WHO/FOOD ADD./69.35



    Issued jointly by FAO and WHO

    The content of this document is the result of the deliberations of the
    Joint Meeting of the FAO Working Party of Experts and the WHO Expert
    Committee on Pesticide Residues, which met in Geneva, 9-16 December,



    Geneva, 1969



    Chemical name

         2,3-p-dioxanedithiol-S,S-bis-(O,O-diethyl phosphorodithioate)


         Delnav (R), AC 528



    Other information on identity and properties

    The technical product contains 70 per cent cis- and trans-
    isomers of phosphorodithioate at a 1:2 ratio. The remaining 30 per
    cent contains related compounds described by Arthur and Casida (1959)

    (a) ca. 10 per cent ethylphosphorothioates and

    (b) ca. 1 per cent bis (diethyoxyphosphinothioyl) disulfide

    (c) ca. 5 per cent 2-p-dioxenethiol-S-(O,O-diethylphos-phorodithioate)

         (diethylphosphorothioic acid and salts

    (d)  (diethylphosphorodithioic acid and salts
         (oxygen analogues of the principal component, and of (c) above


    Biochemical aspects

    Rats were treated orally with P32-labelled dioxathion which possessed
    a ratio of cis- to trans-isomers approximating that of the
    technical product. At levels of 1 and 5 g/kg over a 10-day period,
    metabolites occurred primarily in the urine and to a lesser extent in

    the faeces. More than 95 per cent of the radioactivity found in the
    urine was in the form of hydrolysis products. The hydrolytic products
    excreted in the urine during the first 12 hours after treatment with
    15 mg/kg were identified as diethyl phosphoric acid, 0,0-diethyl
    phosphorothionic and 0,0-diethyl phosphorodithioic acid. A similar
    group of metabolites was encountered after in vitro metabolism
    studies were made using rat liver slices. The presence of these
    hydrolytic metabolites indicates that enzymatic cleavage of the
    phosphorothiolate grouping occurs at the carbon-sulfur as well as at
    the phosphorus-sulfur bonds, along with considerable oxidation before
    and after hydrolysis. A daily dose of 5 mg/kg of dioxathion given
    orally to rats over seven consecutive days resulted in a maximum
    accumulation of 0.6 ppm in the fat. After 10 days' withdrawal the
    level in the fat fell to 0.1 ppm or lower. At daily doses of 10 mg/kg
    a level of 0.6 ppm was reached in the fat after three days, and this
    level held constant through continued feeding for 21 days. When rats
    were given an oral dose of dioxathion (25 mg/kg body-weight), the only
    tissues containing detectable dioxathion were: fat 0.34 ppm cis- and
    0.68 ppm trans-isomer; kidney 0.08 ppm cis- and 0.14 trans- and
    muscle 0.05 ppm cis- and 0.05 trans-. All other tissues contained
    less than 0.01 ppm dioxathion. The cis- and trans-isomers were
    stored in tissue to a greater extent than the other technical product
    components. In the biological systems studied the disulfide compound
    was the least stable of the radioactive components used. The
    cis- and trans-isomers were always similar in stability and much
    more stable than the dioxene component. The single exception was with
    human plasma where the dioxene was the most stable of the technical
    dioxathion components studied (Arthur and Casida, 1959)

    Dioxathion possesses in vitro anticholinesterase activity; it has
    a molar I50 of 7.6  10-7 for isolated beef erythrocyte
    cholinesterase. Oxidation, chemically, with bromine water, or,
    biologically, with fortified rat liver homogenates increased the in
    vitro activity approximately 20-fold. The enzyme system responsible
    for the activation was found in the supernatent layer of the liver
    homogenates following centrifugation (Frawley et al., 1963).

    Dioxathion is also an inhibitor of aliesterase and experiments
    relating to this property are described under "Special Studies, (c)

    As has been observed in other organo-phosphorus compounds, acute doses
    of dioxathion stimulate the pituitary-adrenal system and increase the
    production of adaptive liver enzymes. A single dose of 50 mg/kg
    administered intraperitoneally, to rats, produced, after 15 hours, a
    maximum average cholinesterase depression of 33 per cent of control
    which was accompanied by a four-to five-fold increase in hepatic
    alkaline phosphatase and tyrosine-alpha-ketoglutarate transaminase.
    Daily intraperitoneal injections of 15 mg/kg for 10 days reduced brain

    cholinesterase to 20 per cent of control and produced a significant,
    but much lower increase in hepatic adaptive enzymes than in the group
    receiving the single 50 mg/kg dose (Murphy, 1966).

