PESTICIDE RESIDUES IN FOOD - 1979
Sponsored jointly by FAO and WHO
Joint meeting of the
FAO Panel of Experts on Pesticide Residues
in Food and the Environment
WHO Expert Group on Pesticide Residues
Geneva, 3-12 December 1979
Edifenphos was evaluated by the 1976 Joint Meeting (FAO/WHO 1977) and
a temporary ADI and temporary MRLs were allocated.
Further studies were required on the hepatic involvement observed in
several animal species. Information was also desired with respect to
observations in man, the residues of edifenphos and its main
metabolites on rice in husk at harvest and a method of residue
analysis suitable for edifenphos together with its main metabolites.
Although all of these needs had not been met, additional data on acute
and short-term toxicity and on the residue aspects had become
available and they are evaluated in this monograph addendum.
EVALUATION FOR ACCEPTABLE DAILY INTAKE
Absorption, Distribution and Excretion
Edifenphos is rapidly absorbed, metabolized and eliminated from mice
and rats following oral administration within six hours after
administration. At dosage levels of 10 mg/kg in female rats and 20
mg/kg in mice and male rats, only 15-30% of the administered
radioactivity was detected in digestive organs. After 72 hours, only
trace amounts of radioactivity were found in the whole body. The
major quantity of radioactivity was excreted in the urine (75-90%) and
faeces (5-20%). Accumulation of edifenphos in essential organs can be
excluded as observed with data from single and multiple dose
administration (Ueyama, et al, 1978).
The main metabolite of the rat is ethyl hydrogen S-phenyl
phosphorothiolate (54-57%) and that of mice is dihydrogen S-phenyl
phosphorothiolate (31-42%). Diphenyl disulfide was found in faeces of
both species. No qualitative differences were found in the metabolic
pattern of edifenphos between males and females (Ueyama, et al.,
Effects on Enzymes and Other Biochemical Parameters
The cholinesterase inhibiting ability of malathion, fenitrothion, and
edifenphos was compared using both plasma (pseudo-cholinesterase) and
erythrocytes (acetylcholinesterase) from buffalo calves. Malathion
caused the greatest inhibition of erythrocyte cholinesterase, followed
by edifenphos and fenitrothion. For plasma cholinesterase, malathion
was again the strongest inhibitor followed by fenitrothion and
edifenphos (Malik, et al, 1978a).
Special Studies on Potentiation
Oral administration of single equitoxic doses (LD50) of edifenphos
with fenthion and "Bassa-Wirkstoff" to male rats did not result in
potentiation of the acute toxicity of the combination (Thyssen,
Simultaneous oral administration of edifenphos and azinphos-ethyl to
male rats revealed a greater than additive toxicity, suggesting a
potentiating effect with these two organophosphorus compounds
Short Term Studies
Water Buffalo (Babalus bubalis)
The effect of edifenphos on serum transaminase and alkaline
phosphatase activity was studied after repeated oral dosing of 4 or 8
mg/kg/day for 28 days. Doses of 4 mg/kg/day brought on anorexia,
depression, increased salivation, lacrimation and diarrhea, being
initiated between days 11 and 14. SGOT activity was elevated
significantly, and this increased activity was both dose- and
time-dependent. No significant changes were observed for SGPT and
alkaline phosphatase (Malik, et al, 1978b).
Additional information confirming the rapid absorption, metabolism and
elimination following oral administration have been evaluated.
However, the required information about the liver involvement
requested by a previous meeting has not been provided.
The meeting was informed that a carcinogenicity study with mice was
underway. While the data reviewed did not increase the concern
relating to edifenphos, the absence of the previously requested
information precluded the allocation of an ADI. However, the data
permitted extension of the existing temporary ADI.
Level Causing No Toxicological Effect.
