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    PESTICIDE RESIDUES IN FOOD - 1981


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    EVALUATIONS 1981







    Food and Agriculture Organization of the United Nations
    Rome

    FAO PLANT PRODUCTION AND PROTECTION PAPER 42

    pesticide residues in food:
    1981 evaluations

     the monographs

    data and recommendations
    of the joint meeting
    of the
    FAO panel of experts on pesticide residues
    in food and the environment
    and the
    WHO expert group on pesticide residues

    Geneva, 23 November-2 December 1981

    FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS
    Rome 1982

    ISOFENPHOS

    IDENTITY

    Common name:             isofenphos

    IUPAC chemical name:     O-ethyl- 0-2-isopropoxy-carbonylphenyl
                             isopropylphosphoramidothioate

    Synonyms: C.A. name:     1-methylethyl 2-[ethoxy [1-methylethyl)-
                             amino] phosphinothionyl] oxy] benzoates BAY
                             SRA 12869, OFTANOLR, AMAZER

    Structural formula:

    CHEMICAL STRUCTURE 1

    Other information on identity and properties

    Empirical formula:       C15H24NO4PS

    Molecular weight:        345.4

    Appearance:              colourless oil (pure active ingredient)
                             yellow-brown liquid (technical active
                             ingredient)

    Boiling point:           not distillable (pure active ingredient)
                             at and above 200°C decomposition (techn.
                             a.i.)

    Vapour pressure:         4 × 10-6mbar at 20°C (pure active
                             ingredient)

    Specific gravity:        1.134 at 20°C
                                        
                                       4°

    Solubility:              water 0.002 (pure active ingredient)
    (g/100 g solvent
    at 20°C)                 cyclohecanone            > 60
                             isopropyl alcohol        > 60
                             methylene chloride       > 60
                             ligroin (80 -110°C)      > 60
                             toluene                  > 60

    Minimum degree of
    purity:                  88%

    DATA FOR ESTIMATION OF ACCEPTABLE DAILY INTAKE

    BIOCHEMICAL ASPECTS

    Absorption, distribution, elimination and biotransformation

    Rat

         After a single oral dose of 15 mg/kg bw of ring-uL-(14C)-
    isofenphos, male and female rats excreted 88% of the administered
    radioactivity in the urine and 4% in the faeces within 72h of dosing.
    No 14CO2 was detectable in the expired air. The rate of elimination
    during the first 24 h appeared to be slower in females than in males,
    but the total percentage of administered radioactive dose recovered in
    the excreta of both sexes in the 72h post-treatment period was
    comparable. The major urinary metabolite identified was isopropyl
    salicylate (in conjugated and non-conjugated forms), which accounts
    for 52% of the 14C-dose given to the animals. Other metabolites found
    in the urine included conjugated and unconjugated O-hydroxyhippuric
    acid, salicylic acid and deaminated isofenphos oxygen analogue, which
    together amounts to approximately another 10% of the administered
    radioactivity. There was no significant sex difference in metabolites,
    either qualitatively or quantitatively (Shaw  et al 1977).

         Female rats were intubated with ring uL(14C)-isofenphos at
    15 mg/kg bw/day for 6 consecutive days. Radioactive residues in the
    tissues analysed (liver, fat, kidney and muscle) remained fairly
    constant during the dosing period and declined sharply upon cessation
    of treatment. At the end of an 8-day withdrawal period, the kidney was
    the only tissue still containing small amounts of 14C-residues. In
    the rats sacrificed 1 1/2 h after the sixth dose, unchanged
    isofenphos, isofenphos oxygen analogue, isopropyl salicylate and
    O-hydroxyhippuric acid were detected in all four tissues examined. The
    major identified component in liver, kidney and fat was unchanged
    isofenphos and in muscle was isopropyl salicylate. Deaminated
    isofenphos oxygen analogue was also found in liver, kidney and muscle
    (Strankowski  et al 1977a).

    Pig

         A male pig was fed ring-uL-(14C)-isofenphos-treated feed pellets
    at a rate of 2 mg/kg bw/day for 5 consecutive days. Consumption of the
    treated feed was monitored to ensure complete intake of the daily
    dose. Within 24h of the first dose, 81% of the administered 14C was
    eliminated in the urine and 14% in the faeces. Of the total
    radioactivity administered during the 5-day dosing period, 56% was
    recovered in the urine and 21% in the faeces. At sacrifice 3h after
    treatment on day 5 of the study, the kidney had the highest
    14C-residue level and the brain had the lowest, as compared to 5
    other tissues (liver, heart, bacon, ham and fat) examined.

         A total of 76% 14C-residue in a composite urine sample was
    identified. The urinary metabolites included conjugated and
    unconjugated isopropyl salicylate (55%), deaminated isofenphos oxygen
    analogue glucuronide (10%), salicylic acid (5%), O-hydroxyhippuric
    acid (4%) and cyclic isofenphos (2%). Unchanged isofenphos, found in
    all 4 tissues (liver, kidney, muscle, fat) analysed was the primary
    component identified in muscle and fat. Isopropyl salicylate
    (conjugated and unconjugated) was the major metabolite in liver and
    kidney. Other tissue metabolites detected included deaminated
    isofenphos oxygen analogue O-hydroxyhippuric acid, isofenphos oxygen
    analogue and des N-isopropyl isofenphos (Strankowski  et al 1977b).

    Cow

         In a lactating dairy cow treated acutely, via bolus, with ring-uL
    (14C)-isofenphos at 0.2 mg/kg bw, 14C concentration in the blood
    plasma peaked 2h after the dose and then dropped sharply to one fourth
    of the maximum level within 24h of treatment. Approximately 90% of the
    administered radioactive dose was eliminated in the urine and 4% in
    the faeces within 48 h of dosing. Less than 1% of the 14C dose was
    recovered in the milk. The major metabolite in both urine and milk was
    O-hydroxyhippuric acid. Other metabolites identified in urine and milk
    were salicylic acid, isopropyl salicylate and deaminated isofenphos
    oxygen analogue. In urine and the milk, about 78% and 66% respectively
    of the radioactive residues were identified (Strankowski and Murphy
    1977a).

         A lactating cow receiving, via bolus, 0.2 mg/kg bw/day of ring-
    uL(14C)-isofenphos for 5 days had excreted 63% of the total
    administered radioactivity in the urine at sacrifice 2 h after the
    fifth dose. No 14C residues were indicated in the report to
    "accumulate" in the milk. Of the 11 tissues analysed for radioactive
    tissues at sacrifice, liver and kidney were the only tissues found to
    have significant radioactivity. The major metabolite in liver and
    kidney was salicylic acid, both conjugated and unconjugated. Other
    metabolites identified in both tissues were isofenphos oxygen analogue
    and conjugated and non-conjugated forms of O-hydroxyhippuric acid and
    isopropyl salicylate (Strankowski and Murphy 1977b).

         Figure 1 presents the proposed metabolic pathways for isofenphos
    in mammals (rat, pig and cow) (Shaw  et al 1977; Strankowski  et al 
    1977b; Strankowski and Murphy 1977a).

    Hen

         In hens orally treated with ring-uL(14C)-isofenphos at 4 mg/kg
    bw/day for 3 consecutive days, 41% of the total administered dose was
    stated to be eliminated in the excreta "within 51 h" presumably of the
    last dose. The major components found in the excreta were isofenphos
    and isopropyl salicylate. The total organphosphate residue in tissues
    and eggs accounted for less than 3% of the total dose administered. In
    the tissues and eggs, isofenphos was also the major compound
    identified, with isopropyl salicylate, isofenphos oxygen analogue and
    des N-isopropyl isofenphos being the minor metabolites (Kurtz and Shaw
    II 1977).

    Fish

         Channel catfish were exposed continuously to ring-uL (14C)-
    isofenphos in water at a concentration of about 10 ppb for 28 days.
    During the exposure period, the 14C residues accumulated to a peak of
    approximately 75 times the level of the water in 7 days and slowly
    declined thereafter. About 88% of the total 14C-residues in the whole
    fish could be extracted by acetonitrile, and the non-edible portion of
    the fish contained 76% of the extractable residues. One hundred
    percent of these 14C-residues were identified as unchanged
    isofenphos. Upon treatment withdrawal, approximately 63% of the
    accumulated 14C-residues were excreted within 5 h and 87% within one
    day. Ten days after discontinuation of exposure only 4% of the
    accumulated radioactivity still remained in the fish (Nelson and Roney
    1977).

    Effects on enzymes and other biochemical parameters

         The 50% depression of cholinesterase activity in serum,
    erythrocyte and brain of male rats by isofenphos ("technically pure
    grade") was determined in vitro. The I50 values were: serum:
    8.2 × 10-4 mole, erythrocyte: 3.1 × 10-4 mole, and brain:
    2.87 × 10-4 mole (Solmecke and Kimmerle 1972b).

         Groups of 5 male rats were given acute oral doses of isofenphos
    at levels ranging from 0.5 to 35 mg/kg bw. Activity of plasma and
    erythrocyte cholinesterase in the males was depressed (20 to 81%), in
    a dose-dependent pattern, above 0.5 mg/kg bw at both 2 and 24 h post-
    treatment, with maximum inhibition being noted at 2 h. Plasma
    cholinesterase was no longer affected at any dosage level 3 days after
    dosing, while erythrocyte cholinesterase was still inhibited by 26% at
    35 mg/kg bw 7 days after treatment. In female rats, a single oral dose
    of isofenphos at 2.5 mg/kg bw but not at 0.5 mg/kg bw caused a

    FIGURE 1

    reduction in erythrocyte cholinesterase level by 20% at 2 h post
    dosing but not thereafter. Plasma cholinesterase was not affected.
    Male rats sacrificed 24h after acute oral doses of isofenphos above
    1 mg/kg bw displayed a dose-related decrease (23-74%) in activity of
    brain cholinesterase (Solmecke and Kimmerle 1972b).

         Groups of 5 male and 5 female rats (Wistar II strain of
    unspecified age) were intubated acutely with the oxygen analogue of
    isofenphos at 0.1, 0.25, 0.5, 1.0, 2.5 or 5 mg/kg bw (males) or at
    0.25, 0.5 or 1 mg/kg bw (females). Cholinesterase in plasma and
    erythrocytes was assayed at 2, 5, 24, 48h and 7 days (in males only)
    post treatment. No control groups were included in the experiment and
    the pre-treatment cholinesterase activities of individual groups were
    used as the basis of comparison. In males, there was a depression
    (>20%), generally dose-dependent, of plasma cholinesterase at
    0.5 mg/kg bw and above and of erythrocyte cholinesterase at 2.5 mg/kg
    bw and above after 2, 5 and 24 h. (Males at 1 mg/kg bw showed a
    decrease (29%) of erythrocyte cholinesterase after a single interval
    of 5 h.) Complete or almost complete recovery to pre-treatment values
    was seen after 48 h for plasma cholinesterase and after 7 days for
    erythrocyte cholinesterase. In females, both plasma and erythrocyte
    cholinesterase was inhibited (20 to 43%) at 0.25 mg/kg bw and above
    during the first 24 h. Activities of cholinesterase in plasma and
    erythrocyte were close to the pre-treatment values 48 h after dosing.
    The inhibitory effect of the oxygen analogue on plasma and erythrocyte
    cholinesterase appeared to peak about 2 or 5 h after treatment in both
    sexes (Thyssen 1974a).

