxicity of the
fonofos isomers is in the right direction.
Although  metabolic  conversion  of
fonofos to the oxon occurred predom-
inantly with retention of configuration,
a significant amount of inversion appar-
ently also took place, leading to partial
racemization of the oxon. This result un-
doubtedly contributed  to the smaller
toxicity  ratios between the fonofos iso-
  Studies  on the relative  rates  of
metabolism revealed  that while (R)p-
fonofos was  metabolized at a rate
approximately  2-fold greater than (S)p-
fonofos, analysis of the  products
showed that (/?)P-fonofos was converted
in greater proportion to the detoxication
product diphenyl disulfide (DPDS) than
its enantiomer. Thus, even though (/?)P-
fonofos was metabolized by mouse liver
MFO at a faster rate, the difference in
amounts of fonofos oxon formed from
the fonofos isomers was not large.
  Rat and mouse serum were effective
in degrading  fonofos oxon but not
fonofos.  The in vitro degradation of
fonofos oxon by rat or mouse serum was
relatively slow compared to the oxidative
reactions, but  results clearly showed
that (S)p-oxon was degraded at a faster
rate than the (/?)p-oxon (approximately
2-fold). The greater susceptibility of
(S)p-oxon to enzymatic degradation may
account for  the smaller difference in
toxicities between  the fonofos-oxon
enantiomers relative to their differences
in anticholinesterase activities (50- to
60-fold).  Further, since (/?)p-fonofos is
preferentially metabolized to (S)p-oxon
and (S)p-fonofos to (/?)?- oxon, the faster
rate  of degradation of (S)p-oxon may
contribute to  the even  smaller dif-
ferences in toxicities  of the fonofos
enantiomers  Thus, the  smaller dif-
ference  in  toxicities between the
fonofos enantiomers compared to the
relative toxicities of the oxons and their
anticholinesterase activities may be ac-
counted for by the partial racemization
in the oxidative desulfuration reaction
and faster rate of degradation of (5)p-

Studies on the Mode of
Action  of Methamidophos
  O,S-Dimethyl phosphoramidothioate
(methamidophos),  although  highly
effective  with a bimolecular inhibition
constant of (k,) 9.2 * 102 M'1 min'1 for
the inhibition  of fly-head AChE, is a
relatively weak anticholinesterase.
Studies were conducted on the toxico-
logical properties, in vitro and in vivo
anticholinesterase behavior, and me-
tabolism  of methamidophos
  Evaluation of the kinetics of housefly
ChE  inhibition  showed that its affinity
for the enzyme and its phosphorylation
and bimolecular inhibition rates are all
relatively low.  In  vivo  metabolism
studies in houseflies provided evidence
that it is not activated to a more effective
ChE inhibitor and indirect evidence also
was obtained for its slow degradation In
vitro  metabolism studies  in housefly
and mouse tissues provided  additional
evidence for its lack of activation and
slow metabolic degradation. Compared
to other effective organophosphorus
insecticides, methamidophos was slow
in producing acute symptoms of poison-
ing and ChE inhibition and required the
accumulation  of comparatively high
internal levels for toxic effects. However,
in vivo ChE inhibition  studies provided
evidence for the interrelationships of
ChE inhibition and toxic effects. Thus,
its  relative stability  and low  in  vivo  |
degradation appeared to be  of critical
importance in accumulating and main-
taining a sufficient internal concentra-
tion for a long enough period of time to
permit the development of  its slowly
expressed toxicity.

Insecticide Selectivity

Selective Toxicity of N,N'-
  A series of /V-(alkyl alkylcarbamoyl-
sulfenyl)  derivatives of the methyl-
carbamate insecticides carbofuran, m-
isopropylphenyl methylcarbamate, pro-
poxur, aldicarb, methomyl, and oxamyl
were prepared and examined for toxicity
to  houseflies,  mosquito larvae,  and
white mice. Compared  to the parent
methylcarbamate, the derivatives were
generally  of  equal  toxicity  to the
housefly and substantially more toxic to
mosquito larvae With the exception of
the oxamyl derivatives, all other deriva-
tives were much less acutely toxic to the
white  mouse  The  toxicities of the
derivatives to mosquito  larvae and the
white mouse were correlated with their
octanol-water partition coefficients

Insecticidal Properties  of the
N-Sulfonyl Derivatives of
Propoxur and Carbofuran
  A series of substituted /V-benzene-
sulfonyl derivatives of carbofuran and
propoxur containing electron-with-
drawing  substituents of the benzene
ring was synthesized and examined for
toxicity to houseflies  and  mosquito
larvae The derivatives were generally
nonmsecticidal  when tested  alone, but
some of  the compounds were toxic to
houseflies when synergized with piper-
onyl  butoxide.  The  poor insecticidal
activity and low mammalian toxicity of
these derivatives  are attributable to
their high stability and their inability to
be  converted into  the parent  methyl-

Selective Toxicity of N-
Arylsulfenyl Derivatives of
Methylcarbamate Insecticides
  The lexicological  properties of a
series of /V-sulfenylated derivatives of
commercial  methylcarbamate  insecti-
cides were studied with special focus on
the toxicological response observed in
honeybees. Work on the comparative
metabolism of one of these derivatives
was conducted in the  honeybee and
housefly to determine the basis for its
selective toxicity.