Mouse: 10 ppm in the diet, equivalent to 1.53 mg/kg body weight
Dog: 20 ppm in the diet, equivalent to 0.58 mg/kg body weight
Rat: 5 ppm in the diet, equivalent to 0.25 mg/kg body weight
ESTIMATE OF TEMPORARY ACCEPTABLE DAILY INTAKE FOR MAN
0 - 0.003 mg/kg body weight
RESIDUES IN FOOD AND THEIR EVALUATION
RESIDUES RESULTING FROM SUPERVISED TRIALS
Edifenphos was applied either as emulsifiable concentrate (EC) or dust
(DP) to rice plants cultivated in five geographically different paddy
fields in Japan (Kamochi, 1979). The harvest rice grains were
separated into hulled rice, polished rice, bran, and chaff for
analysis. The residues of edifenphos in each part were determined by
gas chromatography utilizing a flame photometric detector. The
minimum detectable limits for this method were 0.005 mg/kg for hulled
and polished and 0.01 mg/kg for chaff and bran. Recoveries averaged
80-93% for the various rice parts analysed.
Application methods and cultivation conditions for the tests are shown
in Table 1. Table 2 shows the analytical results for concentration
and distribution of edifenphos in rice grains. Some edifenphos was
found in control samples, presumably due to spray drift. Residues
resulting from dust applications were about 40% of those resulting
from emulsifiable concentrate applications.
There was no confirmative relation between residue concentration and
the application frequency or waiting period after last application.
Most of the residue in rough rice was found in the chaff (98.4%) with
average values of 2.8 mg/kg for DP and 6.2 mg/kg for EC. Only 1.6% of
the total residue was found in the hulled rice with average values of
0.015 mg/kg for DP and 0.047 mg/kg for EC. The distribution of the
residue in hulled rice was 88.5% in the bran and 11.5% in the polished
rice. Average residue levels in the bran were 0.14 mg/kg for DP and
0.43 mg/kg for EC whereas in polished rice the levels were 0.005 mg/kg
for DP and 0.010 mg/kg for EC. In the case of EC application, the
range of edifenphos residues in polished rice was from <0.005 to
0.016 mg/kg. For DP application, the residue in polished rice was
scarcely detectable, <0.005 to 0.005 mg/kg.
FATE OF RESIDUES
In a study involving rats and mice, 35S-edifenphos was fed orally at
1/10th of the LD50 level (Ueyama, 1978). It was rapidly metabolized
and excreted, and only trace amounts of labelled materials were
detectable in whole body after 72 hours. The metabolites isolated
were similar to those found in the goat.
Table 1. Site, cultivation conditions and application methods for edifenphos trials on rice
Site Rice Applications Harvest Days from last appl.
(Prefecture) Variety Test Plot1 Dates (day/mo.) Date to harvest
Miyagi Sasanishiki DP (7/8, 14/8) 18/9 21
Monoo EC (21/8, 28/8 18/9 21
Miyagi Sasanishiki DP (8/7, 2/8, 9/8,) 15/9 21
Furukowa EC (18/8, 25/8) 15/9 21
Fukai Nihonbare DP 28/7, 21/8, 29/8 25/9 27
Sakai EC 31/7, 23/8, 31/8 26/9 26
Kochi Jukkoku DP 29/8, 5/9 9/10 21
Agawa EC 11/9, 18/9 9/10 21
Saga Reiho DP (4 applications)
1 DP: 2.5% dust, 40 kg/ha (a.i. 1 kg/ha)
EC: 30% Emulsifiable Concentrate, 1000 times dilution, 1.5 kg/ha (a.i. 0.45 kg/ha).
Table 2. Residual Concentration and Distribution of Edifenphos in Rice Grains (Kamochi, 1979)
Residual Concentration (ppm) and Distribution (%) of edifenphos
Site1 Test plot1 rough rice1 chaff hulled rice bran polished rice
ppm ppm %3 ppm %3 ppm %4
Miyagi DP 0.463 2.00 (97.2) 0.017 (2.8) 0.15 (100) <0.005 (0)
Monoo EC 1.67 7.10 (96.6) 0.074 (3.4) 0.56 (89.2) 0.010 (10.8)
Miyagi DP 0.070 0.38 (100) 0.005 ( 0 ) <0.01 (-) <0.005 (-)
Furukawa EC 0.068 0.37 (100) 0.005 ( 0 ) <0.01 (-) <0.005 (-)
Fukui DP 0.561 2.97 (98.4) 0.011 (1.6) 0.09 (67.2) 0.005 (32.8)
EC 0.839 4.00 (98.1) 0.020 (1.9) 0.22 (100) <0.005 (0)
Fochi DP 0.395 2.03 (96.5) 0.017 (3.5) 0.14 (100) <0.005 (0)
EC 1.54 7.74 (96.1) 0.074 (3.9) 0.42 (81.6) 0.016 (18.4)
Saga DP 1.30 6.44 (98.6) 0.023 (1.4) 0.32 (84.7) 0.005 (15.3)
EC 2.80 12.0 (98.3) 0.062 (1.7) 0.94 (84.9) 0.014 (15.1)