         Groups of 5 male and 5 female rats were orally treated with
    oxygen analogue of isofenphos and then sacrificed 24 h later for
    determination of brain cholinesterase activity. It was stated (no data
    were provided) that brain cholinesterase activity in males was
    moderately depressed at both 10 and 20 mg/kg bw but was unaffected at
    5 mg/kg bw. In the case of females, brain cholinesterase activity was
    stated to be within physiological ranges at dosages ranging from 1 to
    5 mg/kg bw (Thyssen 1974a).

    TOXICOLOGICAL STUDIES

    Acute toxicity

         In experiments with mice, rats, hamsters and rabbits exposed in
    inhalation chambers to isofenphos (dissolved in a mixture of alcohol
    and polyethylene glycol 400) as a spray at 30-minute intervals during
    a 4-h period, rats and hamsters were found to be more sensitive than
    mice and rabbits as shown in Table 1 (Solmecke and Kimmerle 1972c).

        TABLE 1.  Acute toxicity of isofenphos in animals
                                                                                                                           
    Species             Sex       Route               Vehicle        LD50
                                                                     (mg/kg bw)          Reference
                                                                                                                           

    Mouse               M         Oral                PG             127                 Solmecke and Kimmerle 1972c
                        F                                            91.3
                        M         Inhalation          Ethanol &      > 0.272 mg/l        Solmecke and Kimmerle 1972c
                                  (4-hour             PG (1:1)
                                  exposure)

    Rat                 M         Oral                PG             38.7-45 )           Solmecke and Kimmerle 1972c
                        F                                            28 - 32 )           Lamb et al 1977
                        M         Oral                Water &
                                                      Cremophor EL   33 - 48.2           Flucke 1980a;Flucke 1981
                        M         Oral                "              19.9                Flucke 1980b

                        M         I.P.                PG             35.8                Solmecke and Kimmerle 1972c
                        F                                            29.5

                        M         Dermal              None           >1000 µl/kg bw)     Solmecke and Kimmerle 1972c
                                  (4-hour
                                  exposure)           None

                        M         Dermal(24-h)        None           705 µl/kg bw        Kimmerle 1972a

                                  Dermal              None           188 µl/kg bw        Solmecke and Kimmerle 1972a
                                  (7-day
                                  exposure)           (undiluted)

                        M&F       Inhalation          Ethanol &      >1300 mg/l air      Kimmerle 1976
                                  (1-hour             PG (1:1)
                                  exposure)

                        M         Inhalation          Ethanol &      0.21 mg/l air       Solmecke and Kimmerle
                        F         (4-hour             PG (1.1)       0.144 mg/l air      1972c
                                  exposure)
                                                                                                                           

    TABLE 1.  (con't)
                                                                                                                           

    Species             Sex       Route               Vehicle        LD50
                                                                     (mg/kg bw)          Reference
                                                                                                                           

    Hamster             M         Inhalation          Ethanol &      0.23 mg/l air       Solmecke and Kimmerle
                                  (4-hour             PG (1:1)                           1972c
                                  exposure)

    Rabbit              F         Oral                PG             approx. 150         Solmecke and Kimmerle 1972c

                        M         Dermal              Ethanol &      162                 Nelson and Burke 1977a
                        F         (24-hour            PG (1:1)       315
                                  exposure)

    Dog                 F         Oral                PG             > 25                Solmecke and Kimmerle 1972c

    Bobwhite quail      M&F       Oral                PG             8.7                 Lamb and Burke 1979

    Mallard duck        M         Oral                PG             36.0                Nelson and Burke 1977b
                        F                                            32.0

    Quail               M         Oral                Water &        10                  Thyssen 1978
                                                      Cremophor EL   10

    Starling                      Oral                Corn oil       972                 Ross et al 1976

    Hen                 F         Oral                Water &        16                  Thyssen 1978
                                                      Cremophor EL
                                                                                                                           

    PG - polyethylene glycol 400.
        Signs of oral poisoning in mammals

         Cholinergic symptoms (e.g. muscle twitching, breathing disorders)
    induced by toxic acute oral doses of isofenphos were typical of those
    of cholinesterase inhibitors and were similar in mice, rats and
    rabbits. Toxic signs usually developed within 2 h of dosing in mice
    and rats and 4 h in rabbits and lasted for 2 to 10 days in survivors.
    Mortalities from lethal doses usually occurred 1 to 4 days after
    treatment. Cholinergic signs were not observed in dogs treated at
    25 mg/kg bw, the top dosage level tested (Solmecke and Kimmerle
    1972c).

    Short-term studies

    Rat

         Groups of 15 male and 15 female rats (SPF Wistar II strain of
    unspecified age) were orally treated by gavage with isofenphos ("pure
    technical grade") in polyethylene glycol 400 at 0, 0.1, 0.25, 1.0 or
    2.5 mg/kg bw/day for 30 days. Mortality, behaviour, growth, organ
    weights and liver and kidney function were unaffected. Dose-related
    inhibition (>20%) of cholinesterase in plasma and erythrocytes was
    apparent at and above 1 mg/kg bw in females at 9, 16 and 30 days of
    the study. In males, depression (dose related, >20%) of erythrocyte
    cholinesterase was noted at both 1 and 2.5 mg/kg bw at 16 and 30 days
    and of plasma cholinesterase at and above 1 mg/kg bw at 16 days and at
    2.5 mg/kg bw also at 9 and 30 days (Solmecke and Kimmerle 1972a).

         Groups of 10 male and 10 female rats (SPF Wistar W74 strain, 8
    weeks old) were fed isofenphos (91.9% pure) in the diet at the dosage
    levels of 0, 0.3, 1.0 or 3 ppm for 4 weeks. There was no mortality.
    Behaviour, body weight and food consumption were not significantly
    different between control and treated animals. Determinations of blood
    cholinesterase activity after 3, 7, 10, 14, 17, 21, 24 and 28 days of
    dietary feeding indicated dose-related inhibition (>20%) of plasma
    cholinesterase in females at 1 ppm after 7, 17 and 28 days and at
    3 ppm at all intervals. Only a slight depression (13-16%) of plasma
    cholinesterase was noted in males even at 3 ppm. Activity of
    erythrocyte cholinesterase determined over the course of the study and
    of brain cholinesterase measured terminally was not affected in all
    treated groups (both sexes). The no-effect level on plasma
    cholinesterase was 0.3 ppm, which was equivalent to 0.024 mg/kg bw,
    according to body weight and food consumption data (Löser 1978).

    Groups of 15 male and 15 female rats (SPF Wistar strain, 28 to 32 days
    old) were fed isofenphos (86.6% pure) in their diet at levels of 0.5,
    1, 5, 25 or 125 ppm for 3 months. The control group consisted of 30
    males and 30 females. One male at 125 ppm died after 9 days, due to
    pneumonia. Toxic signs, such as salivation and muscle twitching, were
    observed in animals receiving 125 ppm during the first 2 weeks of

    feeding. Body weight was adversely affected in the highest dosage
    group (both sexes). Food consumption was comparable to controls.
    Haematological and biochemical studies and urinalysis conducted after
    one and three months of the study indicated no significant dose-
    related findings other than an increase in SGOT in males at 125 ppm at
    the 1-month interval. Measurements of cholinesterase activity after 1,
    4, 8 and 13 weeks of feeding revealed depression (dose-related >20%)
    of the enzyme in both plasma and erythrocytes in females at 5 ppm and
    above and in males at 25 ppm and above at all intervals. (Males at
    5 ppm showed a 29% decrease of plasma cholinesterase level after one
    week of feeding, but not thereafter.) The brain cholinesterase level
    determined terminally was reduced at 25 ppm and above in both sexes.
    At sacrifice upon termination of the study, an increase in absolute
    weight and organ/body weight ratio of thymus was seen in females at
    125 ppm. There were also variations in absolute weights of several
    organs in males of the top dosage group. No compound-induced
    histopathological alterations were observed in a variety of tissues
    evaluated, including the thymus (Löser 1973; Urwin and Newman 1973).

    Dog

         Groups of dogs (4 males and 4 females per group, 5 months old)
    were fed isofenphos (of unspecified purity) in the diet daily at 0,
    0.3, 1.0, 10 or 30 ppm for 3 months. There were no treatment-related
    effects on mortality, clinical symptoms, reflexes, body weight, food
    consumption, ophthalmoscopic, haematological, clinical chemistry and
    urinalysis parameters. Cholinesterase in plasma and erythrocytes was
    depressed (by >20%), in a dose-dependent manner, at and above
    10 ppm in both sexes after 3, 6 and 13 weeks of the study. (Plasma
    cholinesterase in these dosage groups was inhibited also after 1 week
    of the study.) At the conclusion of the study, absolute weight and
    organ/body weight ratio of the liver was elevated at 10 ppm and
    above in males (non-dose related) and females (dose related).
    Histopathological examination of about 30 tissues, including the
    liver, from each dog revealed no abnormalities or variations
    associated with inclusion of isofenphos in the diet. The no-effect
    level for the study was 1.0 ppm (Murmann 1973; Thomson and Newman
    1973).

         Groups of dogs (4 males and 4 females per group) were fed a daily
    diet containing the following levels of isofenphos (89.3% pure) in a
    2-year study: Control: 0 ppm; Group 1: males - 3 ppm from week 1
    to week 83 and 2 ppm from week 84 to week 104, and females - 3 ppm
    from week 1 to week 104; Group 2: 15 ppm throughout the 104 weeks,
    and Group 3: 75 ppm from week 1 to week 53, 150 ppm from week 54 to
    week 99 and 300 ppm from week 100 to week 104. No compound-related
    findings were noted with respect to general condition (reflexes, body
    temperature and pulse rate), water intake, ophthalmoscopic and
    urinalysis parameters. Assay of cholinesterase 12 times over the
    course of the study indicated dose-dependent inhibition (>20%) of

    plasma cholinesterase in Groups 2 and 3 (both sexes) and of
    erythrocyte cholinesterase in Group 3 at all intervals. Depression
    (approx. or equal to 20%) of plasma cholinesterase was also found in
    Group 1 males at weeks 7, 39, 66 and 79 but was not evident at weeks
    92 and 103 after the dietary level was reduced from 3 ppm to 2 ppm at
    week 84. Animals of Group 3 also showed depression (>60%) of brain
    cholinesterase activity measured terminally.