  Of the 34 different sulfenylated de-
rivatives of propoxur, carbofuran,  and
carbaryl evaluated, only three showed a
toxicity ratio greater than one although
many were safer to bees than the parent
methylcarbamate. The most notable
exception was the 2-methyl-4-f-butyl-
phenylsulfenyI derivative of propoxur (7)
which was more than 178-fold safer to
the honeybee than propoxur Although
the 4-f-butyl moiety in the ring appeared
to introduce the  greatest amount of
honeybee safety in these derivatives, no
obvious relationship between the struc-
ture of  the  carbamate derivative  and
honeybee toxicity  was apparent
  The pathways in the honeybee  and
housefly, while qualitatively similar,
contain enough  quantitative differences
to account for  the difference in  the
toxicity  of  (7) to the two insects.
Penetration  of  (7) and  its subsequent
conversion into  propoxur are apparently
the rate-limiting steps in the metabolism
of (7) in the honeybee  Rapid conversion
of propoxur into secondary metabolites
results  in its accumulation in a  low,
steady-state level in  the  bee  Conse-
quently, intoxication does not occur. In
houseflies, however,  penetration  and
conversion of (7) into propoxur was fast.
Intoxicating levels of propoxur accumu-
lated within a short period of time after

Selective  Toxicity of
Phosphoramidothioate Esters
  A study of the comparative metabolism
of the hexanoyl  derivatives and  the
smaller  chain acyl derivatives of meth-
amidophos in a mammal and insect was
undertaken to  establish the basis for
their lexicological  properties Metabolic
and lexicological data gathered for 0,5-
dimethyl propionyl- (2) and hexanoyl-
phosphoramidolhioaie (3)showlhat the
metabolism of the two compounds in
the while mouse and  housefly is
qualilalively and quaniitalively different
  The major difference in the metabolism
of (2) and (3) in the mouse appears lo be
in the larger amounts of methamidophos
formed  from  (3) compared  to  (2).
Melhamidophos is highly toxic to  the
mouse (LDso 14  mg/kg), and is probably
the agent responsible for intoxication
when the mouse  is trealed with either
(2)  or (3).  The  small  amounts of
melhamidophos formed from (2) readily
accounl for ils safety to mice. Similarly,
ihe relalively high toxicily of (3) to mice
may be  atlribuled to  the subslantial
quantily of melhamidophos  formed in
Ihe mouse treated with (3)

Hydrolytic and Toxicological
Properties of Ethyl a-
Phosph oramidates
  The desired order of  selectivity
demonstraled by  phoxim (0,0-diethyl
O-or-cyanobenzaldoxime phosphorothi-
oate)  and the limited knowledge of the
relationship between chemical structure
and biological activily of phosphorami-
date esters led to sludies on phosphor-
amidates containing the  a-cyanobenz-
aldoxime leaving group which is present
in phoxim.
  A series of ethyl alkylphosphoramidale
eslers of a-cyanobenzaldoximes were,
Iherefore, synlhesized and examined
for alkaline hydrolysis, anlicholinester-
ase activity, and toxicity to houseflies
and  mice  The compounds  showed
variable toxicily lo bolh houseflies and
mice,  and were effective inhibitors of
  Satisfactory correlalion was obtained
between alkaline  hydrolysis rale con-
stants and  anticholmesterase  activity
with Tafl's a* and Es values Toxicity to
houseflies was related to anticholine-
sterase activity and Hansch's  n con-
stants Several of the compounds were
of low toxicity to Ihe while mouse but
were  highly effective  against  the

Insecticidal Properties of
Phosphonamidothioate Esters
and Derivatives
  A series of S-alkyl methyl- or ethyl-
phosphonamidothioate eslers analogous
lo melhamidophos (O,S-dimethyl phos-
phoramidothiale) and  acephate (O,S-
dimelhyl /V-acetylphosphoramido-
thioate) was synthesized and evaluated
for lexicological properties. Although S-
methyl methyl- and ethylphosphonami-
dothioate were more effeclive against
the housefly than  methamidophos, the
acylated denvalives showed variable
loxicily bui were all subslanlially less
effective than acephate.  The acylated
derivatives, however, showed improved
mouse toxicity. S-methyl methyl- and
ethylphosphonamidodithioate were
less active  against the housefly  than

Insecticidal Activity of DDT
  A series  of  DDT analogs whose
structures were based on a hypothetical
model for the DDT receptor site was
synthesized  and examined for insecti-
cidal  activity.  Several of these com-
pounds showed activity; one of them,
2,2-bis(p-ethoxyphenyl)-1,1 -dichloropro-
pane  showed  activity equal to that of
DDT against houseflies  and mosquito
larvae. This is the first case  in which a
DDT analog with an a-methyl moiety in
place of hydrogen  has been shown to
have insecticidal activity.