    Acute toxicity

    Animal     Route   Solvent     mg/kg body-weight   References

    Mouse (M)  Oral    Maize oil          176          Frawley et
                                                       al., 1963

    Rat   (M)  Oral    Peanut oil         43           Gaines, 1960

    Rat   (F)  Oral    Peanut oil         23           Gaines, 1960

    Rat   (F)  i.p.    Ethanol-propylene  30           Frawley et
                       glycol                          al., 1963

    Dog        Oral    None              10-40         Frawley et
                                                       al., 1963

    The cis-isomer has approximately four times the toxicity of the
    trans-isomer (subcutaneous rat LD50: cis, 66-86 mg/kg; trans,
    230-290 mg/kg). The symptoms following acute exposure to dioxathion
    are typical of parasympathetic stimulation and death is usually
    preceded by clonic convulsions. The rate of onset of symptoms was
    observed to be slower than with some other organo-phosphorus
    compounds, presumably due to less rapid conversion to the oxygen
    analogue. Maximum inhibition of cholinesterase at all sources of the
    enzyme occurred within one hour following an intraperitoneal injection
    of 13 mg/kg given to rats, with a recovery time of from two to three
    weeks (Frawley et al., 1963).

    Short-term studies


    Groups, each of 25 male and 25 female rats, were fed 0, 1, 3, 10, 100
    and 500 ppm of dioxathion in their diet. Duration of the test diet was
    13 weeks, except that the animals fed 500 ppm were sacrificed after
    one week because of marked food refusal and loss of body-weight. In
    addition, groups of five male and five female rats were sacrificed at
    given intervals prior to termination of the 13-week feeding period,
    for determination of erythrocyte, plasma and brain cholinesterase
    activity. Of the rats fed 100 ppm, only the females showed minimal
    symptoms of parasympathetic stimulation. At all the lower doses no sex

    difference due to dioxathion could be observed with respect to any of
    the parameters considered. Marked brain, plasma and erythrocyte
    cholinesterase depression occurred at the 100 ppm level. At the 10 ppm
    level, brain cholinesterase was normal, but plasma and erythrocyte
    cholinesterase were significantly depressed. At the 3 ppm and 1 ppm
    levels, cholinesterase activity was normal in all tissues examined.
    Recovery at all levels was rapid for plasma and slow for brain and
    erythrocyte cholinesterase. Gross and histological examinations
    revealed no pathological changes in the animals fed 100 ppm or lower


    Dioxathion was administered to dogs, five days a week for a one- to
    two-week period, at dosages from 0.25 mg/kg to 8.0 mg/kg. Three of the
    four dogs given 8.0 mg/kg displayed the typical syndrome of
    parasympathetic stimulation, which gradually subsided after
    withdrawal, and all dogs were free of symptoms 10 days after the last
    dose. No such effects were evident in the dogs receiving doses below
    8.0 mg/kg. Plasma cholinesterase was inhibited at doses of 0.8 mg/kg
    and above and erythrocyte cholinesterase at doses of 2.5 mg/kg and
    above. Rapid recovery of plasma cholinesterase occurred, the level
    being normal two weeks after withdrawal, however there was only slow
    recovery of erythrocyte cholinesterase and it was still not complete
    after five weeks. Another group of dogs fed 0.075 mg/kg and lower
    doses, five days a week for 90 days showed no inhibition of either
    plasma or erythrocyte cholinesterase when periodic tests were made
    (Frawley et al., 1963).

    Special studies

    (a) Reproduction

    Rat. A three-generation reproduction study was conducted on groups
    of weanling rats fed 3 ppm and 10 ppm for 79 days before mating. No
    abnormal pathologic changes were found in any of the parental
    generations after 39 weeks. No adverse effect on reproductive
    performance, fertility, lactation or litter size was found at either
    level. The progeny were viable, normal in size and anatomical
    structure. Findings among all test animals, three parental generations
    and six litters of progeny, were comparable to control animals for all
    parameters (Kennedy et al., 1968).

    (b) Neurotoxicity

    Using TOCP (triorthocresyl phosphate) as a positive control, oral
    doses from 10 to 1000 mg/kg and subcutaneous doses from 25 to 200
    mg/kg of dioxathion were administered to a total of 75 mature hens.
    The hens treated with TOCP at 500 mg/kg developed typical neurological
    symptoms associated with myelin degeneration whereas the hens treated
    with dioxathion either died from the acutely toxic dose or recovered
    without development of the neurological symptoms (Frawley et al.,

    (c) Potentiation

    Four organo-phosphorus insecticides, including dioxathion, and one
    carbamate insecticide, carbaryl, were administered both individually
    and in combination to rats. Potentiation, based upon percentage
    mortality, occurred when malathion and dioxathion were administered
    simultaneously, but it was greatly enhanced when dioxathion was
    administered four hours before malathion. Dioxathion and carbaryl also
    potentiated each other, but only when an interval existed before the
    administration of the carbaryl (Hagan et al., 1961).