         Adverse effects on parameters other than cholinesterase activity
    in plasma and erythrocytes were observed only in Group 3. In this
    group, one male died at week 104 and another male was sacrificed
    terminally in moribund condition. Toxic symptoms, such as salivation,
    vomiting, diarrhoea, weakness in the hind extremities, unsteady gait,
    etc., began to appear after 78 weeks and increased in severity with
    time. The symptoms were more marked in males than in females.
    Depression of body weight gain (both sexes) and of food consumption
    (females) was obvious after 100 weeks. An increase in the SAP level at
    several intervals during the study and a reduction of the terminal
    serum A/G ratio were observed in males and females. The one male that
    died at 104 weeks showed significant changes in several haematological
    parameters. At terminal sacrifice, elevated organ/body weight ratios
    of lung, liver, kidney, prostate, brain and pituitary and depressed
    organ/body weight ratios of adrenal and testis were detected in the
    males but, except in the testis, microscopic abnormalities of the
    tissues in question were not noted. Histopathologically, hypoplasia of
    the testis and erosion of esophageal mucosa, partially accompanied by
    cellular reaction of propria mucosa, were observed in several males of
    Group 3 but none in the other groups, including the control. Two
    animals (Group 3) also exhibited foci of softening ("degeneration
    processes") in the brain stem with a degree of severity greater than
    the one case seen in the control group. The no-effect level in the
    study was 2 ppm (Hoffman and Kaliner 1977).

    Hen and quail

         In adult hens and female quail intubated with isofenphos as an
    emulsion in water and polyethylene glycol 400 daily for 5 consecutive
    days, and then observed for 14 additional days, the 5 day LD50 was
    found to be 6 mg/kg bw/day and 5 mg/kg bw/day respectively. Major
    symptoms observed were inactivity in both species, ruffled feathers
    and breathing disorders in the quail (Thyssen 1978).

    Bobwhite quail and mallard duck

         The dietary LC50 in bobwhite quail and mallard ducks fed
    isofenphos in their diet for 5 days, followed by untreated diet for
    3 days, was determined to be 145 ppm (Nelson and Burke 1977) and
    >1000 ppm respectively (Nelson and Burke 1977c; Lamb and Burke 1977).

    Rabbit - dermal

         Groups of 6 male and 6 female rabbits (3 males and 3 females per
    group with intact skin and the remainder per group with abraded skin)
    were exposed dermally to isofenphos (as an emulsion in water and
    Cremophor EL) at 0, 1 or 5 mg/kg bw/day 7 h per day, 5 times per week
    for a total of 15 applications in a 21-day period. The treated sites,
    not covered with bandages, were washed with soap and water after each
    daily exposure period. One male at 5 mg/kg bw died of a cause
    unrelated to treatment after 10 applications. There were no toxic
    signs or adverse effects on body weight or on terminal haematological
    and clinical chemistry parameters. Local irritation on treated sites,
    generally transitory in nature and characterized by erythema on intact
    skin and erythema, oedema and eschar formation on abraded skin, were
    seen in some animals at 5 mg/kg bw. Changes in weights of several
    organs were seen at 5 mg/kg bw without any accompanying
    histopathological changes. Plasma and erythrocyte cholinesterase
    activities measured after the 10th and 15th exposures and brain
    cholinesterase level determined terminally in both sexes were
    significantly depressed at 5 mg/kg bw (Thyssen and Kaliner 1977).

    Rat - inhalation

         In male and female rats exposed to isofenphos as an aerosol (of
    unspecified particle sizes) 4h per day for 5 days, the LC50 was
    >0.055 mg/l in males and 0.029-0.055 mg/l in females ((Solmecke and
    Kimmerle 1972c).

         Groups of 10 male and 10 female rats were exposed to an aerosol
    of isofenphos at 0, 0.72 or 2.93 mg/m3 air 6h per day for 5
    consecutive days per week over a 3-week period. (No information was
    given on the particle size of the aerosol.) The treated animals were
    not different from the controls as judged by general condition, body
    weight gain, terminal haematological values, kidney and liver
    function, gross pathological changes and organ weights. Cholinesterase
    in plasma and erythrocytes was inhibited (>20%) at 2.93 mg/m3 air at
    each weekly determination (Solmecke and Kimmerle 1972a).

    Long-term studies

    Mouse

         Groups of 40 male and 40 female mice (6 to 7 weeks old, SPF NMRI
    strain) were fed diets containing technical isofenphos (89.3% pure) at
    0, 1, 10 or 100 ppm for 108 weeks. Additionally, each group included 3
    sub-groups of 10 males and 10 females. One sub-group, fed the treated
    diet for 108 weeks, was used for haematological tests and urinalysis
    every 3 months over the course of the study. Another sub-group
    maintained on dietary feedings for 9 months, was subjected to liver

    function tests (plasma alkaline phosphatase and GPT) every 3 months.
    The third sub-group was given isofenphos in the diet for 24 weeks and
    activity of cholinesterase in plasma and erythrocyte was measured
    after 2, 4, 8, 12 and 24 weeks and in brain after 24 weeks. All
    animals that died during the study or were sacrificed terminally were
    subjected to gross and histopathological examination. Mortality, which
    was high in all groups, including the control, especially in the
    females, was not attributed to treatment. At the end of the study,
    only 42 to 57% of the males and 12 to 20% of the females per group
    were still alive. Nevertheless, 53 to 73% of females per group
    survived at least 80 weeks. There were no significant differences
    between control and treated groups in clinical symptoms, body weight
    and food consumption. Data on differential white blood cell counts
    showed an increase at 100 ppm of eosinophil (males) and segmented cell
    counts (females) after 21 months and of segmented cell counts in males
    after 24 months. Liver function tests and urinalysis parameters were
    normal. Dose-related inhibition (72 to 94%) of plasma cholinesterase
    occurred in both sexes at and above 10 ppm at all intervals.
    Erythrocyte cholinesterase was unaffected in the treated groups, with
    depression consistently below 20%. Terminal brain cholinesterase
    activity was reduced at 100 ppm by 46% in males and 31% in females. At
    terminal sacrifice, absolute weight of lung in males was significantly
    elevated at 100 ppm but no concomitant histological lesion of the
    particular tissue was evident. An apparent dose-related increase, as
    compared to concurrent controls, in frequency of mild gastritis (in
    glandular mucosa of the stomach) was observed in males of all treated
    groups which was, however, not statistically significant, especially
    in the case of the 1 ppm and 10 ppm groups. No histopathological
    changes related to treatment was observed in a variety of other
    tissues evaluated.

         Based on the tumour data, isofenphos was not carcinogenic in the
    mouse, as indicated by the absence of a dose-related increase in
    incidence of a) any particular type of tumours, b) animals with
    tumours, c) animals with multiple tumours, and d) animals with
    malignant tumours. The time of appearance of tumours was comparable
    between control and treated groups.

         The no-effect level for the study was 1 ppm (Brune  et al. 
    1978).

    Rat

         Groups of 50 male and 50 female rats (SPF Wistar strain, 28 to 32
    days old) were fed diets containing isofenphos (of unspecified purity)
    at 1, 10 or 100 ppm for 2 years. The control group consisted of 100
    males and 100 females. Mortality was unaffected by treatment, and
    between 69 and 90% of animals per group were still alive at the end of
    the study. At 100 ppm, cholinergic signs were seen during the first
    week and depression of growth in both sexes and of food consumption in

    males was apparent throughout the feeding period. Haematological and
    biochemical studies and urinalysis conducted at five intervals over
    the course of the study failed to reveal any consistent effect related
    to presence of isofenphos in the diet. Assay of cholinesterase after
    1, 2, 4, 13, 26, 52, 78 and 105 weeks of the study indicated dose-
    related inhibition (>20%) of both plasm and erythrocyte
    cholinesterase at 10 ppm and above in both sexes at all intervals and
    of plasma cholinesterase in males at 1 ppm after 4 and 78 weeks. The
    extent of depression of cholinesterase in plasma and erythrocytes
    remained fairly constant over the course of the study. At the
    conclusion of the experiment, brain cholinesterase activity was
    reduced at 100 ppm in both sexes. Variations in absolute weights of
    several organs were seen at 100 ppm but histological changes of the
    particular organs were absent. Except for an apparent increase in
    incidence of ulceration of the forestomach in females at 100 ppm,
    as compared to the concurrent controls, no treatment-related
    alterations were evident in a set of about 30 tissues examined
    histopathologically. Analysis of tumour data revealed no indications
    suggestive of carcinogenic activity of isofenphos under the conditions
    of the experiment.

         The study demonstrated 1 ppm as a marginal no-effect level with
    respect to plasma cholinesterase. On other parameters monitored,
    10 ppm was without any significant adverse effect (Bomhard and Löser
    1977).

    Special studies on reproduction

         Groups of 20 male and 20 female weanling rats (SPF CFY strain)
    were fed dietary levels of technical isofenphos (of unspecified
    purity) at 0, 1, 10 or 100 ppm for 60 days prior to mating to initiate
    a reproduction study originally designed to cover 3 generations with
    one litter per generation. Three litters (F1a, F2a and F3a) were
    obtained as expected for control, 1 ppm and 10 ppm groups. In the case
    of the highest dosage group, owing to poor survival of pups, there
    were insufficient F1a weanlings to form an adequate second parental
    generation (F1). F0 animals at 100 ppm, as well as those of other
    groups including the control, were therefore re-mated. This second
    mating again failed to provide sufficient F1b offspring at 100 ppm as
    potential parents. In an attempt to determine the causes of the
    initial adverse effects at 100 ppm, F0 animals of control and top
    dosage groups were allowed two additional matings, with the fourth
    mating using treated males and untreated females and vice-versa. In
    the third mating trial, the number of litters (F1c) at 100 ppm
    obtained was too small to permit any meaningful interpretation of
    results. Based on results of the fourth mating, fertility appeared to
    be adversely affected, primarily in the females. This summary will
    refer only to data on control, 1 ppm and 10 ppm groups.

         In parental generations, there were no mortality and clinical
    symptoms attributable to treatment. Body weight gain during pre-
    mating, gestation and lactation periods, mating performance, duration
    of gestation and terminal gross pathological findings were not
    significantly different from controls. Pregnancy rate was 90%, 70% and
    60% respectively in control, 1 ppm and 10 ppm groups in the F0
    generation at the second mating suggesting a slight effect at 10 ppm
    and a marginal effect at 1 ppm. (The pregnancy rate at 100 ppm was 40%
    for the second mating trial.) Assay of plasma and erythrocyte
    cholinesterase in F0 animals after 37 weeks of dietary feeding
    revealed inhibition (>20%) at 10 ppm of erythrocyte cholinesterase in
    both sexes and of plasma cholinesterase in females. Over the three
    progeny generations (F1a, F2a and F3a litters), litter size, pup
    mortality and mean pup weight from birth through lactation to weaning
    were comparable to controls at both 1 and 10 ppm. In F1b litters, pup
    weight at 10 ppm seemed to be slightly decreased at days 12 and 21.
    Pups observed grossly at weaning displayed no apparent compound-
    induced abnormalities. The available data tend to indicate 1 ppm as a
    marginal no-effect level with respect to reproductive capability
    (Palmer et at 1977).

    Special studies on teratogenicity

    Rat

         Groups of 20 to 21 pregnant rats (Long Evans FB30 strain) were
    intubated with isofenphos (86.6% pure) in polyethylene glycol 400 at
    doses of 0, 0.3, 1 or 3 mg/kg bw/day from gestation day 6 to day 15.
    The dams were sacrificed on gestation day 20 and foetuses were removed
    by caesarean section for external, visceral and skeletal examination.