Effect of Ring Substituents
on the Inhibition of
Cholinesterase by Carbamate
  A  series of 27  substituted aryl  N-
methoxy-/V-methyl carbamates was
synthesized. The ability  of these com-
pounds to reversibly inhibit housefly-
head (HFAChE) and bovine erythrocyte
acetylcholmesterase (BAChE) and horse
serum  cholinesterase   (HSChE) was
determined.  All  were compelitive re-
versible inhibitors of BAChE, but some
showed mixed competitive inhibition
against HFAChE and HSChE. Dissocia-
tion constants (K,) as small as 9 9 * 1CT9
M and as large as 1.4 * 10~4 M were
observed.  Satisfactory correlation be-
tween log K, for the inhibition of fly-head
acetylcholinesterase by the /V-methoxy-
/V-methylcarbamates and -log Uoforthe
inhibition  of the same enzyme by ihe
corresponding  methylcarbamates was

Biological and Environmental
Alteration of Insecticides

Characterization of the Bound
Phenthoate Residues in Citrus
  The  metabolism of phenthoate  or
phosphorodithioate (Cidial®) in Valencia
orange fruit was examined with empha-
sis on the characterization  of bound
phenthoate residues in  the  fruit peel.
Data for the distribution of radioactivity
in the surface wash, peel, and pulp after
treatment with l4C-phenthoaie showed
a gradual decline  in external fruit
surface wash with a gradual increase in
percentage of radioactivity recovered in
the internal  acetone  extract and peel
residue. Radioactive labeling was also
used to indicate the nature and distribu-
tion of metabolites from the different
fractions  of orange fruit.  The five
principal metabolites obtained were
phenthoate oxon, demethyl phenthoate,
phenthoate acid, ethyl mandelate, and
mandelic acid, along with unchanged

Metabolism of
Dibutylaminosulfenyl and
Carbofuran in Plants, Animals
and Soil

Metabolism in Corn and Cotton
  The  metabolism of 2,3-dihydro-2,2-
dimethyl-7-benzofuranyl (d\-n-buty\-
aminosulfenyl)  (methyl)-carbamate
(DBSC or dibutylaminosulfenyl carbo-
furan) and 2,3-dihydro-2,2-dimethyl-7-
yl)-carbamate (MSC or morpholinosul-
fenyl carbofuran), two potentially useful
insecticides, in corn and cotton plants
was studied.
  DBSC  metabolites in  cotton plants
were  identified 1, 3, 6, and  10 days
following stem injection of ring-14C-
DBSC or carbonyl-14C-DBSC and con-
firmed by chromatography  in  several
solvent systems.  Both sets  of labeling
experiments  showed similar results.
The principal speciesfound after 1 and 3
days was carbofuran, but 3-hydroxycar-
bofuran was equally important at 6 and
10 days.
  The  metabolism of MSC  in cotton
plants was determined following  stem
injection  of carbonyl-14C-MSC. Overall,
results were  similar to those found in
the metabolism of labeled DBSC.
  DBSC  metabolites in  cotton plants
were also identified by treatment of the
foliage with carbonyl- and ring-labeled
DBSC. Absorption  occurred  steadily
over the  10-day  test period; the same
metabolites as those identifiedfollowing
stem  injection were observed. Overall,
the rate  of metabolism  after foliage
treatment appeared  more  rapid  than
after stem injection
  Stem injection of carbonyl-labeled
DBSC was used  to identify the various
metabolites of DBSC in corn  plants. The
same metabolites found in cotton were
found in corn,  although DBSC  was
converted to  carbofuran more rapidly
than in cotton, and the carbofuran was
converted into subsequent metabolites.
The percentage of radioactivity remain-
ing in the residual straw was higher in
corn compared to cotton.

Metabolism of DBSC in the Rat
  The metabolism of DBSC in the rat
was examined to determine the fate of
this compound  in mammals and to
establish the basis for its lower mam-
malian toxicity relative to carbofuran.
Ring-labeled,  carbonyl-labeled, or butyl-
labeled DBSC was administered in two
doses orally in male and female rats
The most important means of elimination
was via the urine for the ring-labeled
and dibutyl-labeled  DBSC  and via
expired  carbon dioxide and  urine for
carbonyl-labeled DBSC. The distribution
and  identity of the  metabolites was
determined by calculating percentages
of radioactivity recovered.
  DBSC is initially  metabolized by two
major pathways in the rat: via oxidation
of the sulfur to give sulfonyl DBSC, and
via N-S bond cleavage to give the actual
toxicant,  carbofuran. Following the
initial metabolic degradation of DBSC to
carbofuran,  carbofuran was detoxified
primarily via oxidation at the 3-carbon
position of the dihydrofuranyl ring. The
primary excreted  metabolite  was 3-
ketocarbofuran phenol, followed by 3-
  While DBSC is converted to carbofuran
in the rat, it appears that the rate of
formation  of carbofuran is slower than
expected from studies on its degradation
under different pH conditions. Based on
the high levels of DBSC present in the
blood 3 h after treatment, it appears that
carbofuran is released slowly and this
slow release, in part, accounts for the
lower mammalian toxicity of DBSC

Metabolism  in Soil
  The alteration of MSC and DBSC in
Cosad® sandy loam soil under aerobic
and anaerobic conditions was studied,
and  the degradation of  the major
primary metabolites,  carbofuran and
dibutylamine, investigated.
  Metabolism of ring-labeled carbonyl-
labeled DBSC occurred rapidly in both
types of soil conditions, a half-life of 2-3
days and a first-order thiolysis of DBSC
are reported.  The  major radioactive
component during the 30-day test
period remained carbofuran, which was
either oxidized at the 3-position of the
ring, or hydrolyzed at the carbamate
ester  to form  carbofuran phenol  Bis-
carbofuran-disulfide, dibutylamine, and
at least 7 unidentified minor compounds
were also detected. Phenolic degradation
products appeared  to be bound to the
soil  humus by an oxygen-dependent
process. Ring cleavage was alsooxygen-