Average residue DP 0.558 2.76 0.015 0.14 <0.005
(ppm) EC 1.38 6.24 0.047 0.43 0.010
Average 100 98.43 1.63 1.33 0.23
distribution 88.54 11.54
1 As Table 1;
2 This item was calculated by summing up the other four items;
3 Percentage distribution of edifenphos against rough rice;
4 Percentage distribution of edifenphos against hulled rice.
Table 3. Residual concentrations (mg/kg) and distribution of three
metabolites of edifenphos in rice grain1
(Kamochi, 1979) (All samples of hulled rice contained <0.01 mg/kg or
any of the three metabolites)
Test plot2 Chaff Rice Grain3
diphenyl m-hydroxy p-hydroxy p-hydroxy
DP <0.02 <0.02 0.07 <0.02
EC <0.02 <0.02 0.24 0.05
DP <0.02 <0.02 <0.02 <0.02
EC <0.02 <0.02 <0.02 <0.02
DP <0.02 <0.02 0.10 0.02
EC <0.02 <0.02 0.13 0.03
DP <0.02 <0.02 0.14 0.03
EC <0.02 <0.02 0.40 0.08
DP <0.02 0.02 0.15 0.03
EC <0.02 0.04 0.44 0.09
Average DP <0.02 0.02 0.10* 0.02
EC <0.02 0.02 0.25* 0.05
1 Average of duplication;
2 Sites as Tables 1 and 2;
3 Rice grain = rough rice; weight ratios of hulled rice and chaff
are 80% and 20%, respectively
* Significance = 5%
In plants (rice)
In an experiment to determine the distribution and residue levels (if
any) of three presumed metabolites of edifenphos: (I) diphenyl
disulfide, (II) ethyl S-p-hydroxyphenyl S-phenyl phosphorodithiolate
(p-hydroxy edifenphos), and (III) ethyl S-m-hydroxy phenyl S-phenyl
phosphorodithiolate (m-hydroxy edifenphos) in the various parts of
rice grain (rough rice) from the five regions in Japan and treatment
programs shown in Table 1, samples were analysed using a
newly-developed method to be described later (Ueyama, 1979a). The
results of these analyses are shown in Table 3 and can be summarized
as follows: 1) the amounts if diphenyl disulfide and m-hydroxy
edifenphos in rough rice were less than the detectable limit, 2)
p-hydroxy edifenphos was found only in the chaff and was not found in
hulled rice. By comparing the residue levels of edifenphos (Table 2)
and p-hydroxy edifenphos in chaff it was found that a linear
relationship was obtained giving the formula Y = 0.037 × 0.007 (Y =
p-hydroxy, mg/kg and X = edifenphos, mg/kg) with a correlation co-
efficient r of 0.93. This result could have significance for the
regulatory analysis of rough rice, since 98% of the edifenphos residue
is also found in the chaff and therefore it would be possible to
calculate the total residue, edifenphos plus m-hydroxy edifenphos,
based on a determination of edifenphos alone in a multi-residue
Behaviour in soil
Absorption and translocation
The movement of 35S-labelled edifenphos was studied in three types of
soil. The vertical movement in the soil column differed with soil
type and the order of mobility was: sandy loam > alluvial clay loam
> volcanic ash loam. (Timme, 1977; Tomizawa, et al., 1976).
The degradation of edifenphos in alluvial soil was more rapid under
flooded than under non-flooded conditions (Rajaram and Sethunathan,
In studies with 35S-edifenphos it was found that the main degradation
products at the initial stage of soil metabolism were S,S,S-triphenyl
phosphorotrithioate, O,O-diethyl S-phenyl phosphorothioate, S-phenyl
phosphorothioate and diphenyl disulfide. The sulphur atom of
edifenphos was converted to sulfuric acid through diphenyldisulfide
and benezenesulfonic acid (Timme, 1977; Tomizawa et al, 1976).