         No treatment-related mortality or abnormal behavioural reactions
    were noted, and maternal body weight was normal during the dosing
    period. An increase in the average number of resorbed foetuses per
    litter occurred at 3 mg/kg bw, but a dose-related elevation in the
    percentage of litters with rasorptions was not apparent at this dosage
    level. There were no significant differences between control and
    treated groups with respect to fertility rate, mean number of
    implantation sites, mean litter size, foetal weight, placental weight
    and incidence of stunted foetuses. The frequency of foetuses showing
    slight bone variations and/or malformation was very low and not
    significantly different from that in the control group (Machemer
    1972).

         Groups of 21 to 23 pregnant rats (Long Evans FB30 strain) were
    exposed dermally to isofenphos (dissolved in polyethylene glycol 400)
    at 0, 0.3, 1.0, 3 or 10 mg/kg bw/day for 5 h per day from gestation
    day 6 to day 15. The treated (clipped) sites were washed with soap
    solution after each daily exposure period. Animals' necks were

    immobilized. On day 20 of pregnancy, the dams were sacrificed and
    foetuses were removed by caesarean section. A decrease in maternal
    body weight gain during and after treatment period was observed at
    10 mg/kg bw. Dams at this level also showed an increased incidence of
    ruffled fur. There was a slight increase in the mean number of
    resorptions per pregnant dam at 10 mg/kg bw, but the percentage of
    pregnant dams with resorbed foetus(es) was not significantly elevated.
    No treatment-related effects were observed on other tested parameters,
    viz. fertility rate, mean number of implantations, mean litter size,
    foetal weight, placental weight, frequency of stunted foetuses and
    incidence of skeletal variations. A number of foetal malformations
    were seen in all groups, including the control, but they were
    generally non-dose-related. The only abnormalities observed at
    10 mg/kg bw, but not in the lower dosage groups or controls, were
    multiple defects manifested as hypoplasia of telencephalon and
    unspecified eye malformation in 3 foetuses from a single litter. These
    particular findings were not believed to be compound induced. Under
    conditions of the experiment, isofenphos was not teratogenic, although
    a dosage level of 10 mg/kg bw was maternally toxic (Schlüter 1981).

         Groups of 22 to 25 pregnant rats (Long Evans FB30 strain) were
    exposed in an inhalation chamber to an aerosol of isofenphos
    (formulated with a 1:1 mixture of polyethylene glycol 400 and ethanol)
    at actual chamber concentrations of 0, 0.25, 0.75 or 3.1 mg/m3 air,
    for 6 h per day from day 6 through day 15 of gestation. More than 97%
    of the particles counted were of sizes within respirable range in the
    rat. On day 20 of gestation, the dams were sacrificed and the foetuses
    were removed for gross, skeletal and visceral examination. No maternal
    toxicity was observed. Pregnancy rate, mean number of implantations,
    mean number of resorptions, mean litter size, foetal weight and
    incidence of stunted foetuses were not adversely affected. An increase
    in placental weight was noted at 3.1 mg/m3 air, which was of doubtful
    biological significance and unlikely to be treatment-related in view
    of the absence of any embryotoxic activity. There was no dose-related
    effect on the incidence of skeletal and visceral malformations. Based
    on the data, isofenphos was not teratogenic under the conditions of
    the experiment (Schlüter and Thyssen 1981).

    Rabbit

         Groups of pregnant (Himalayan) rabbits (11 to 13 rabbits per
    group) were orally treated with isofenphos at 0, 1, 2 or 5 mg/kg
    bw/day from gestation day 6 through day 18. The females were
    sacrificed on gestation day 29 and uterine contents were examined.
    Foetuses were subjected to evaluation for external, skeletal and
    internal malformations.

         Compound-related mortalities and toxic symptoms (diarrhoea) were
    seen in does at 5 mg/kg bw. A marked decrease (non-dose-related) in
    maternal body weight gain during the treatment period occurred in all

    treated groups. One of the 13 does aborted at 2 mg/kg bw. There were
    no significant differences between control and treated groups in
    pregnancy rate, litter size, mean number of implantations, mean number
    of resorptions, sex ratio, foetal weight, placental weight and
    incidence of stunted foetuses. "Slight bone variations" were not
    indicated to occur in any foetuses from control or treated groups. The
    only malformation observed in the study was indicated to be
    arthrogryphosis of both front extremities in a foetus at the dose
    level of 5 mg/kg bw (Machemer 1975).

    Special studies on mutagenicity

         In two similar but separate studies, isofenphos was evaluated for
    its mutagenic activity using two microbial assay systems (rec-assay
    and reversion test). Indicator micro-organisms used in the former were
     Bacillus subtilis strains N1G17, N1G45, H 17 and M 45. For the
    latter,  Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA
    98 and TA 100 and  Escherichia coli strain WP2 hcr were employed.
    The reversion test was conducted in the presence or absence of a
    mammalian metabolic activation system (S-9 homogenates of liver from
    rats or mice induced by phenobarbital or Aroclor 1254) while the
    rec-assay was carried out without the activation preparation. Under
    the conditions of the experiments, isofenphos was demonstrated not to
    be mutagenic at concentrations up to 0.02 ml/disc of undilated
    isofenphos and 5000 µg/plate (Shirasu  et al 1980) or 300 µg/disc
    and 1000 µg/plate (Inukai and Iyatomi 1977), respectively, in the
    rec-assay and the reversion test. Mutagenic responses were obtained
    with several positive control compounds, such as mitomycin C,
    9-amino-acridine HCl, furylfuramide, acetylaminofluorene and
    beta-propiolactone (Inukai and Iyatomi 1977; Shirasu  et al 1980).

         Another Ames test conducted with  S. typhimurium strains
    TA 100, TA 1537, and TA 98, with or without the S-9 mix and using
    concentrations as high as 3150 µg/plate, also confirmed the non-
    mutagenicity of isofenphos (Oesch 1977).

         In a dominant lethal assay, groups of 20 male NMRI mice (10 weeks
    old) were given by gavage a single dose of 0 or 15 mg/kg bw of
    isofenphos. Each male was then mated with three untreated female mice,
    which were replaced weekly for 8 consecutive weeks. The females were
    sacrificed around the 14th day of gestation or 14 days after
    separation from the males and the uteri were removed for examination.
    The treated males exhibited no signs of toxicity. There were no
    significant differences between the control and the treated groups
    with respect to fertility rate, living implants and dead implants.
    Pre-implantation loss (no. of corpora lutea and no. of implantations)
    was significantly increased at 15 mg/kg bw during the first week after
    dosing, but not thereafter. It was indicated in the report that the
    values for pre-implantation loss observed in the treated group were
    "completely within the norm of the strain" (Machemer 1973).

    Special studies on carcinogenicity

         See under "Long-term studies".

    Special studies on neurotoxicity

    Hen

         Groups generally comprising 10 adult hens (15 to 18 months old)
    were given acute oral or intraperitoneal doses of technical
    isofenphos. The oral and intraperitoneal LD50 was found to be
    21 mg/kg bw and 11.4 mg/kg bw respectively. No delayed neurotoxic
    signs were observed in any of the treated animals during a 6-week
    post-exposure observation period. Pre-treatment of hens with atropine
    (50 mg/kg bw) intraperitoneally elevated oral LD50 of isofenphos to
    74 mg/kg bw. Five hens surviving an oral dose of 20 mg/kg bw of
    isofenphos and 6 atropinized hens that were still alive after
    receiving 74 mg/kg bw of the insecticide were sacrificed 21 days after
    dosing for histopathological examination of the brain, spinal cord and
    peripheral nerve. These hens showed no compound-related morphological
    change or variation from normal in any of the nervous tissues
    examined. Hens treated with an oral dose of TOCP at 350 mg/kg bw
    displayed typical neurotoxic signs and showed degenerative nerve
    fibres in the spinal cord and the sciatic nerve (Kimmerle 1972; Cherry
     et al 1972).

    Special studies on antidotes

         In 2 separate experiments, atropine sulphate (50 mg/kg bw), 2-PAM
    (50 mg/kg bw) or Toxogonin (20 mg/kg bw) was given singly or in
    combination (atropine plus PAM or Toxogonin) i.p. to male rats: a) 45
    to 120 minutes after single oral toxic doses of isofenphos (and before
    onset of severe toxic symptoms), and b) 5 minutes after acute oral
    toxic doses of the oxygen analogue of isofenphos and before onset of
    toxic symptoms. Results indicated that, under the conditions of the
    experiments, none of the three compounds tested was effective as an
    antidote for isofenphos or its oxygen analogue (Solmecke and Kimmerle
    1972b, Thyssen 1974a).

         Simultaneous administration of atropine sulphate at 100 mg/kg bw
    and obidoxime at 20 mg/kg bw to male rats i.p. after onset of toxic
    symptoms (at 25 minutes) resulting from acute oral toxic doses of
    isofenphos elevated LD50 of the insecticide from 43.1 mg/kg bw to
    84.2 mg/kg bw. No data were provided on the effect of the antidotes on
    the toxic signs (Kimmerle and Gröning 1975).

    Special studies on potentiation

         When the acute oral LD50 values in male rats, determined
    experimentally for equitoxic mixtures of isofenphos and malathion or
    of isofenphos and EPN, were compared with the expected LD50 values
    for the mixtures, the acute toxicity of isofenphos was found to be
    potentiated by malathion. An additive effect resulted when isofenphos
    was combined with EPN (Solmecke and Kimmerle 1972b).

         In further studies with male rats, it was demonstrated with
    isofenphos did not potentiate the acute oral toxicity of phenamiphos
    or phoxim (both organophorous insecticides) (Thyssen 1976; Thyssen
    1977).

    Special studies on the acute toxicity of the oxygen analogue

    Rat

         The LD50 of the oxygen analogue of isofenphos was determined in
    6 female and 6 male rats and is reported in Table 2. Thyssen and
    Kimmerle 1974).

        TABLE 2.  Acute toxicity of the oxygen analogue of isofenphos in rats
                                                                                         

    Sex       Route               Vehicle                       LD50
                                                                (mg/kg bw)
                                                                                         

    M         Oral                Water & Cremophor EL          30.8
    F                                                           16.1

    M         i.p.                Water & Cremophor EL          15.9
    F                                                           7.8

    M         Dermal              None                          117.6 µl/kg bw
    F         (24 h)              (undiluted)                   25 µl/kg bw

    M         Dermal              Water & Cremophor EL          97.9
    F         (24 h)                                            29.5

    M         Inhalation          Ethanol & polyethylene        >353 mg/m3 air
              (1 h)               glycol 400 (1:1)
    F                                                           approx. 353 mg/m3 air

    M         Inhalation          Ethanol & polyethylene        >195 mg/m3 air
              (4 h)               glycol 400 (1:1)
    F                                                           63.8 - 131 mg/m3 air
                                                                                         
    
    Signs of oral poisoning

         Acutely toxic doses of the oxygen analogue of isofenphos to
    rats produced cholinergic symptoms typical of those seen with
    cholinesterase inhibitors. These toxic signs occurred within 1 h of
    dosing and persisted for a maximum of 2 days in survivors. Deaths from
    lethal doses were observed from about 30 minutes to 3 days after
    treatment (Thyssen and Kimmerle 1974).