    To evaluate further the potentiation of dioxathion and malathion a
    subacute feeding study was made whereby dogs were administered oral
    doses of 0.1, 0.2 and 0.4 mg/kg of dioxathion or 0.2, 0.4 and 0.8
    mg/kg of malathion daily, for a pre-treatment period of six weeks. The
    dogs pre-treated with dioxathion were then given malathion and those
    pre-treated with malathion given dioxathion for an additional six
    weeks. When administered according to this schedule, dioxathion and
    malathion did not show any enhanced inhibition of plasma or
    erythrocyte cholinesterase (Zaratzian of al., 1961).

    In another experiment, dioxathion was fed to rats along with 14 other
    organo-phosphorus compounds and also with the carbamate, carbaryl. No
    significant potentiation, on the basis of the LD50 was observed when
    the compounds were administered simultaneously. However, when
    dioxathion was administered four hours prior to malathion, significant
    potentiation occurred (Frawley et al., 1963).

    A direct measurement of aliesterase activity was made, using diethyl
    succinate and tributyrin as substrates. Rats were fed dietary levels
    of 0, 0.2, 1, 5, and 25 ppm of dioxathion for given periods up to 13
    weeks. Four groups were sacrificed at separate intervals during the
    feeding period, and a fifth group was sacrificed four weeks after
    return to a normal diet. The rates of hydrolysis of the substrates
    were used as a measure of aliesterase activity in the liver and serum.
    Cholinesterase activity was also measured in these tissues. A dietary
    level of 1 ppm caused slight inhibition of aliesterase in the liver,
    whereas at 5 ppm no cholinesterase inhibition was evident. Similar
    difference in activity was found for serum. Maximum inhibition of
    aliesterase occurred early in the feeding period and complete recovery
    of activity had resulted by four weeks after return to a normal diet.
    These results indicate that aliesterase is much more sensitive to
    inhibition by dioxathion than is cholinesterase (DuBois et al., 1968).

    Observation in man

    Five female and five male adult human volunteers received 0.075
    mg/kg/day of dioxathion, orally for 28 days. Plasma and erythrocyte
    cholinesterase measurements were made at frequent intervals and no
    significant change from pre-treatment levels was found. After 28 days,
    the treatment was varied and during the following 28 days two subjects
    continued to receive 0.075 mg/kg/day, two received 0.150 mg/kg/day and
    the remaining six received 0.075 mg/kg/day of dioxathion along with

    0.150 mg/kg/day of malathion. Erythrocyte cholinesterase levels were
    not affected by any of these doses. Plasma cholinesterase was not
    affected by the continuing dose of 0.075 mg/kg/day but 10-20 per cent
    inhibition was observed in the two subjects which received the higher
    dose of 0.150 mg/kg/day. This depression was thought to be significant
    because during a 17-day post-treatment period the activity returned to
    pre-treatment level. Subjects receiving the combined
    dioxathion-malathion treatment did not appear to display any
    cholinesterase depression related to the experimental treatment. All
    clinical findings were similar to those observed in a control group of
    subjects which received placebos. Blood counts, coagulation times and
    prothrombin times were normal in all subjects (Frawley et al., 1963).


    The toxicological studies reported above have been conducted on
    technical dioxathion which contains 70 per cent dioxathion.
    Short-term studies in rats and dogs including reproduction studies in
    rats demonstrated that all test doses failed to produce any
    morphologic changes. Conventional two-year chronic toxicity studies
    have not been conducted on dioxathion. The toxic action of this
    compound is restricted to inhibition of cholinesterase enzymes. The
    reported studies prove similar susceptibility in man and dogs. The
    significance of aliesterase inhibition with regard to metabolism of
    some chemicals is apparent, and the higher sensitivity to aliesterase
    inhibition was taken into account when estimating the acceptable daily


    Level causing no significant toxicological effect

         Rat: 3 ppm in the diet, equivalent to 0.15 mg/kg per day 
         Dog: 0.075 mg/kg,per day 
         Man: 0.075 mg/kg per day

    Estimate of acceptable daily intake for man

         0-0.0015 mg/kg body-weight


    Use pattern

    Pre-harvest treatments

    The major uses of dioxathion as an agricultural insecticide are given
    in the following table. Dioxathion is formulated as an emulsifiable