Alteration of DBSC in  a  Water
  A study  was undertaken to determine
the reactions involved in the degradation
of DBSC and the resulting breakdown
products under different environmental
conditions. The breakdown of DBSC in
different buffer solutions (hydrolysis), in
buffer  solutions containing sulfhydryl
reagents (thiolysis), and on the surface
of silica gel TLC plates was investigated.
  In buffer solution and on thm-layer
plates, N-S bond cleavage readily  oc-
curred to  give carbofuran as a  major
product, with  minor amounts of bis-
carbofuran-/V,/V'-disulfide and -trisul-
fide. First-order kinetics was observed
for the hydrolysis reaction in buffer. An
unknown  polar compound was also
obtained in these systems in significant
amounts.  Thiolytic N-S bond cleavage
occurred  in the  presence of excess
cysteine and  glutathione  at pH  7.0 to
give carbofuran as the sole identifiable
product, and  first-order  kinetics was

Acid-Catalyzed Alteration of
  During the course of a  study on the
behavior  of  DBSC  under different
solvent  conditions,  DBSC was  found
unstable in a  dichloromethane-acetic
acid (9:1) mixture.  In  addition  to
carbofuran, TLC analysis  showed that
other  reaction products were  the
polysulfide derivatives of DBSC (CF-Sn-
NBu2 where n =2-4), biscarbofuran-/V,/V'-
disulfide  (CF-S2-CF) and polysulfides
(CF-Sn-CF  where n = 3-5), and dibutyl-
  NMRand massspectralanalysiswere
used to establish the overall structure of
each component in CF-Sn-NBu2and CF-
Sn-CF.  It was not possible, however, to
determine the  exact nature of the
polysulfide moiety. Poor insecticidal
activity was observed for CF-S2-CF, a
finding that was unexpected in light of
the good activity observed for the other
  Analysis of carbonyl- and dibutylami-
no-14C-labeled DBSC degradation pro-
ducts showed that the amount of CF-Sn-
NBu2 gradually increased to a steady-
state level after 60-72 h, while amounts
of CF-S2-CF and CF-Sn-CF continued to
increase throughout the entire reaction
period  of  120  h This finding suggests
that CF-Sn-NBu2 is an intermediate and
is slowly transformed into CF-Sa-CF and
  Silica gel  KCis reversed-phase TLC
proved to  be a simple and convenient
method for separation of individual
components in the polysulfide mixtures
of DBSC  and  biscarbofuran disulfide
Separation of  CF-Sn-NBu2 yielded at
least 8 components, and  separation of
CF-Sn-CF,  the biscarbofuran derivatives,

7 components.  A general correspon-
dence between R( values andthe number
of sulfurations was observed.

Effect of Impurities on  the
Potentiation  of

Identification and
Toxicological Properties of
  The major  impurities commonly
present or those that may be developed
during storage  in technical malathion
and acephate were studied. An evalua-
tion  of the impurities'  effect on the
mammalian toxicity  of purified insecti-
cides was made
  Impurities present in technical mala-
thion (—95% pure) and technical ace-
phate  (—95% pure) were isolated by
column and thin-layer chromatography
Eleven  structures  in malathion  and
seven  in acephate  were identified by
nuclear magnetic resonance, infrared
spectroscopy, and mass spectroscopy.
Eight of the compounds  in  technical
malathion were examined for their tox-
icologica I effects when added to purified
malathion.  Data show that several of
the impurities potentiated rat toxicity.
The highest potentiation was observed
with 0,S,S-tnmethyl phosphorodithi-
oate and  the S-methyl  isomende of
malathion. Potentiating effects were
also decidedly greater in  the rat than in
the mouse  Of  the  impurities isolated
from acephate,  O,O,S-trimethyl  phos-
phorothioate showed slight potentiation
of mouse toxicity when added to purified
acephate  but an antagonizing  effect
was observed  with 0,O-dimethyl-/V-
  Storage of technical malathion for 3
to 6  months  at 40°C resulted in
materials  that  were noticeably more
toxic to mice. A significant reduction in
mouse toxicity was observed after
storage of technical acephate under the
same conditions.
Mode of Potentiating Action
of Impurities Present in
  Results provided in vitro and in  vivo
evidence that the impurities described
above dimmish  the  activities of serum
carboxylesterase and liver  carboxylest-
erase (the enzymes involved in malathion
detoxication) and  also  diminish  the
activity  of  serum cholinesterase  The
compounds  tested  were,  in  order of
increasing  potency, 0,0,5-tnmethyl
phosphorodithioate, O,0,S-tri methyl
phosphorothioate, O,S,S-tri methyl phos-
phorodithioate,  and O,S-dimethyl S-
(1,2-dicarboethoxy)ethyl   phosphorodi-
thioate.  In  vivo, the diminutions of
serum esterase activity caused by these
compounds  were transient, with the
exception of those caused by 0,0,5-tri-
methyl  phosphorothioate, which  per-
sisted for at least 54 h.
  The titer of malathion carboxylesterase
in liver and sera of rats, three strains of
mice, and five groups of pretreated mice
was correlated with  the malathion LD5o
values measured in these groups. The
equation of a regression line relating
enzyme  titer to  toxicity  was used to
predict  the  median  lethal  dosage of
purified  malathion  to humans.  The
mean value, 3655 mg/kg, was compared
with dosages ingested in actual cases of
human  malathion  poisonings  A  dis-
crepancy was noted between LD5ovalue
predicted in  this study and  the  life-
threatening doses of commercial mala-
thion reported in the clinical literature
  A soluble, stable, and partially purified
preparation  of human liver  malathion
carboxylesterase  was  obtained by
detergent extraction and  fractionation
of whole-liver homogenate. The activity
of  this fraction was  susceptible to
concentration-dependent mactivation
by isomalathion, the most potent of the
esterase inactivators, but  was resistant
to three  other impurities  isolated from
technical malathion.