Edifenphos was degraded by UV light in hexane, water and as a film,
cleavage of the P-S bond being the main reaction (Murai, 1977). The
products isolated were O-ethyl, S-phenyl phosphorothioic acid,
S-phenyl hydrogen phosphorothioic acid, ethyl and hydrogen phosphoric
acid. The phenylthio radicals were oxidized to sulfuric acid except
in hexane where diphenyl di- and mono-sulfide and benzene sulfinate
and sulfonate were isolated. No transesterification occurred.
METHODS OF RESIDUE ANALYSIS
An analytical method was developed for determining the three
organosoluble metabolites of edifenphos (1) diphenyl disulfide, (2)
p-hydroxy edifenphos, and (3) m-hydroxy edifenphos in rough rice
(Ueyama, 1979b). These metabolites were extracted overnight by 80%
acetone in water from hulled rice or chaff and the extracts
partitioned with dichloromethane. A second partition between
acetonitrile/hexane was needed for hydroxy edifenphos in hulled rice.
After a suitable cleanup procedure for all metabolites employing
Florisil column chromatography, the hydroxy metabolites were
derivatized with diazomethane and all compounds determined by gas
chromatography employing a flame photometric detector. The limit of
detection by this method was 0.01 mg/kg for hulled rice and 0.02 mg/kg
for chaff. Recoveries ranged from 64 to 86%. The method appears to
be quite suitable for regulatory analysis.
A very sophisticated method of analysis for the major aqueous
metabolite des-3-phenyl edifenphos in rice grains was developed
(Kurogochi, 1979). The procedure involves the use of stable isotope
dilution analysis by fortifying the sample with d5-des-S-phenyl
edifenphos at the initial blending step, methylation with
diazomethane, and measuring the residue by GC-MS with selected ion
current monitoring. The minimum detectable amount was 0.1 ng and
detection limits in hulled rice and chaff were 0.01 mg/kg and 0.05
mg/kg, respectively. Although the method would certainly seem
reliable and useful in the hands of a research chemist with proper
experience and equipment, its utility for routine regulatory analysis
NATIONAL MRLs REPORTED TO THE MEETING
The following information was received for edifenphos on rice (Bayer,
Country MRL, mg/kg Pre-harvest interval in days
Italy 0.05 60
Japan 0.1 21
In response to requests from the 1976 Meeting, information was
received on the residues of edifenphos and its main metabolites on
rice in husk at harvest and on a method of residue analysis suitable
for edifenphos together with its main metabolites.
Emulsifiable concentrate (diluted) and dust of edifenphos were applied
in accordance with good local practice to rice plants in paddy fields
at five geographic locations in Japan. After harvest the rice grains
were separated into hulled rice, chaff, polished rice, and bran for
analysis by gas chromatography using a flame photometric detector.
Residues of edifenphos in rough rice ranged from 0.068 to 2.80 mg/kg
with 98.4% of the residue distributed into the chaff. In hulled rice
the residues ranged from <0.005 to 0.074 mg/kg, while in polished
rice the residues ranged from <0.005 to 0.016 mg/kg. Further
analysis for the three main metabolites of edifenphos revealed that
only the p-hydroxy edifenphos could be detected in rough rice, ranging
from <0.02 to 0.09 mg/kg and concentrated almost entirely in the
chaff. These results indicate a need to increase the maximum residue
limit for rice (in husk) from 0.2 to 5 mg/kg for rice (hulled) from
0.05 to 0.1 mg/kg, and for rice (polished) from 0.01 to 0.02 mg/kg.
Data from analytical experiments on rice grains from the five
geographic locations in Japan, mentioned above, showed that field
incurred residues of the metabolites diphenyl disulfide and m-hydroxy
edifenphos were below the detectable limit (<0.02 mg/kg) in rough
rice whereas p-hydroxy edifenphos could be found mainly in the chaff
at levels ranging from <0.02 to 0.44 mg/kg (<0.02 to 0.09 mg/kg in
rough rice). A linear relationship obeying the equation Y= 0.037 ×
0.007, r = 0.93 was found between residue levels of edifenphos and
p-hydroxy edifenphos in chaff.
Additional information on the fate of 35S-edifenphos fed orally to
mice and rats at 1/10th of the LD50 showed that the compound was
rapidly metabolized and excreted within 72 hours.