                                              Rate of             Pre-harvest
    Crop                  Pest              application             interval
                                              (kg/ha)                (days)

    Citrus: grapefruit,   mites             0.03-0.06 kg/100 1         0
    oranges,              citrus thrips
    limes, lemons,

    apples,* pears,       mites             0.06 kg/100 1              7

    quinces               coddling moths    (max.7.56 kg/ha)
                          apple maggots

    grapes                mites, grape      0.95 kg/ha (spray)        14
                          leafhoppers       1.5 kg/ha (dust)

    *  Apple pomace from treated fruit should not be fed to dairy or
       meat animals.
    Dioxathion is applied to beef cattle, sheep, goats and swine in the
    form of sprays and dips of 0.15 per cent concentration and by
    backrubber. Use is not permitted on dairy animals. It is effective in
    controlling ticks, lice, horn flies, screw worms and sheep ked. Repeat
    applications can be made after 2-week intervals.

    Post-harvest treatments

    No use is known for application on stored products.

    Other uses

    In addition to the major uses listed above, dioxathion is used for
    controlling mites on walnuts, on stone fruit prior to fruit formation,
    ornamentals and beans grown for shelled dry beans.

    Residues resulting from supervised trials

    Data in the form of unpublished reports (Hercules, 1958-1961),
    retained at FAO headquarters in Rome, indicate residues likely to
    occur from dioxathion application to plants and animals. These data
    show the rate of decline of residues from various application rates
    including that representative of the use pattern (above). Unless
    otherwise stated the residues are expressed in terms of technical
    dioxathion (which contains 70 per cent cis- and trans-isomers) in
    contrast to United States and Canada tolerances which are for the
    total of cis- and trans-isomers.


    Residues in citrus, confined to the peel, are persistent with an
    indicated half-life of 75-100 days. No residues exceeding 0.03 ppm
    were detected in the pulp. Gunther et al. (1958) studied the deposit
    and persistence of dioxathion on field sprayed mature navel oranges
    and lemons in California. Maximum residues occurred from 2 to 21 days
    after application. Residues measured on the day of spraying were
    slightly lower, probably due to loss in handling. Data are summarized
    in the following table:

                  Rate of     Pre-harvest      Residue range
    Crop        application     interval       (at harvest)
                (kg/100 l)       (days)            (ppm)

                                             peel          fruit

    Navel       0.02          0-21           1.0-3.5       0.2-0.8
    oranges     0.04          0-21           3.8-4.8       0.8-1.1
                0.09          0-21           6.3-9.7       1.4-2.1

    temple      0.06          0-28           5.4-11.7      1.1-2.3

    lemons      0.02          0-28           4.6-9.0       1.4-2.7
                0.04          0-28           8.4-14.7      2.5-4.4
                0.09          0-28          13.2-23.4      4.0-7.0

    * Calculated on basis of average peel weights 22 per cent and
      30 per cent for oranges and lemons, respectively.


    Two spray studies were made in the United States of America (one in
    California and one in Delaware) to determine dioxathion residues in
    grapes. In the California study Thompson seedless grapes (varying from
    fruit ready for harvest to fruit two months prior to harvest) were
    treated with one application of dioxathion at 0.95 kg/ha. None of the
    resulting residues, even those on samples taken three hours after
    application, exceeded 0.4 ppm. Residues from double the application
    rate did not exceed 1 ppm. In the Delaware study grapes were treated
    with two applications at 0.95 kg/ha. The first application was made
    when small fruit was present and the second application was made eight
    weeks later, a few weeks prior to harvest. Residues from this study

    showed a rapid initial decrease from an average of 13.5 ppm three
    hours after the initial spraying to an estimated 2.0 ppm 10 days
    later. Thereafter the residues exhibited a high degree of persistence
    decreasing to an average of 1 ppm 56 days after the spray application.
    A second application on grapes which contained a 1 ppm residue from
    the first application resulted in an initial average residue of 5.3
    ppm which again rapidly declined in the first nine days to an
    estimated 2 ppm. Again this residue showed persistence during the
    remaining days until harvest, reaching a 1.8 ppm average at 21 days.
    At the recommended pre-harvest period of 14 days, the Delaware study
    showed a maximum residue of 2.4 ppm and an average residue of 2.0 ppm
    (technical dioxathion).

    Pome fruits

    Tests were conducted in Indiana, New Mexico and North Carolina on
    apples using five to eight cover sprays at various dosages. In apples,
    as with citrus, residues remained in the peel. Samples taken one week
    after the last application, where the maximum recommended use
    programme was followed (seven cover sprays using 0.06 kg/100 1) had
    the following residues.