         The acute LD50 of the oxygen analogue of isofenphos in hens was
    >10 <25 mg/kg bw by the oral route and about 10 mg/kg bw i.p. No
    neurotoxic signs were observed in any of the treated hens over a
    28-day post exposure observation period (Thyssen 1974c).

    Special studies on the subacute oral toxicity of the oxygen analogue
    and of a mixture of isofenphos and its metabolites

         Groups of 15 male and 15 female rats (Wistar II strain) were
    treated daily by gavage with the oxygen analogue of isofenphos (HOL
    0654) (as an emulsion in water and Cremophor EL) at 0, 0.2, 0.6 or
    1.8 mg/kg bw for 30 consecutive days. Activity of cholinesterase in
    plasma and erythrocytes was determined five times over the course of
    the study and brain cholinesterase was assayed at terminal sacrifice.
    Physical appearance, behaviour and body weight were comparable to
    controls. Dose-dependent inhibition (>20%) of plasma cholinesterase
    was observed in males and females at 1.8 mg/kg bw throughout the
    feeding period, at 0.6 mg/kg bw in males after 3 weeks only and
    in females at 3, 10, 17 and 23 days of the study. The level of
    erythrocyte cholinesterase was reduced by >20% in both sexes at
    1.8 mg/kg bw. In general, the degree of depression of both plasma and
    erythrocyte cholinesterase was greater in the females than in the
    males. Terminal brain cholinesterase was unaffected in both sexes.
    There were no significant gross pathological alterations in treated
    animals. The no-effect level on plasma cholinesterase was 0.2 mg/kg bw
    (Thyssen 1974b).

    Cow

         Groups of 3 dairy cows were fed alfalfa pellets treated with a
    mixture of isofenphos, isofenphos oxygen analogue and des-N-isopropyl
    isofenphos oxygen analogue (1:8:1) at 2, 6 or 20 ppm for 28 days and
    then sacrificed for analyses of tissue residues. One cow was used as a
    control. Whole blood cholinesterase was assayed at weekly intervals.
    Other than the observation of a more sedated or docile appearance in
    cows at 6 ppm and above, as compared to the control animal or animals
    at 2 ppm, there were no adverse effects noted with respect to body
    weight, feed consumption and milk production. A reduction (>20%) of
    whole blood cholinesterase activity occurred at 20 ppm at days 14, 21
    and 28 of the study and at 6 ppm at days 21 and 28. The inhibition of
    whole blood cholinesterase was dose-related and increased with time
    over the course of the study (Strankowski  et al 1977c).

    Hen

         Groups of 4 hens were fed diets containing a mixture of
    isofenphos, isofenphos oxygen analogue and des N-isopropyl isofenphos
    oxygen analogue (1:8:1) at the levels of 0, 5, 15, 50 or 150 ppm for
    28 days and then sacrificed for analyses of tissue residues. Body
    weight and food consumption at both 50 and 150 ppm and egg production
    at 150 ppm were adversely affected. Dose-related depression (22 to
    98%) of plasma cholinesterase determined at terminal sacrifice was
    observed in all treated groups (Strankowski 1977).

    OBSERVATIONS IN HUMANS

         In 3 male workers (wearing standard protective clothing) involved
    for 5 to 11 working days (generally 7.5 to 8 h per day and not more
    than 3 consecutive days at one time) in the application of a 7.5%
    granulated formulation of isofenphos through a standard applicator
    fixed to carrot seed drilling equipment, the whole blood
    cholinesterase activity was not significantly depressed, as compared
    to 2 male and 1 female control subjects. There was no mention in the
    report with respect to adverse effects or symptoms of exposure. No
    other clinical measurements were made (Bagnall 1976).

    RESIDUES IN FOOD

    USE PATTERN

         Isofenphos is an insecticide possessing good activity against
    soil insects, such as Diabrotica spp., wireworms, chafer grubs,
    vegetable flies and flea beetles. It has proved to be highly effective
    against leaf-eating pests, such as pear sucker and Colorado potato
    beetle, and also against rice stem borers.

         The compound acts as a contact and stomach poison. It has a root-
    systemic action, i.e. the active ingredient is absorbed by the roots
    of the plants and translocated to a limited degree within the plant.

         The active ingredient is notable for its long residual activity
    and its good dispersibility in soil. In numerous trials, isofenphos
    has displayed generally good crop plant tolerance, whether used as a
    seed treatment or applied as granular and emulsifiable concentrate
    formulations. Isofenphos is formulated as an emulsifiable concentrate,
    granular formulation and seed dressing powder.

         It is mainly applied in row at or immediately after seeding. Two
    applications are registered only for corn in the United States. The
    crops, pests and recommended or registered dose rates are listed in
    Table 3.

        TABLE 3.  Recommended application rates for isofenphos
                                                                                                    

    Crop                          Pests               Formulation    Dose (a.i.)
                                                                                                    

    Onion, carrot, brassica
      leafy vegetables            vegetable flies     GR             1.5-2.5 kg/ha in row

    Onion                         onion fly           GR             5 kg/ha broadcast or spray
                                                      and            overall
                                                      EC             0.076 g/m in row

    Brassica leafy vegetables     cabbage root fly    GR             0.05 % a.i. (at 100 cm3/plant
                                                      and            as soil drench after
                                                      EC             transplanting)

                                                                     0.0375-0.075 % a.i. at a rate
                                                                     of 0.0375-0.15g/m row

    Potato                        wireworm            GR             5 kg/ha broadcast overall
                                                                     before planting

    Maize                         Diabrotica sp.      GR             0.11 g/m in row at seeding +
                                                      and            lay by (50 cm row spacing)
                                                      EC

    Oilseeds, rape,               flea beetle         Seed           12-16 g/kg seed
    swedes, marrowstem                                dressing       seed treatment
    kale, turnips                                     powder
                                                                                                    
    
    RESIDUES RESULTING FROM SUPERVISED TRIALS

         Experiments were run on various plants at different locations in
    Canada, Europe and USA. Isofenphos was applied at about the
    recommended, and in certain cases somewhat higher, dosage rates.

         The residues looked for included isofenphos, isofenphos oxygen
    analogue (IOA) in European experiments and des-N-isopropyl isofenphos
    (DNI) and des N-isopropyl isofenphos oxygen analogue (DNIOA) were
    additionally included in the analyses carried out in USA. The residues
    measured in brassicas, oilseed, rape, root and tuber vegetables were
    low, generally at or about the limit of determination for the
    recommended pre-harvest interval, as well as those found in kernel of
    corn at the milk and dry stages. In green forage, however, residues
    were as much as 2 mg/kg. Onions and potatoes also contained detectable
    residues. The major part of the residue consisted of isofenphos and
    IOA in all crops.

    Brassica leafy vegetables

         Brussels sprouts were treated by applying a soil drench at a rate
    of 0.05 g a.i./plant shortly or 1 day after planting. No residue was
    measurable 99-119 days after treatment (Bayer 1978). isofenphos was
    applied on cabbages, savoy and white, and cauliflower either in row or
    with overall treatment at dose rates of 0.075 g a.i./m in a 10 cm band
    or 1.5 to 3 kg/a.i./ha, respectively. The experiments were carried out
    in Federal Republic of Germany (FRG) and UK and results are summarized
    in Table 4 (Bayer 1973-74, 1977).

    Celery

         Oftanol 200 EC was worked in the soil at rate of 4 kg a.i./ha one
    day before planting. Samples were taken 84 and 90 days after treatment
    and analysed for isofenphos and IOA. The residues were below the limit
    of determination (0.01 mg/kg) (Netherlands 1981).

    Maize

         Supervised trials were carried out in various states of the USA
    in 1979 and 1980.

         AMAZE 15 G or 20 G were used for the treatment of the plots in a
    15 cm band at planting plus either a double side dress at the 4-leaf
    growth stage or band treatment at the base of the plant at growth
    stage of 45 cm. A dosage rate of 0.19 g a.i./m was applied in all
    treatments (equal to 2.5 kg a.i./ha). Samples were taken from green
    forage, kernel, cob and husk in the milk stage and from the dried
    parts of plant. Tests were made for residues of isofenphos and its
    three metabolites. The limit of determination was 0.01 mg/kg for all
    compounds individually.

        TABLE 4.  Residues of isofenphos in brassica leafy vegetables
                                                                                                                                    

                                               Application                Residues 1 (mg/kg) at intervals (days) after application
                                                                                                                                    
    Crop           Country   Year      No.     Rate
                                            (kg a.i./ha)   Formulation    0/42      49        55-56     60        64-70     80-87
                                                                                                                                    

    Cabbage        FRG       1974      1    3 kg/ha        5 G                      0.01                          0.01     <0.01
                   FRG       1974      1    "              5 G                      0.08                          0.02      0.01
      savoy        FRG       1974      1    "              5 G                     <0.01                         <0.01     <0.01
                   FRG       1977      1    )0.075 g/m     500 EC         0.02                0.01                0.01     <0.01
                   FRG       1977      1    ) in row       500 EC         0.12                0.03                0.02     <0.01
                   FRG       1977      1    )              500 EC         0.09                0.03                0.02      0.01

    Cabbage        UK        1974      1    1.5 kg/ha      5 G                                                    0.007
                   FRG       1973      1    2.5 kg/ha      5 G                                          0.1                 0.01
      white        FRG       1973      1    2.5 kg/ha      5 G                                          0.06                0.03
                   FRG       1973      1    2.5 kg/ha      5 G                                          0.08                0.02

    Cauliflower    FRG       1977      1    0.075 g/m      500 EC         0.11                0.04               <0.01     <0.01
                                            in row
                   FRG       1977      1    0.075 g/m      500 EC         0.01               <0.01               <0.01
                                            in row
                   FRG       1977      1    0.075 g/m      500 EC         0.02               <0.01               <0.01     <0.01
                                            in row
                   FRG       1973      1    2-2.5 kg/ha    5 G                               <0.012              <0.011    <0.013
                                                                                                                                    

    1  Sum of isofenphos and its oxygen analogue;
    2  Results were obtained by analysing 3 samples taken from different plots;
    3  Results of analyses of 6 samples.
             Isofenphos, IOA and DNIOA were detected in forage. The results
    are summarized in Table 5. Levels of DNIOA ranged from 0.01 to
    0.08 mg/kg, with an average of 0.04 mg/kg. No measurable residues were
    found in kernels either in tile milk or dry stage. Residues in the
    cob were at or below the limit of determination (total residue
    < 0.02 mg/kg). The husk contained residues of less than 0.2 mg/kg
    at all times and stages (Mobay 1979-80).

    Sweet corn

         Experimental plots were treated in a 15 cm band at planting and
    with either a 15 cm band at the base of the plant or a basal spray at
    lay by 4 to 8 weeks later. OFTANOL 6 EC and 15 G formulations were
    used in the trials (Mobay 1978a).