A gas chromatographic method of residue analysis suitable for
edifenphos and its main metabolites diphenyldisulfide, m-hydroxy
edifenphos, and p-hydroxy edifenphos in rice grains was developed. The
method is acceptable for regulatory purposes and probably could be
adopted or modified for use on other cereal and vegetable food crops
if the need arises.
A method of analysis for the major aqueous metabolite des-S-phenyl
edifenphos was also developed, but is primarily of research or
academic interest since it requires the use of stable isotope dilution
techniques and GC-selected ion current monitoring mass spectroscopy.
The temporary residue limits on rice (in husk), rice (hulled) and rice
(polished) recommended by the 1976 Meeting are increased as follows.
The limits refer to the parent compound.
Preharvest interval on which
Commodity Limit (mg/kg) recommendation is based (days)
Rice (in husk) 5 21
Rice (hulled) 0.1 21
Rice (polished) 0.02 21
FURTHER WORK OR INFORMATION
Required by June 1981:
1. Further studies to examine the hepatic involvement observed in
several animal species;
2. Results of the carcinogenicity study currently in progress.
1. Observations on man relative to occupational exposure;
2. Information on residues of edifenphos and its p-hydroxy metabolite
in food animals arising from the use of rice straw and bran in
Kamochi, Sachiko, Ueyama, Isao and Talsase, Iwao. Determination of
residues of edifenphos (HINOSAN(R)) in rice grains. (1979) Report No.
1096 (RA), Nihon Tokushu Noyaku Seizo K.K., Japan
Kurogochi, Shin, Ueyama, Isao, Kamochi, Sachiko and Takase, Iwao.
Determination of ethyl-S-phenyl hydrogen phosphorothiolate by gas
chromatography selected ion current monitoring (GC-SICM) coupled with
stable isotope dilution analysis. (1979) Report No. 1107(R), Nihon
Tokushu Noyaku Seizo K.K., Japan.
Malik, J.K., Gupta, R.C. and Paul, B.A. - In Vitro study on the
Comparative Inhibitory Effect of Malathion, Sumithion, and Hinosan on
Blood Cholinesterase on Bubalus bubalis. (Reviewed in summary
only). Indian J. Exp. Biol. 16: 496-497.
Malik, J.K., Garg, B.D., Verma, S.P. and Ahmad, A. - Serum
Transaminases and Alkaline Phosphatase Activities During Subacute
Toxicity of Hinosan in Bubalus Bubalis. (Reviewed in summary
only). Indian J. Exp. Biol., 16: 497-499.
Murai, T. Agr. Biol. Chem. 41: 71 - 1977, abstracted in report of
IUPAC Commission on Pest. Chemistry, 1979.
Thyssen, J. Untersuchungen zur Kombinationstoxizatät von difenphos,
enthion und Bassa-Wirkstoff. (1977a) Unpublished report from Instituut
für Toxikologie, submitted by Bayer, AG.
Untersuchungen zur Kombinationstoxizatät von Azinphos-Athyl und
edifenphos. (1977b) Unpublished report from Instituut für Toxikologie,
submitted by Bayer, AG.
Timme, G. - Hinosan metabolic fate behaviour in the environment.
(1977) Unpublished report Pflanzenschutz Anwendungstechnik Biologische
Forschung, submitted to the WHO by Bayer, AG.
Tomizawa, C., Uesugi, Y., Ueyama, I., Yamamoto, H. - Movement and
metabolism of S-benzyl O,O-diisopropyl phosphorothiolate (Kitazin P)
and O-ethyl S,S-diphenyl phosphorodithiolate (edifenphos) in various
types of soil. J. Environ. Sci. Health, 11: 231-251. (Cited by Timme,
Ueyama, I., Takase, I., Tomizawa, C. - Metabolism of Edifenphos
(O-ethyl Diphenyl Phosphorodithiolate) in Mouse and Rat. Agric. Biol.
Chem., 42: 885-887.
Ueyama, Isao, Kamochi, Sachiko and Takase, Iwao. Results of the
residual determination in hulled rice and chaff. (1979a) Report No.
1108(RA), Nihon Tokushu Noyaku Sezio K.K., Japan.
Determination of diphenyl disulfide and p- and m-hydroxy edifephos
by gas chromatography (FPD). (1979b) Report No. 1106 (RA), Nihon
Tokushu Noyaku Seizo K.K., Japan.