         Location            Range          Average
                             (ppm)          (ppm)

         Indiana             4.3-4.9        4.6
         New Mexico          5.7-8.2        6.5
         North Carolina      5.6-6.6        6.0

    The samples taken above were picked one to five weeks prior to normal
    harvest time. Dioxathion residues are quite persistent, growth
    dilution being the primary cause of residue decline. The average
    half-life indicated in the three studies above is about 11 weeks.


    Studies conducted by the USDA and summarized below furnish information
    on the site of residue deposition, magnitude of residue, build-up and
    decline pattern, and metabolic fate of dioxathion in livestock.
    Chamberlain et al. (1960) treated steers with 32P-labelled technical
    dioxathion using a 16.2 mg/kg dermal application and 4.56 mg/kg oral
    administration. Plapp et al. (1960) gave steers an 8.8 mg/kg dermal
    application of 32P-labelled technical dioxathion. Studies on the
    deposition of residue in tissues were conducted by Jackson et al.
    (1962) using dermal applications of 0.15 per cent and 0.25 per cent

    In all studies the largest residue was found on the hair of the animal
    and to a lesser but still appreciable degree on the hide. The only
    significant accumulation of absorbed residue in animal tissue occurred

    in the fat with only traces (0.1 ppm or less) present in the liver,
    kidney and muscle. The residue in the fat reached a peak two to seven
    days after spraying and then declined to 0.1 ppm or less two to three
    weeks after spraying with the recommended 0.15 per cent spray. No
    accumulative residue build-up was noted in the fat of cattle after six
    applications with the 0.15 per cent spray following the recommended
    two-week interval between applications.

    The following table indicates the level of residue found in the fat
    from the three studies.

    Dermal application       Days after     Residues in fat
          rate               application         ppm

    0.15 per cent spray          2         0.37-0.95 (0.73 av.)

    0.25 per cent spray          2         0.91-1.15 (1.05 av.)

    8.8 mg/kg                     7         0.2

    16.2 mg/kg                    7         1.5

    The radio-labelled studies showed that the bulk of absorbed dioxathion
    was rapidly metabolized and that 10-20 per cent of that dermally
    applied was excreted in the urine in one week principally as diethyl
    phosphoric, diethyl thiophosphoric and diethyl dithiophosphoric acids.
    A higher percentage of the dose was eliminated in the urine and faeces
    of orally treated animals. The maximum radioactivity in the blood
    occurred at three hours in the dermally treated steer with a maximum
    blood cholinesterase inhibition of 32 per cent, whereas the orally
    treated steer exhibited a maximum cholinesterase depression of 83 per
    cent and a maximum absorption of radioactivity in the blood after 12

    The residue pattern in sheep, hogs and shorn goats is the same as for
    cattle with the following decreasing order of residues in the fat:
    cattle > shorn goats > sheep > hogs. Residues in the fat of hogs
    did not exceed 0.1 ppm even with exaggerated treatment. The maximum
    decrease in blood cholinesterase activity in sheep and goats (60-80
    per cent of normal) occurred two days after one dermal application
    but returned to normal in about one week. The depression in shorn
    goats was the most severe (20-40 per cent of normal, two days after
    application) and returned to about normal two weeks after the

    Three dairy cows were fed dioxathion daily for 28 days at a rate of
    0.33 to 0.4 mg/kg body-weight. This is the maximum amount which would
    occur in a diet of 50 per cent citrus pulp which had a residue of 23
    ppm. Residues of 0.06-0.12 ppm (0.08 ppm average) were found in the

    fat. It can therefore be estimated that the total residue in the fat
    of cattle from a combination of dermal treatment and ingestion of
    treated citrus pulp would not be likely to exceed 1.0 ppm based on the
    cis- and trans-isomers.

    No residues were found in the meat of cattle in any of the studies.

    Determinations for dioxathion in the milk of the dairy cows fed
    0.33-0.4 mg/kg body-weight daily for 28 days were negative with an
    analytical sensitivity of 0.01-0.02 ppm.