Properties of Rat Malathion
  Two distinct esterase fractions isolated
from rat liver microsomes were exam-
ined for enzymatic hydrolysis of [meth-
oxy-14C] malathion with special emphasis
on product analysis The effect of O,S,S-
tnmethyl phosphorodithioate  and iso-
malathion as inhibitors of these ester-
ases was also investigated
  Two  malathion  carboxylesterase
fractions, designated as esterase frac-
tions A and B, which hydrolyze malathion
were purified 13- and 18-fold, respec-
tively. Although the  two enzymes could
not  be distinguished from  each other
kmetically, fraction A contained at least
one electrophoretic species not present
in fraction  B. The molecular weight of
esterase fraction A  was  estimated at
50,000-60,000, but  that of B was about
twice this value
  Incubation of [methoxy-14C] malathion
with either fraction resulted ma mixture
of  malathion or- and  /3-monoacids, but
the composition of the mixture produced
by fraction A (a/ft ratio = 1.5) differed
from that produced by fraction B (a/ft
ratio - 0.2), indicating the presence of
multiple species of carboxylesterases in
mammalian liver microsomes.
  Isomalathion was substantially more
potent as an inhibitor of both rat liver
and rat serum malathion carboxyl-
esterases than O,S,S-trimethyl phos-
phorodithioate. Isomalathion appeared
to be equipotent  in  inhibiting the rat
liver carboxylesterase-catalyzed reac-
tions leading to either a- or/3-monoacid,
while 0,S,S-trimethyl phosphorodithi-
oate preferentially diminished those
reactions.  In contrast, the rat  serum
carboxylesterase-catalyzed reactions
leading  to either a- or /6-monoacid were
inhibited to approximately an equal  de-
gree by  either isomalathion or O,S,S-tri-
methyl  phosphorodithioate

Delayed Toxicity of Trialkyl

Delayed Toxicity of O,O,S-
Trimethyl Phosphorothioate
  The delayed toxic effects observed in
rodents after treatment with simple
trialkyl  phosphorothioates  and phos-
phorodithioates were investigated
0,0,5-Tnmethyl phosphorothioate,  an
impurity present in several technical
organophosphorus insecticides, admin-
istered  orally to rats,  caused delayed
mortality at single doses as low as  15
mg/kg, with death occurring 4-22 days
following treatment. Delayed toxic signs
were also observed in mice but in less
severity than  in rats. 0,0,S-Tnethyl
phosphorothioate  and O,S,S-tnmethyl
phosphorodithioate induced the same
signs of intoxication at slightly higher
  Rats  treated with O,O,S-tnmethyl
phosphorothioate  refused food and
water within 24 h after treatment until
time of  death.  Neither injection of nu-
trient solution  nor atropine  reduced or
blocked intoxication  The isomeric
0,0,0-trimethyl phosphorothioate was,
however,  a potent  antagonist of the
toxicity  of O, O,S-tnmethyl phosphoro-
thioate  As little as 1% of the 0,0,0-tn-
methyl isomer protected rats from the
intoxicating effects of the O,0,S-tri-
methyl isomer  at doses as high as 200
mg/kg. Rat serum carboxylesterase and
ChE were inhibited for prolonged per-
iods following  a  single  oral  dose of
0,0,S-tnmethyl phosphorothioate but
the duration of inhibition was signifi-
cantly less when the toxicant contained
1% of the 0,0,0-trimethyl isomer