         The active ingredient was applied at a dose rate of 0.19 g/m,
    equal to 3.72 kg a.i/ha. The residues (parent compound and
    3 metabolites) were analysed separately in 10 milk stage kernel
    samples. The oxygen analogue was detected twice at levels of
    0.02 mg/kg and 0.05 mg/kg; the other residues were below the limit of
    determination (0.01 mg/kg for all compounds). No residues were
    detected in dry kernels (3 samples).

         The green and dry forages were also analysed, and levels of
    isofenphos and IOA are listed in Table 5. The cob in the milk stage
    did not contain detectable residues, with the exception of 0.02 mg/kg
    of oxygen analogue. The oxygen analogue was also the only detectable
    residue in husk at the milk stage, with a maximum level of 0.16 mg/kg.
    The dry husk contained both IOA and DNIOA. Maximum levels were
    0.27 mg/kg and 0.02 mg/kg respectively.

    Onion

         At various locations in the USA in 1978, 17 experimental plots
    were treated in furrow at planting with either 15 G, 10 G, 5 G or 4 EC
    formulations at a dose rate of 0.1 g isofenphos/m. The dose rate
    expressed in kg/na varied depending on the row spacing, which was
    between 30 to 100 cm (Mobay 1978b). Both mature bulbs and immature
    bulbs were sampled and analysed for isofenphos, IOA, DNI and DNIOA.
    The residues were generally low in the bulb.

         DNI and DNIOA were only detected in one sample at levels of
    0.01 mg/kg and 0.08 mg/kg respectively. Supervised trials were also
    carried out in Federal Republic of Germany, France and in The
    Netherlands. Five kg a.i./ha was applied from both 5 G and 500 EC
    formulations one day before sowing or at the 4-leaf stage. Samples
    were taken 92 to 158 days after application and the levels of
    isofenphos and IOA were measured (Bayer 1972-73, 1975, 1978). The
    number of samples taken from experimental plots both in USA and in
    Europe are listed in Table 6.

        TABLE 5.  Residues of isofenphos in forage of maize and sweet corn
                                                                                                                                                

                                                                     Residues (mg/kg) at intervals (days) after application
                                            Application              in green forage
                                                                                                                                                
    Crop        Country      Year    No.     Rate                                                                                         in dry
                                          (kg a.i./ha)  Formulation  14-15     20        25-26     29-31     35-39     42-45     55-69    forage
                                                                                                                                                

    Maize       USA                                                  0.03                          0.02                0.02
                             1980    2       2.5        15 G
                Indiana                                              0.141                         0.071               0.111
                "            1980    2       2.5        15 G                                                 0.05                0.04      0.04
                                                                                                             0.141               0.191     0.111
                "            1980    2       2.5        20 G                                                 0.03                0.07     <0.01
                                                                                                             0.111               0.231    <0.011

                Nebraska     1979    2       2.5        20 G        <0.01                         <0.01               <0.01     <0.01     <0.01
                                                                    <0.011                        <0.011              <0.011    <0.011    <0.011
                "            1979    2       2.5        15 G        <0.01                          0.02               <0.01     <0.01     <0.01
                                                                    <0.011                         0.01               <0.01     <0.01     <0.01

    Sweet corn  USA          1978    2       3.72       6 EC                                       0.22
                Missouri                                                                           0.741

                Oregon       1978    2       3.72       6 EC                  <0.01               <0.01               <0.01     <0.01     <0.01
                                                                              <0.031               0.031               0.041     0.031    <0.011

                Nebraska     78      2       3.72       6 EC         7.86                0.01                         <0.01                0.04
                                                                     9.261               0.251                         0.061               0.051
                Texas        1978    2       3.72       6 EC         0.2                 0.05                0.08                          0.04
                                                                     2.091               1.611               1.991                         0.071
                Florida      1978    2       3.72       6 EC         0.03                          0.02                0.04               <0.01
                                                                     0.751                         1.371               0.671               0.071

                Missouri     78      2       3.72       15 G         0.94                          0.07                                    0.1
                                                                     0.741                         0.231                                   0.191
                                                                                                                                                

    TABLE 5.  (con't)
                                                                                                                                                

                                                                     Residues (mg/kg) at intervals (days) after application
                                            Application              in green forage
                                                                                                                                                
    Crop        Country      Year    No.     Rate                                                                                         in dry
                                          (kg a.i./ha)  Formulation  14-15     20        25-26     29-31     35-39     42-45     55-69    forage
                                                                                                                                                

                Oregon       1978    2       3.72       15 G                   0.02                0.02                0.02     <0.01     <0.01
                                                                               0.081               0.081               0.061     0.06      0.011
                Texas        1978    2       3.72       15 G                                       0.01      0.03                          0.05
                                                                                                   0.721     1.041                         0.151
                Florida      1978    2       3.72       15 G                                                                              <0.01
                                                                                                                                           0.041
                Missouri     78      2       3.72       15 G         0.39                          0.03                                   <0.01
                                                                     0.871                         0.14                                    0.11
                                                                                                                                                

    1  Residues of oxygen analogue.
        TABLE 6.  Supervised trials measuring isofenphos and its oxygen
              analogue in onions
                                                                        

    Compound            Residue (mg/kg)          No. of samples
                                                                        

    Isofenphos          < 0.01                       12

                        < 0.05                        0

                        < 0.1                         1

                        < 0.2                         1

                        < 0.6                         3

    IOA                 < 0.01                        4

                        < 0.05                        8

                        < 0.1                         2

                        < 0.2                         2

                        < 0.6                         1

    Isofenphos +        < 0.01                        2
    IOA  1              < 0.05                        4

                        < 0.1                         3
                                                                        

    1  Result of experiments in Europe.

         The sum of the residue detected was always below 1 mg/kg in
    mature bulb samples. Immature bulbs were analysed in two occasions and
    the residues found were: isofenphos-0.32 and 0.95 mg/kg; isofenphos
    oxygen analogue - 0.18 and 0.26 mg/kg.

    Rapeseed

         Seeds of spring and autumn varieties of rape were seed dressed
    with BAY 6643 B at a rate of 15 g isofenphos/kg seed in 1973-74. The
    crops were sampled at various locations in the FRG. Residues were
    below the limit of determination (0.01 mg/kg) in all of the 13 trials
    (Bayer 1974a).

         Supervised trials with three varieties were carried out at three
    locations in Canada in 1989 (Mobay 1980a). AMAZE 40% was used for seed
    dressing prior to seeding at a rate of 25 g a.i./kg seed. The seeds
    were treated either with formulation or the powder was applied to seed
    after coating with oil. The harvested seed was analysed for
    isofenphos, IOA, DNI and DNIOA 94-113 days after treatment. Residues
    of active ingredient and its metabolites were below the limit of
    determination (0.01-0.02 mg/kg).

    Root and tuber vegetables

    Celeriac

         Supervised trials, in which 4 kg a.i./ha of Oftanol 200 EC was
    applied before planting, were carried out on celeriac fields at two
    locations of The Netherlands in 1978. The mature crops were samples
    158 days after application. Residues of isofenphos were not detected
    (Netherlands 1981).

    Potato

         In an experiment in FRG, a 5 G formulation of isofenphos was
    worked into the soil at a rate of 5 kg a.i./ha before planting. At 110
    days after application, a residue of 0.11 mg/kg was detected in the
    tubers that had been washed before analysis (Bayer 1973).

         Potato fields were treated with isofenphos at three different
    locations in Spain in 1980. The broadcast applications were made at a
    rate of 2.5 kg a.i./ha at the time of planting. Samples were taken
    111, 112 and 159 days later at the time of harvest and their residue
    contents were found to be 0.45, 0.04 and 0.05 mg/kg respectively
    (Bayer 1980).

    Swedes and turnips

         Oftanol T was used for seed dressing at a rate of 16 to
    20 g a.i./kg seed. Experimental plots from 8 locations in FRG were
    sampled 109 to 179 days after sowing in case of swedes, while three
    plots of turnips were sampled 89 to 103 days after sowing. Leaves and
    roots were analysed separately. No residue was detected (<0.01 mg/kg)
    (Bayer 1974-76).

    Availability of soil residues to rotational crops

         Wheat, green beans and sugarbeets were planted as rotational
    crops 3.5 months after a sandy soil had been treated with ring-µL
    (14C)-isofenphos at a field application rate of 5.6 kg a.i./ha (Kurtz
    1977). Corn had been used as the original crop in this isofenphos-
    treated soil. When the rotational crops were planted, the soil
    contained approximately 4 mg/kg isofenphos. The rotational crops were

    sampled at 2, 8 and 32 weeks after planting. At the time of harvest,
    8 weeks for green beans and 32 weeks for sugarbeet and wheat, the
    residue level was measured in different parts of the crops (Table 7).
    No des-N-isopropyl isofenphos was detected in any of the crops.

    TABLE 7.  Residues of isofenphos detected in rotational
              crops grown in soil treated with isofenphos 1
                                                                

    Crop                Sample               Residue (mg/kg)
                                                                
                                       I         IOA       DNIOA
                                                                

    Green bean          Forage       < 0.01      0.05     < 0.01

    Sugarbeets          Roots          0.04      0.02     < 0.01

                        Tops         < 0.01      0.02       0.06

    Wheat               Heads        < 0.01      0.24       0.03

                        Straw          0.14      0.79       0.2

                        Forage         0.05      1.08       0.23
                                                                

    1  Application rate - 5.6 kg a.i./ha.

         In the investigation of field rotational crops, wheat, oats or
    sorghum, spinach turnips and soybean were planted in plots treated the
    previous year with isofenphos (Mobay 1980b). The plots had been used
    previously for performance tests and hence were cropped using normal
    agricultural practices. Rotational crop planting dates ranged from 214
    to 390 days (7 to 13 months) post-treatment. Treatment was with either
    AMAZE 20 G in an 18cm band over the row at 1.2 kg a.i./ha to 1.5 kg
    a.i./ha broadcast equivalent-with presswheel incorporation or with
    AMAZE 6 E or 20 G applied in furrow at 1.1 to 2 kg a.i./ha broadcast
    equivalent. Prior to planting the rotational crops, the plots were
    rototilled or disced. The crops were then planted across and
    perpendicular to the direction of the originally treated rows. To
    insure homogeneity of sampling, the entire lengths of the crop rows
    were harvested. Each of the harvested crops were analysed for residues
    of isofenphos, IOA, DNI and DNIOA. The results of the study are given
    in Tables 8 and 9.

        TABLE 8.  Residues of isofenphos in grain (wheat, oats or sorghum) rotational crops
                                                                                           
                               Treatment       Plant Back     Residue (mg/kg 1
    Crop           Location    Rate            Interval
                   (USA)       (kg a.i./ha)    (days)         Forage      Grain       Straw
                                                                                           
    Winter
    Wheat          Kansas          1.5            77         <0.01       <0.01       <0.01

    Sorghum        Texas           1.2           214         <0.012      <0.01        0.02
                                                             <0.033

    Spring
    wheat          Kansas          1.5           295          0.064       NA5         NA
                                                              0.026

    Spring
    Wheat          Nebraska        1.5           350         <0.014      <0.01       <0.01
                                                             <0.016

    Oats           Illinois        1.2           362         <0.014      <0.01       <0.01
                                                             <0.016
                                                                                           
    1  Sum of isofenphos, IOA, DNI and DNIOA expressed in isofenphos equivalents;
    2  Immature crop, 42 days post-planting;
    3  Immature crop, 57 days post-planting;
    4  Immature crop, 45 days post-planting;
    5  NA - no mature crop available for analyses;
    6  Immature crop, 60 days post-planting.
    