    In plants

    Casida and Ahmed (1959) studied the behaviour of 32P-labelled
    technical dioxathion components on lima bean, cabbage, cotton and
    tomato plants. They showed that the cis- and trans-isomers were
    the least susceptible components to volatization from plant surfaces.
    Hydrolysis of dioxathion components on plant surfaces did not occur
    readily. Hydrolysis that did occur was primarily with absorbed
    materials and in this case the cis- and trans-isomers hydrolyzed
    more slowly than the other components. The rapid hydrolysis of
    absorbed dioxathion components was further demonstrated with bean
    seedlings which readily absorbed radioactive components through the
    roots from water containing 40 ppm of each of the dioxathion
    components. After 1.5 days absorption time through the roots, the
    cis- and trans-isomers in the bean foliage were about 70 per cent

    The formation of more polar derivatives and more potent in vitro
    cholinesterase inhibitors does occur on exposure to sunlight or after
    application to plants. However, Casida and Ahmed (1959) found that the
    amounts of unhydrolyzed but more polar materials in aged plant
    residues were but a small fraction (10 per cent on cabbages, 1 per
    cent on beans) of the unchanged cis- and trans-isomers. The more
    polar non-hydrolyzed derivatives from the dioxene component formed
    more readily and disappeared more rapidly than the cis- and
    trans-isomer derivatives.

    Casida and Ahmed (1959) also found that the cis- to trans-isomer
    ratio (about 1:2) held nearly constant after application of technical
    dioxathion to plants. No conversion of one geometrical isomer to the
    other occurred nor did the cis- and trans-isomers on plants from
    any dioxene derivative.

    The confinement of dioxathion residue in citrus to the peel is
    associated with its solubility in the waxes and oils in that portion
    of the fruit. Any conversion of the cis- and trans-isomers to a
    metabolite must occur rather slowly since dioxathion disappears from
    the peel at a slow rate. No appreciable cholinesterase inhibiting
    material was detected in lemon pulp (edible portion) 71 days after an
    excessive 0.18 kg/100 1 spray treatment in a field trial.

    In animals

    The general metabolic pattern described for rats in the section
    entitled "Evaluation for Acceptable Daily Intake" appears to be
    similar for livestock. The only significant residue stored in cattle
    occurred in the fat with 75 per cent of the residue as the cis- and
    trans-isomer and 21 per cent as the dioxene fraction. The metabolic
    products in rat and cattle urine and faeces were similar following
    oral administration.

    Evidence of residues in food in commerce or at consumption

    Total diet and food survey data for a limited number of
    organo-phosphorus pesticide residues collected by the United States
    Food and Drug Administration were obtained using a GLC method which
    includes a Florisil column cleanup as described by Mills et al.
    (1963). Pardue and Watts (1968) found that a standard of dioxathion
    added to a Florisil column was not recovered in either the 6 per
    cent, 15 per cent or 50 per cent ethyl ether in petroleum ether
    eluate. Consequently, no applicable surveillance data are currently
    available for dioxathion. (See discussion below under "Methods of
    residue analysis".)

    In storage and processing

    Apples and pears are usually pared and cored before dehydration. Since
    dioxathion residues in these fruits are confined to the peel, no
    residue would be expected in the dried fruit.

    The solubility of dioxathion is such that no appreciable residue would
    be expected in fruit juice; however, small residues may result from
    physical carry-over.

    In the preparation of cattle feed from citrus, a 62 per cent residue
    loss was demonstrated in the drying operation. This plant scale test
    involved adding 4 ppm dioxathion to the press cake just prior to
    drying with combustion gas at 200-300F. An average residue of 22.6
    ppm was found in the finished product. A small scale laboratory test
    with lemon peel demonstrated an over-all residue loss of 42 per cent
    as a result of liming, pressing and mild drying at 122F.

    In a controlled experiment with dioxathion added to wet apple pomace,
    a 60 per cent residue loss occurred during drying in a commercial
    processing unit. No milk-residue studies have been carried out with a
    feeding level as high as that expected from the use of dried pomace
    from treated apples; therefore such feed should not be used for dairy
    or meat animals.

    Methods of residue analysis

    The analytical method described here is specific for the major
    components, the cis- and trans-isomers of

    2,3-p-dioxanedithiol-S,S-bis-(O,O-diethyl phosphorodithioate). These
    are the most persistent and abundant constituents and their
    determination is the most effective measurement of the toxic hazard of
    the residue. Unhydrolyzed metabolic conversion products or other
    components of the technical product which are excluded by the
    analytical procedure used have been absent or found only in small
    amounts in the vegetable and animal systems investigated and at a much
    lower level than the unchanged cis- and trans-isomers of
    dioxathion and completely hydrolyzed components.

    The method described by Dunn (1958) extracts the residue with hexane
    or isopropyl alcohol. Cleanup is achieved by use of an acid alumina
    column. Waxes are readily eluted with hexane and dioxathion is eluted
    with benzene. Final cleanup is done by partition chromatography on
    Celite 545 with a solvent pair such as acetonitrile and hexane.
    Dioxathion is determined by cleaving the molecule with mercuric
    chloride to yield 2,3-dichloro-p-dioxane as one of the reaction
    products. Hydrolysis of this product yields ethylene glycol and
    glyoxal, the latter being determined colorimetrically as glyoxal
    2,4-dinitrophenylosazone. The intensity of the colour formed in basic
    dimethyl formamide solution is measured at absorbance peak 614 m and
    the quantity estimated by comparison with a standard absorbance curve
    prepared from technical or pure dioxathion.