Structure-Delayed Toxicity
  Analogs of 0,O,S-tnmethyl phos-
phorothioate were examined for acute
and delayed toxicity to rats as well as for
anticholinesterase activity. The O,0-
dimethyl S-alkyl phosphorothioate
esters containing small S-alkyl moieties
(methyl, ethyl, ^-propyl) were highly
toxic to the rat. An increase in the S-
alkyl  chain length  beyond  propyl,
however, caused a precipitous drop in
rat toxicity Of the smaller S-alkyl 6,0-
dimethyl esters, the S-methyl and S-
ethyl  analogs clearly  caused  delayed
toxic effects with death occurring at
long time intervals after treatment Rats
treated with the S-/7-propyl  analog, the
most toxic ester, died relatively quickly
and it was difficult to differentiate acute
from delayed effects.
  Anticholinesterase  measurements
show that the dimethyl  esters are
inactive as cholmesterase inhibitors,
being 105-to 106-fold less effective than
such strong inhibitors as paraoxon and
tetraethyl pyrophosphate  Bimolecular
rate  constants (k,) for the inhibition of
either bovine erythrocyte acetylcholme-
sterase (BAChE) or rat serum  cholme-
sterase (RSChE) were essentially the
same for  all the  straight-chain 0,0-
dimethyl S-alkyl esters (CH3 to /?-C6Hi3),
and the range in rat  toxicity for these
compounds was  12 to > 750 mg/kg.
The  striking  difference between  rat
toxicity and anticholinesterase activity
suggests a  noncholmergic  mechanism
of intoxication.
Delayed Toxicity and  Delayed
Neurotoxicity of Phosphoro-
and Phosphonothioate Esters
  Simple dialkyl phenylphosphonothi-
oates such as O,0-diethyl phenylphos-
phonothioate, its corresponding oxon,
and 0,O-diethyl 4-chlorophenylphos-
phonothioate, impurities  present in
technical-grade ethyl leptophos [O-(4-
bromo-2,5-dichlorophenyl)  O-ethyl
phenylphosphonothioate], caused de-
layed neurotoxicity in hens The mode of
action of delayed  neurotoxic  com-
pounds is still  uncertain  but clinical
symptoms associated with delayed
neurotoxicity are not seen until 8 to 14
days after exposure to the  toxic agent
The delayed toxic response described in
rats treated with the simple trialkyl
phosphorothioates suggested the pos-
sibility that some of those  compounds
may cause  delayed neurotoxicity in
hens. It was also possible  that dialkyl
phenylphosphonates  may cause  de-
layed  toxicity in rats. Therefore, tri-
methyl  phosphate, trimethyl phos-
phorothioates, and  a series of methyl
and ethyl esters of methyl-, ethyl-, and
phenylphosphonates  and  phosphono-
thioates were examined  for delayed
neurotoxicity to hens and delayed toxic-
ity to rats.
  Comparison of the toxic properties of
those compounds that are delayed-toxic
to rats  with those that are delayed-
neurotoxic to hens suggests that the
two types of toxicity are different. At
least compounds that cause  delayed
toxicity in rats are not delayed-neurotoxic
to hens and vice  versa   Of the  15
compounds examined in  the  present
study, only 0,0-diethyl phenylphos-
phonothioate and its oxon, 0,0-diethyl
phenylphosphonate, were delayed-
  0,S-Diethyl ethylphosphonothioate
produced cholmergic symptoms in rats
and most of the rats lost weight for the
first two orthree days. Asthecholinergic
symptoms subsided, rats that lost less
than 25% of their  initial body weight
completely recovered while  those that
lost more than 25% of  their initial
weight  died  Death  occurred up to 6
days after treatment when the weight
loss was 34 to 37% The symptomology
of poisoning appears  to  indicate a
possible similarity between the effects
caused by the two types of compounds

Mode  of Action of Delayed-
Toxic Action of O,O,S-
Trimethyl Phosphorothioate
  Efforts to determine the  mode(s) of
action  of  O,O,S-trimethyl  phosphoro-
thioate  have focused on the following
areas the effects of this compound on
serum  electrolyte; serum  enzyme and
urinary electrolyte levels, determinations
of elimination,  respiration, and tissue
distribution of  radiochemical  label in
animals treated with  14C-O, O,S-tn-
methyl phosphorothioate (labeled in the
OCH3 moiety), pathological  studies of
tissue damage following treatment with
the compound; and  the definition of
pharmacokinetic parameters through
the use of cannulae implanted in the
exterior jugular  vein of test animals.
  Animals intoxicated with  0,O,S-
trimethyl phosphorothioate showed
several  physiological  and  pathological
changes, the most important of which
are lowered serum K+ levels, severe
hemato-concentration resulting from
diarrhea and excessive urination, and
damage to the liver and  kidneys at
moderate-to-high doses of the material
  Initial 14C-labeling studies showed  I
rapid  uptake and stabilization  in  the
body with slow decline of total radioac-
tivity up to and including 3 h following
treatment. Relative levels were  much
higher following i.p. and i.v. treatments
when compared to oral treatment. At 24
h, however,  a higher percentage of the
initial maximum 14C-level in the blood
remained following oral treatment
when compared  to  levels from  i.p
  Metabolism leads primarily to excre-
tion in the urine with a half-lifeof 20-24
h. Of the 63%of total applied radioactivity
excreted in  the urine, only 1.6% was
isolated as unchanged material Little of
the 14C-label was excreted as C02 or in
fecal matter
Delayed  Neurotoxicity of
Organophosphorus Esters