    TABLE 9.  Residues of isofenphos in rotational crops
                                                                        
                                Treatment    Plant back   Residue
    Crop           Location        rate      Interval     (mg/kg) 1
                               (kg a.i./ha)  (days)
                                                                        
    Spinach        Arizona         2           279         <0.01
    Spinach        Arizona         2           279         <0.01
    Turnip tops    Kansas          1.5         316         <0.01
    Spinach        Nebraska        1.5         363         <0.01
    Turnip tops    Nebraska        1.5         363         <0.01
    Turnip root    Arizona         2           279         <0.01
    Turnip root    Arizona         2           279         <0.01
    Turnip root    Kansas          1.5         316         <0.01
    Turnip root    Nebraska        1.5         363         <0.01
    Soybean        Kansas          1.5         295          0.03
    Soybean        Nebraska        1.5         363         <0.01
    Soybean        Illinois        1.14        390         <0.01
                                                                        
    1  Sum of isofenphos, IOA, DNI and DNIOA expressed in isofenphos
       equivalents.

         Residues in all rotational crops studied consisted mainly of
    isofenphos and IOA and were <0.05 mg/kg; most were <0.01 mg/kg. In
    the four mature grain crops, the grain had no detectable residue and
    the straw showed a detectable residue of 0.02 mg/kg in only one
    sample. In the five vegetable crops, only one had a detectable residue
    (0.01 mg/kg) was seed in only one of the bean samples and in two of
    the dry vine samples. Based on these results, little or no isofenphos
    residue would be expected in grain, vegetable, root or oil seed crops
    grown in fields treated 9 or more months earlier with isofenphos at
    rates up to 2 kg a.i./ha.

    FATE OF RESIDUES

    General comments

         The degradation and metabolism of isofenphos was studied in
    animals, plants, soils, water and during frozen storage. The
    structural formula, chemical name, identifying symbol and occurrence
    of its degradation products are shown in Figure 2. Metabolic pathways
    for isofenphos in animals (A) plants (P) and Soil (S) are shown in
    Figure 3.

    In plants

         The metabolism of isofenphos was studied in maize grown in ring -
    µL (14C)-isofenphos treated soil both outdoors and in a greenhouse
    (Stanley  et al 1977).

         For the outdoor experiment the labelled isofenphos was prepared
    as a 6 EC formulation and incorporated at the rate of 5.6 kg a.i./ha
    (ca 4 mg/kg soil) into soil contained in a tub. Sweet corn was planted
    in the soil. Plant samples above the soil line were harvested at 28,
    56 and 94 days after planting. The 94-day crop was divided into
    kernels, cobs, husks and stalks in the laboratory.

         In the greenhouse experiment the labelled isofenphos was
    incorporated into soil at a rate of 4 mg/kg. The soil was placed in
    pots and field corn was planted in it. The crop was harvested at
    maturity, 141 days after treatment, and the plants were divided as in
    the other experiment. The root samples were washed with water to
    remove soil particles.

         The compounds identified in the corn stalks were qualitatively
    the same throughout the study, although their relative quantities
    changed as the plant matured.

    FIGURE 2

    FIGURE 3

         Over 70% of the radioactivity appeared to be non-conjugated and
    readily released from the plant matrix of the 28- and 56-day corn
    stalk samples by methanol chloroform extraction, while in the 94-day
    corn stalks, only 19% of the radioactivity was organosoluble without
    hydrolysis. The distribution of radioactivity among isofenphos and its
    metabolites in corn stalk grown outdoors is shown in Table 10.

        TABLE 10.  Distribution of radioactivity among isofenphos and its metabolites inmaize grown
               outdoors in ring-µL (14C)-isofenphos-treated soil
                                                                                            

                                          Percent Radioactivity Distribution
                                                                                            
                                 Organic/Aqueous                Water/Solid Fractions
    Days after     Sample            Extract                          Acid released
    Treatment
                                                                                            
                             I         IOA       DNIOA     SA        DBHA      DAIOA    Unk.
                                                                                            

    28             stalk     19        52          3       nd        nd        nd        nd

    56             stalk      8        63          6      <1         12       <1        2

    94             stalk      2        15          2       5         35        3         5

                   cob        0        17          0       nd        nd        nd        nd
                                                                                            
    
         The primary organosoluble metabolite was isofenphos oxygen
    analogue, but isofenphos and des N-isopropyl isofenphos oxygen
    analogue were also detected. Acid hydrolysis of the aqueous and soil
    fractions from corn stalk liberated 2,5-dihydroxybenzoic acid (DHBA)
    SA and DAIOA.

         As the plant matured, DHBA became the predominant metabolite. The
    only component identified in the husk and cob samples was IOA.

         The small quantity of radioactivity in the organosoluble fraction
    of the kernel prevented adequate identifications to be made, but none
    of the intact phosphate esters could have been present in an amount
    higher than 4% of the kernel's total residue, which accounted for 6.1%
    of the activity measured in the whole plant. The concentration of
    radioactivity and its distribution in the tissues of mature corn grown
    in the greenhouse was similar to that seen in the outdoor study. The
    compounds identified were exactly the same.

         Bermuda onions were grown in a greenhouse for 60 and 91 days in
    soil in which 4 mg/kg ring-µL (14C)-isofenphos had been incorporated.
    The harvested plants were divided into tops (above the soil level) and
    bulbs (Stanley 1977a). In mature plants, the concentration of residues
    increased in the tops and decreased in the bulb, in comparison with
    levels measured in 60-day plants, mainly as a result of the changes in
    the weight of the plant portions.

         Over 84% of the radioactivity in each sample was extracted by
    blending and acid hydrolysis. Over 80% of the radioactivity in the
    tops and over 50% in the bulbs was organosoluble, indicating the
    amount of non-conjugated residues.

         The compounds detected in the organosoluble fractions after
    blending were isofenphos, IOA and DNIOA. IOA was the major compound in
    the tops and the 91-day bulbs, 78-79% and 35% of total activity
    respectively, while isofenphos and IOA ratio was 44% and 24% in the
    60-day bulbs. DNIOA was present in all samples in small amounts but
    was less (<1%) in bulbs and in tops (2-3%) at both intervals. Acid
    hydrolysis of the aqueous fraction from the organic extract liberated
    salicylic acid (1 to 2%) and an unknown (<1 to 3%). The remaining
    radioactivity (6 to 12%) on the TLC plates was at the origin,
    indicating very polar compounds, or was present as streaking and not
    as discrete spots.

         The radioactivity remaining in the solids after organic
    extraction constituted about 5 to 8% in tops and 14 to 16% in bulbs of
    the total activity of 60 and 91-day samples, respectively. Acid
    hydrolysis of the solids released over one half of the radioactivity
    remaining in the solids after methanol chloroform extractions. The
    radioactivity of solids was not further characterized.

    In animals

    Pig

         The feeding study of a male pig is described previously under
    "Biochemical aspects". Four tissues, kidney, liver, muscle and fat
    were analysed for isofenphos and its metabolites. The amount of
    organosoluble metabolites are given in Table 11.

         Enzymatic hydrolysis of neutralized aqueous fractions from kidney
    and liver released most of the radioactivity remaining in these
    tissues. The major enzyme-released metabolite was IPS. Small amounts
    of HHA and DAIOA were also found. Between 30-42% of the kidney and
    liver radioactivity extracted after enzyme hydrolysis could not be
    identified. Unknown (a) consistuted between 19 and 27% of the total
    tissue radioactivity.

    TABLE 11.  Organosoluble metabolites of isofenphos in pig tissues
                                                                        

    Compounds      % Radioactivity in tissues on 5th day of feeding
                                                                        
                   Liver          Kidney         Muscle         Fat
                                                                        

    Isofenphos      13               2             52            61

    IOA             <1              <1             16            10

    IPS              1               2             17            15

    HHA             <1              ND              3            ND

    DNI             ND              ND              5             5

    DAIOA            4               1              3            <1

    Unknown (a)      9              ND             ND            ND

    Total           30               6             97            92
                                                                        

    Cow

         Under "Biochemical Aspects", feeding studies are reported in two
    lactating dairy cows. In the first animal the only milk sample with
    a radioactive residue, >0.01 mg/kg isofenphos equivalents, was
    analysed for isofenphos and its metabolites. The 7.5h sample contained
    0.014 mg/kg isofenphos equivalents and accounted for 0.07% of the
    administered radioactivity. DAIOA and HHA were the main metabolites in
    the organosoluble fraction and accounted for 13% and 10% of total
    activity in milk, respectively. Small amounts (2 to 4%) of IPS, SA and
    several unknowns (12%) were also detected. Enzymatic hydrolysis of the
    solvent-extracted, pH 5 milk released an additional 45% of the milk
    radioactivity. HHA (27%) was the major constituent in this fraction of
    the milk. DAIOA, SA, IPS and several unknowns were also found in small
    amounts.

         In the second experiment milk was collected prior to each day's
    dosing and 8 hours after each dosing. Milk production remained at the
    cow's normal levels through the 5 days of the study. The cow was
    sacrificed 2 hours after the fifth dose was administered. The
    following tissues were collected and analysed, their residue content,
    in isofenphos equivalent (mg/kg) were: muscle-loin 0.02, shoulder
    0.02, round 0.02; fat-renal 0.07; omental 0.07; subcutaneous 0.06;
    kidney 0.53; heart 0.04; liver 0.47; udder 0.05 and brain 0.02.

         In kidney and liver tissues, less than 35% of the radioactivity
    was organosoluble and consisted of SA 26 to 14%, HHA 3 to 4%, IOA 2 to
    1%,, IPS 1% to <1%, respectively. Traces (1%) of unknowns were found
    in the liver.

         Sulphatase beta-glucuronidase combination was employed in the
    enzyme hydrolyses, which released 42 to 45% activity from the kidney
    and liver extracts respectively. IPS (7 to 3%), SA (8 to 2%) and HHA
    (6 to 8%) were identified in the enzymatic extracts of kidney and
    liver.

         Several unknowns (20 to 33%) were detected, but a single
    compound, unknown (a), accounted for the 90% of activity. The
    radioactive residues which were not organosoluble or liberated by
    enzyme hydrolysis were not further characterized.