    The cleavage reaction is peculiar to thio acetals and thus is specific
    for the pesticide dioxathion as no other pesticide residues known are
    glyoxal precursors and the cleanup procedures exclude possible
    precursors in the sample material.

    The method is sensitive to 5 g. The following table indicates the
    result of method validation studies:

                    Fortification    Recovery      Blank
    Sample          level, ppm       (per cent.)   values

    Citrus peel     1-10             96-103        0.1

    Citrus pulp     0-03             100           0.008

    Apples          1-30             95-101        0.1

    Grapes          0.1-20           100           0.07 or less

    Animal fat      1.0              84-90         0.2 or less

    A multi-residue method for a large number of organo-phosphorus
    pesticides and alteration products is being developed by the United
    States Food and Drug Administration. It is anticipated that this

    procedure using a charcoal column cleanup prior to determination by
    GLC equipped with a KCl thermionic detector will soon be available for
    the practical accumulation of total diet and surveillance data on a
    large number of organo-phosphorus compounds. Watts et al. (1968a and
    b) described a charcoal column cleanup procedure and determined the
    recovery of about 60 organo-phosphorus pesticides and alteration
    products through this cleanup. In addition they investigated the
    applicability of three widely used GLC columns for determination of
    this large group of pesticides. The columns were packed with 80-100
    mesh, Gas Chrom Q coated with (a) 10 per cent DC 200, (b) 2 per cent
    diethylene glycol succinate (2 per cent DEGS) and (c) a 1:1 mixed
    column with 10 per cent DC 200 and 15 per cent QF1.

    They found that 0.1 ppm dioxathion added to an ethyl acetate extract
    of kale was 95 per cent recovered in the charcoal column cleanup. All
    three GLC columns were adequate for thermionic detection. The 2 per
    cent DEGS column gave the best sensitivity with 1.5 ng dioxathion
    needed for a 30-50 per cent scale deflection.

    National tolerances

    Expressed as the cis- and trans-isomers of
    2,3-p-dioxane-dithiol-S,S-bis-(O,O-diethyl phosphorodithioate).

    Country        Crop                               Tolerance

    Canada         apples, pears, quinces             4.9

                   citrus: grapefruit, oranges
                   lemons, limes, tangerines
                   tangelos                           2.5

                   grapes                             2.0

                   animals: beef cattle, sheep,
                   goats, swine                       1.0

    Netherlands    leaf and sprout vegetables,
                   fruit vegetables, pulses,
                   fruit including grapes             0.4

    United States  apples, pears, quinces             4.9
    of America
                   citrus: grapefruits, oranges
                   lemons, limes, tangerines
                   tangelos                           2.8

                   grapes                             2.1

    Country        Crop                               Tolerance
                   animals: beef cattle, sheep,
                   coats, swine                       1.0 (in fat)
    West           leaf and sprout vegetables,
    Germany        fruit vegetables, pulses,
                   fruit including grapes             0.4



    Dioxathion is a narrow spectrum insecticide and acaricide and its use
    in agriculture is limited to a small number of food commodities. If
    its use were to extend, a reappraisal of the recommendations would be
    necessary. Therefore, the recommendations for limits should be on a
    "temporary" basis.

    Although figures are available for residues at stated times after
    application, no data are available for losses during subsequent
    storage and processing, except for the dehydration of citrus peel in
    which case there is about a 60 per cent loss during drying.

    No data are available on the nature of the residues derived from the
    impurities in the technical product. No results are available from
    total diet studies or from surveillance of food in commerce.

    There are colorimetric analytical methods which are suitable for
    regulatory purposes and for development as referee methods.


    Temporary tolerances

    The following temporary tolerances, to be in effect until 1972, are to
    apply to raw agricultural products moving in commerce unless otherwise
    indicated. In the case of fruit and vegetables the tolerances should
    be applied as soon as practicable after harvest and in any event prior
    to actual retail to the public. In the case of commodities entering
    international trade, the tolerances should be applied by the importing
    country at the point of entry or as soon as practicable thereafter.

              Citrus              3.0

              Pome fruits         5.0

              Grapes              2.0

              Meat                1.0 (not to include poultry)

    These tolerances apply to the total of cis- and trans-isomers of
    the principal active ingredient.