Effect of Structure on the
Delayed Neurotoxicity of O-
Analogs Related to Leptophos
  A systematic  study of pesticidal
activity versus  delayed-neurotoxic
potential  of structural  analogs of
leptophos was conducted. Analogs of   ,
leptophos  [O-(4-bromo-2,5-dichloro-
phenyl)  0-methyl phenylphosphono-
thioate] were examined for  acute
toxicity to mice and houseflies, and for
delayed-neurotoxic  activity in  adult
hens  following  administration of a
single oral dose Development of ataxia
after recovery from acute poisoning was
the criterion for  delayed-neurotoxic
activity. All mono- and dichlorophenyl
analogs were delayed-neurotoxic,  the
2,5-dichlorophenyl analog  being  the
most  potent delayed-neurotoxic com-
pound tested. Substitution for  the
methyl by an  ethyl group  abolished
delayed-neurotoxic activity in both
leptophos  and the 2,5-dichlorophenyl
analog at doses of 1000 mg/kg.  The/?-
propyl and r?-butyl analogs of the latter
were also nondelayed-neurotoxic at
500  mg/kg  and 333 mg/kg,  respec-
tively. Substitution of methyl for ethyl in
EPN  [0-p-mtrophenyl O-ethyl  phenyl-
phosphonothioate] did not alter its  de-
layed neurotoxicity.
Effect of  Chirality on the
Toxicological Properties of
Leptophos and
  The resolution and determination of
the absolute configuration of the chiral

 isomers  of  leptophos and desbromo-
 leptophos and the lexicological prop-
 erties of  these compounds were inves-
  The chiral  isomers  of  0-methyl
 phenylphosphonothioic acid, 0-(4-
 bromo-2,5-dichlorophenyl) 0-methyl
 phenylphosphonothioate (leptophos),
 and 0-(2,5-dichlorophenyl) 0-methyl
 phenylphosphonothioate (desbromo-
 leptophos) were prepared and their
 absolute configurations established by
 x-ray diffraction analysis of the (-)-a-
 methylbenzylammonium salt. The abso-
 lute configurations of the enantiomers
 of  leptophos and desbromoleptophos
 were assigned by relating them to the
 configurations of the corresponding O-
 methyl phenylphosphonothioic acids.
 The (R)p(+) isomers were  more acutely
 toxic to the housefly  and white mouse,
 while  the  (S)p(-) isomers were more
 delayed-neurotoxic to the hen
Effect of Impurities on the
Delayed Neurotoxicity of
Ethyl L ep toph os

  The delayed neurotoxicity of ethyl
leptophos, the effect of organophos-
phorus (OP) impurities in the technical-
grade material on delayed neurotoxicity,
the relationship  of dose and time on
delayed  neurotoxicity of  technical
versus purified material with multiple
doses, and the inherent  delayed neuro-
toxicity of  significant  OP impurities
were investigated.
  The delayed neurotoxicity of technical
and purified 0-(4-bromo-2,5-dichloro-
phenyl) 0-ethyl phenylphosphonothioate
administered orally to hens  was com-
pared under different conditions. The
technical material  was neurotoxic  at
750 mg/kg while  the purified compound
was neurotoxic at  1000-1250 mg/kg.
The'chemical composition of the tech-
nical material was analyzed for organo-
phosphate  contaminants. Impurities
present  as more than  0.1%  were
examined for delayed-neurotoxic activity.
O,O-Diethyl  phenylphosphonothioate,
its oxon analog, and O,O-diethyl 4-
chlorophenylphosphonothioate were 5-
10  times  more potent  as delayed
neurotoxins than  the parent compound.
These  impurities evidently  potentiate
the delayed neurotoxicity  of  ethyl
leptophos  and  may potentiate the
delayed neurotoxicity of other O-ethyl
phenylphosphonothioate pesticides as
Mode of Action of
Phosphoramidothioate Esters
  The mode of action of methamidophos
(O,S-dimethyl  phosphoroamidothioate)
was studied A poor anticholmesterase
agent,  it is yet highly toxic to animals,
which  die of typical cholmergic symp-
toms of poisoning. The  study was
designed to determine the nature of the
phosphorylatmg  moiety when electric
eel acetylcholinesterase (EEAChE) is
inhibited by methamidophos
  Separation of EEAChE from metham-
idophos was  first established using a
Sephadex G-25 column. Enzyme inhi-
bition  studies were  conducted  using
both [0-methyl  14C]- or [S-methyl 14C]-
methamidophos. Results provide strong
evidence for P-S bond cleavage  in the
reaction leading to the inhibition of
acetylcholinesterase by methamidophos