    Further work or information

    Required before 30 June 1972

    1. Determination and identification of impurities.

    2. Data on the disappearance of residues during storage and
       processing, including residues from impurities in the technical

    3. Data on residue levels in raw agricultural products moving in

    4. Data on residue levels in total diet studies.


    1. Estimation of the effect on aliesterase activity in dogs.

    2. Long-term studies in rats.


    Arthur, B. W. and Casida, J. E., (1959) Biological activity and 
    metabolism of Hercules AC-528 components in rats and cockroaches. 
    J. Econ. Entomol., 52: 20-27

    Casida, J. E. and Ahmed, M. K. (1959) Mechanism of residue loss of 
    Hercules 528 components on plant foliage. J. Econ. Entomol., 52:

    Chamberlain, W. F., Gatterdam, P. E. and Hopkins, D. E. (1960) 
    Metabolism of P32-Delnav in cattle. J. Econ. Entomol., 53: 672-675

    DuBois, K. P., Kinoshita, F. K. and Frawley, J. P. (1968) Quantitative 
    measurement of aliesterase inhibition by EPN
    (0-ethyl-0-p-nitrophenyl phenylphosphonothioate) and Delnav.
    Toxicol. appl. Pharmacol. (In press)

    Dunn, C. L. (1958) Determination of 2,3-p-dioxanedithiol S,S-bis (0,0
    -diethyl phosphorodithioate). J. Agr. Food Chem., 6: 203-209

    Frawley, J. P., Weir, R., Tusing, T., DuBois, K. P. and Calandra, J.
    C. (1963) Toxicologic investigations on Delnav(R) Toxicol. appl.
    Pharmacol., 5: 605-624

    Gaines, T. B. (1960) The acute toxicity of pesticides to rats.
    Toxicol. appl. Pharmacol., 2: 88-99

    Gunther, F. A., Jeppson, L. R., Barkley, J. H., Elliott, L. M. and
    Blinn, R. C. (1958) Persistence of residues of 2,3-p-dioxanedithiol
    S,S-bis(0,0-diethyl phosphorodithioate) as an acaricide on and in
    mature lemons and oranges. J. Agr. and Food Chem., 6: 210-211

    Hagan, E. C., Jenner, R. M. and Fitzhugh, O. G. (1961) Acute oral 
    toxicity and potentiation studies with anticholinesterase compounds.
    Fed. Proc., 20: 432

    Hercules. (1958-1961) Hercules Powder Co. Inc. Unpublished reports on
    Delnav residues

    Jackson, J. B., Radeleff, R. D., Roberts, R. H. et al. (1962) Acute
    toxicity of Delnav and its residues in tissues of livestock. J. Econ.
    Entomol., 55: 669-702

    Kennedy, G., Frawley, J. P. and Calandra, J. C. (1968) Multigeneration
    reproduction study in rats fed Delnav, Herban and Toxaphene. Toxicol.
    appl. Pharmacol. (In press)

    Mills, P. A., Onley, J. H. and Gaither, R. A. (1963) Rapid method for
    chlorinated pesticide residues in nonfatty foods. J. Assoc. Offic.
    Agric. Chem., 46: 186-191

    Murphy, S. D. (1966) Response of adaptive liver enzymes to acute
    poisoning by organophosphate insecticides. Toxicol. appl. Pharmacol.,
    8: 266-276

    Pardue, J. R. and Watts, R. R. (1968) U.S. Food and Drug
    Administration. Private communication

    Plapp, F. W., Bigley, W. S. and Darrow, D. I. (1960) Studies on the
    metabolism and residues of P32-labelled Delnav in Hereford steer. 
    J. Econ. Entomol., 53: 60-64

    Watts, R. R., Storherr, R. W., Pardue, J. R. et al. (1968a) A widely
    applicable charcoal column cleanup method for organophosphorus
    pesticide residues in crop extracts. Presented at the 1968 annual
    meeting of the Association of Official Analytical Chemists,
    Washington, D.C., Oct. 14, 1968

    Watts, R. R. and Storherr, R. W. (1968b) Gas chromatographic
    determination of organophosphorus pesticide residues. General aspects
    of potassium thermionic detection, retention times and response data
    for three columns. Presented at the 1968 annual meeting of the
    Association of Official Analytical Chemists, Washington, D.C., Oct.
    14, 1968

    Zaratzian, V. L., Arnault, L. T., Michel, T. C. and Fitzhugh, O. G.
    (1961)  Effects of organic phosphates Delnav and Malathion in the dog.
    Fed. Proc., 20: 432

    See Also:
       Toxicological Abbreviations
       Dioxathion (WHO Pesticide Residues Series 2)