Gas Chromatography of
  The  development  of  a  novel gas
chromatographic column (surface-mod-
ified support), obtained by high-temper-
ature conditioning and subsequent
exhaustive methanol  extraction of 6%
Carbowax 20M on HCI-extracted Chro-
mosorb W, was applied to insecticides
and extended to other pesticides. An
evaluation of other support phases for
the gas Chromatography of pesticides
was also made.
  Forty-four stationary phases  were
prepared as surface-modified supports
and evaluated Several of these supports
exhibit  excellent characteristics for the
direct gas Chromatography of organo-
phosphorus and carbamate insecticides'
low bleed, very good peak symmetry and
resolution,  short retention  times at
relatively low temperatures, and  high
surface  mactivation with little decom-
position of labile compound.
  The most effective supports for both
phosphorothionates and phosphates
were prepared with AN  600,  Reoplex
400, SILAR-9CP, NPGS,  EGSP-Z, and
BDS These  columns are effective in
resolving fenthion, fenoxon, and their
sulfoxides and sulfones at  a single
isothermal temperature. The SILAR-
9CP column is  interesting because it
shows the greatest retention differences
between phosphorothionates and their
oxygen analogs  The NPGS  surface-
modified  support  is the  only support
examined so  far that successfully
chromatographs azinphosmethyl  oxon,
a compound which  pyrolyzes on-
column  above  205°C but requires an
 oven temperature of at least 185°C for
  Surface-modified  supports were
 evaluated for the direct gas Chromatog-
 raphy of 12 carbamate insecticides of
 diverse chemical structures and physical
 properties, and  for the temperature-
 programmed separation of impurities in
 their technical  products. Promising
 results were achieved with a 2-ft x 2-
 mm Nickel 200 column containing  a
 surface-modified Carbowax 1540 sup-
 port Effective isothermal separation of
 Isolan,  aldicarb, 11203,  MIPC,  and
 propoxur with sharp symmetrical peaks
 was obtained at column temperatures
 of 100-130°Cand an injection tempera-
 ture of 110°C. Less volatile carbamates
 carbofuran, mexacarbate, 9699, Pyrolan,
 and carbaryl produced broader, less
 symmetrical peaks at these  tempera-
 tures but,  except for carbaryl, which
 showed  increased  pyrolysis,  higher
 column temperatures appear promising
 for improving peak shape and symmetry.
 Temperature-programmed  Chromatog-
 raphy of the technical insecticides
 from 50 to 130°C at 10°C/mm appears
 promising for the separation of impuri-
 ties for further  identification studies
 Triton  X-305 and  EGSP-Z also show
 promise for the direct Chromatography
 of a number of carbamate insecticides

 Neurophysiological Studies
 on the Mode  of Action  of
  A series of neurophysiological studies
 on  the mode of  action of insecticides
 was conducted.  Pyrethroid insecticides
 were found to poison the nervous sys-
tem by acting at specific sites on neu-
 rons. On motor  nerves,  all poisoning
 was confined to presynaptic nerve term-
 mals, in  sensory nerves the sensory
 endings were poisoned.
  Picrotoxinin, a natural plant convul-
 sant, was toxic to  the desheathed
 housefly central nervous system (CNS),
 and was  much  less toxic to the intact
  A series of substituted /V,/V'-thiodi-
 carbamates was found to poison intact
 houseflies with the same latency from
 application to disruption of flight motor
 neuron  coordination  Evidence sug-
 gested that the sulfenylated carbamate
analogs were converted very rapidly at
the cuticle to the parent carbamate and
that this  conversion was not rate-
  A housefly  strain was obtained
 containing the kdr factor, which rendered
the strain resistant  to pyrethroid and

    DDT  insecticides.  The entire  nervous
    system of the kdr housefly was resistant
    to direct  perfusion of pyrethroids  and
    DDT. Both the central and peripheral
    nervous  systems were  refractory to
    compounds. In addition, the perineunal
    sheath on the CNS made little difference
    to bioassay responses whether intact or
    removed, and, therefore,  played no role
    in  the resistance.  An  experimental
    procedure was outlined which measures
    quantitatively the amount of kdr present
    in mosquitoes and houseflies.
      "Slow" and "fast"  motor  units of
    Dipteran  maggots  are  mediated by
    different neurotransmitters, thataspartic
    acid  mimics the "slow" neurotrans-
    mitter, and glutamic  acid the  "fast"
    neurotransmitter   However,  a  major
    problem was encountered with accepting
    any ammo acid as a neurotransmitter
    when  it  was  shown  that  normal,
    neurally-evoked synaptic transmissions
    occurred in  the presence of  slightly
    elevated calcium concentrations, which
    also  rendered the preparations com-
    pletely  insensitive to  perfused or
    iontophoretically-applied  asparate or
    glutamate under the same conditions.
      Attempts were  made  to distinguish
    between DDT and pyrethroid actions on
    the insect nervous system  A  list of
        criteria was reported which allowed
        analogs to be categorized as DDT-like or
          The consequences of insecticide
        poisoning were investigated by compar-
        ing the histological appearance of the
        thoracic ganglion of houseflies which
        had been treated with doses causing
        mortality  in  50% of a  population
        Lindane treatment was found to cause
        "vacuoles" in the thoracic ganglion that
        were correlated in number  with the
        amount of disability measured  behav-
        iorally. The disability type was correlated
        with the vacuolization site.
  Examination of CNS activity  in the
housefly was accomplished by recording
the comparing  motor unit activity
before,  during,  and after  poisoning.
Measurements  were  made without
dissection and during performance  of
uninhibited movement, the only restric-
tions being a tether and wires inserted
through the cuticle in a few sites on the
thorax. This approach allowed the first
unambiguous description of overt poi-
soning symptoms based on  nervous
           T. R. Fukuto,  R. B. March, and T. A. Miller are with University of California,
             Riverside. CA 92521
           William F. Durham is the EPA Project Officer (see below).
           The complete report, entitled "Chemistry and Mode of Action of Insecticides:
             Phase II," (Order No. PB 83-247 213; Cost: $20.50, subject to change) will be
             available only from:
                  National Technical Information Service
                  5285 Port Royal Road
                  Springfield,  VA 22161
                   Telephone: 703-487-4650
           The EPA Project Officer can be contacted at:
                  Health Effects Research Laboratory
                   U.S. Environmental Protection Agency
                  Research Triangle Park, NC 27711
United States
Environmental Protection
Center for Environmental Research
Cincinnati OH 45268
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