         A feeding study was conducted to study the biological effects and
    to determine the tissue and milk residues after feeding isofenphos and
    its metabolites. The ratio 1:8:1 of isofenphos, IOA and DNIOA in the
    mixture was based on plant metabolism studies of isofenphos and should
    be representative of field-treated samples. Alfalfa pellets containing
    the mixture of compounds at 2, 6 and 20 mg/kg levels were given to
    dairy cattle  ad libitum for 28 days (Strankowski 1977c). Feed
    consumption, body weight and milk production were unaffected by
    consumption of the treated feed as compared to the control cow. The
    milk of cows fed 20 mg/kg contained detectable residues only in the
    range of 0.001 to 0.01 mg/kg of isofenphos equivalents on the 28th day
    of feeding. After feeding for 28 days the animals were sacrificed and
    the following tissues were taken; liver, kidney, muscle (round, flank
    and loin) and fat (omental, renal and subcutaneous). Residues in all
    the tissues analysed, except liver and kidney at the 20 mg/kg level,
    were below 0.0, mg/kg. The liver and kidney samples contained
    residues, in isofenphos equivalents, ranging from 0.01 to 0.02 and
    from 0.01 to 0.04 mg/kg, respectively.

    Hen

         Laying hens were given a single oral dose of ring-µL(14C)-
    isofenphos at 2.0 mg/kg (Dupre 1975). Eggs and excreta were collected.
    At four sacrifice intervals (6, 24, 48 and 96 h post-treatment) blood,
    various organs and tissues were collected from four birds per group.

         In eggs, the level of radioactivity reached a maximum level of
    0.099 mg/kg with an average of 0.047 mg/kg in isofenphos equivalent at
    48 h post-treatment. Approximately 72% and 78% of the radioactivity
    was eliminated in the excreta within 24 and 96 h respectively. A
    maximum level in the blood plasma of 0.274 mg/kg 14C activity at 6 h
    post-treatment fell to 0.009 mg/kg 96 h after treatment; 24 h after
    treatment, residue levels in skin, fat, breast, thighs, heart and
    gizzard muscle were less than 0.05 mg/kg, while liver and kidney
    contained 0.9 mg/kg and 0.68 mg/kg respectively.

         In the feeding study in hens described under 'Biochemical
    Aspects' residues, expressed as mg/kg isofenphos equivalents, found in
    various tissues were: gizzard 17.1, kidney 4.9, liver 3.9, fat 0.83,
    skin 0.67, heart 0.42, muscle 0.14, eggs - 51 h 0.25, 48 h 0.06, 24 h
    0.032. The distribution of the residues in the organosoluble fractions
    of various tissues is given in Table 12.

        TABLE 12.  Distribution of isofenphos and its metabolites in tissues and eggs
    of laying hens
                                                                                               

                                                 Distribution (%)
                                                                                               

    Compound       Gizzard   Kidney    Liver     Fat       Skin      Heart     Muscle    Eggs
                                                                                               

    Isofenphos       90        14         2        74        53        26        31        37
    IDA               0         4         1         4         3         3         4         9
    IPS               0         1         1        18        19        10         8        10
    DNI               0         0         0         3         3         0         0         0
    HHA               0         0         0         0         0         0         5         0
    Unknown           0        47        19         0         8        36        28         5
                                                                                               
    
         Feed treated with a mixture containing isofenphos, IOA and DNIOA
    in the ratio of 1:8:1, at 5, 15, 20 and 150 mg/kg feed was distributed
    to laying hens ad libitum for 28 days (Strankowski 1977d). Feed
    consumption, body weights and production decreased as the amount of
    isofenphos and its metabolites in the feed increased. At sacrifice,
    giblets (heart, gizzard, liver), muscle (equal portions of thigh and
    breast), fat (equal portions of visceral and subcutaneous) and skin
    (without feathers) were collected. No residue was detected in the
    muscle at any dose level. Residues in fat, giblets, skin and eggs are
    summarized in Table 13.

    Fish

         Studies on continuous exposure for 28 days of channel catfish to
    isofenphos are reported under "Biochemical Aspects".

    In soil

         Isofenphos degradation under aerobic conditions was investigated
    by Minor and Murphy (1977) in sandy loam and silt loam, while the
    effects of anaerobic and sterile conditions were studied in sandy
    loam. Ring µ-L-(14C) labelled isofenphos was uniformly incorporated
    into the soil at a level of 8.7 mg/kg. The extractable residue derived

    TABLE 13.  Residues in isofenphos equivalents found in tissues and
               eggs of laying hens1
                                                                        

                                  Residue (mg/kg)
    Dose                                                                
                   Fat            Giblet         Skin           Eggs
                                                                        

    50 mg/kg     <0.01 (4)       <0.01 (2)      <0.01 (2)       0.002

                                  0.01 (2)       0.02           0.003

                                                 0.08           0.003

                                                                0.004

    150 mg/kg    <0.01 (2)        0.01          <0.01 (2)       0.017

                  0.01 (2)        0.02           0.03 (2)       0.013

                                  0.04                          0.015

                                  0.06                          0.003
                                                                        

    1  No. of samples in brackets.


    from isofenphos decreased with time, while the radioactivity bound to
    soil, or lost, increased. Only 10% of the originally applied
    radioactivity could be extracted from silt loam at 240 days post-
    treatment, whereas 23% of the-radioactivity could still be removed
    from the sandy loam one year after traces (<1% of original activity)
    of IPS. The concentration of isofenphos decreased continuously with
    time, and its half life was found to be 127 days in sandy loam and 59
    days in silt loam.

         The concentration of isofenphos oxygen analogue reached a maximum
    between 60 and 120 days post-treatment. The bound radioactivity
    increased to a maximum of approximately 20% and the remainder had
    volatilized. The bound radioactivity was distributed among humin,
    humic acid and fulvic acid fractions in a fashion similar for both
    soils. Even after stringent HCl-NaOH fractionation, approximately 50%
    of the bound residues was still unextractable.

         The volatile radioactivity from soil was composed of isopropyl
    salicylate, 14CO2-isofenphos and cyclic isofenphos. At 134 days
    post-treatment, the total volatile radioactivity trapped was
    distributed among IPS (67%), 14CO2 (30%), cyclic isofenphos (2%)

    and isofenphos (1%) (Minor 1980: The volatile compounds in the
    experiments accounted for 16 to 51% of the total activity applied to
    soil 134 days before, while the soils contained the remaining part of
    activity, indicating the importance of conditions (e.g. temperature
    and/or microorganism activity) on degradation rate of isofenphos in
    soil. In sandy loam and under anaerobic conditions, isofenphos was not
    converted to its oxygen analogue, but some of the radioactivity was
    lost. Under sterile conditions in sandy loam, isofenphos was neither
    altered nor lost. In isolated soil micro-organisms grown in "shake
    culture", isofenphos was not altered. Conversion of isofenphos to its
    oxygen analogue appeared to be strictly a chemical reaction occurring
    in soil under aerobic conditions. The production of isopropyl
    salicylate may also have occurred without microbial activity, but the
    isolated 14CO2 was probably the end result of soil micro-organism
    cleavage of the isofenphos aromatic ring after the phosphate group had
    been removed. Therefore, isofenphos soil degradation appeared to
    involve both chemical and microbial alterations, and the chemical
    reactions of oxidation and hydrolysis appeared to be prerequisite to
    microbial action. Comparison of results from isofenphos treated soil
    kept in a greenhouse or maintained outdoors showed that isofenphos
    degradation qualitatively is the same under both environments, but the
    rate of degradation may vary.

         The persistence of isofenphos in soil was investigated in nine
    tests set up in various locations in the United States and Canada
    (Mobay 1980c). Isofenphos, 6E formulation, was incorporated uniformly
    at a rate of 2 mg/kg wet soil (equivalent to 2.24 kg/ha field
    application) into the upper 7.6 cm of soils of seven different types.
    Samples were taken at 0, 30 to 33, 60 to 61, 89 to 92 and 119 to 124
    days post-treatment from 0 to 15 cm and 15 to 30 cm depths. The
    residue of isofenphos decreased in all nine studies, and its level in
    the upper layer was between 0.11 mg/kg and 0.43 mg/kg 119 to 124 days
    after treatment. IOA reached its maximum (0.19 to 0.5 mg/kg) 32 to 123
    days post-treatment. Depending on the soil types and possibly on
    environmental conditions, the ratio of isofenphos and its oxygen
    analogue varied between 0.37 to 14 at the last sampling. Soil samples
    from 15 to 30 cm did not contain detectable residues (0.01 mg/kg),
    with the exception of rocky sandy silt loam in which 0.02 to 0.15
    mg/kg isofenphos or IOA could be detected.

         The degradation of isofenphos was studied in the Federal Republic
    of Germany (FRG) and in the UK (Bayer 1973-75). The applications were
    made with either EC or granular formulation as surface spray or
    broadcast at rates of 1, 5 and 7.5 kg a.i./ha. The sampling day varied
    from 0 to 534 post-treatment. The results show a pattern similar to
    that obtained in the experiments described previously. The results of
    laboratory experiments on the persistence of isofenphos carried out in
    standard German soil No. 1 and No. 2 (Bayer 1974a) were consistent
    with the results of field trials, as were the mobility tests with soil
    thin layer chromatography (Thornton 1976) or soil column (Obrist and
    Thornton 1977; Bayer 1973-74, 1977b).

         The latter experiments indicated that isofenphos and IOA had low
    mobility and the majority of the residue remained in the upper 12.5 cm
    of the soil, while the leachates contained no intact isofenphos but
    did contain a small amount of IOA and traces of IPS (one experiment).

    In water

         The stability of ring-µL-(14C)-isofenphos in sterile buffer
    solutions of various pH's at various temperatures and two
    concentrations (1 mg/l and 10 mg/l) was determined (Mc Namara 1977).
    Both hydrolysis and volatilization were involved in the dissipation of
    isofenphos from the buffered solutions. Isofenphos was not hydrolysed
    at temperatures of 20°C and below or at pH's near neutrality, but
    volatilization did occur at 20°C and above and increased as the
    temperature and isofenphos concentration increased. Hydrolysis of
    isofenphos was, however, encountered in both strongly acidic and
    alkaline buffers. Hydrolysis products detected in pH3 buffers included
    DNI, IOA, DNIOA, IPS, SA. At pH9, the identified hydrolysis products
    of isofenphos were IOA, IPS, SA and N-isopropyl salicyclamide. At
    37°C, the half life of isofenphos at 1 and 10 mg/kg was 79 and 30 days
    respectively, in pH3 buffer, and 88 and 32 days respectively, in pH9
    buffer. At 50°C, the half life of isofenphos at 1 and 10 mg/kg was 15
    and 9 days respectively, in pH 3 buffers; 34 and 15 days respectively,
    in pH 6 buffer; and 6 days for each concentration in pH 9 buffer.

         Ring-µL(14C)-isofenphos at an initial concentration of 10 mg/kg
    was incubated outdoors in a simulated pond (pH 8) (Mc Namara 1977a).
    Within 70 days, 89% of the radioactivity was lost, and the remaining
    radioactivity was distributed between water and sediment in an
    appropriate 2:1 ratio. The loss of radioactivity was probably due to
    volatilization of isofenphos and some of its metabolites. The major
    radioactive compounds identified in the pond water were isofenphos,
    IOA and cyclic-IOA. Minor components were cyclic isofenphos, IPS and
    SA. The amount of radioactivity adsorbed by the pond sediment rose to
    20% of the initially applied radioactivity within 7 days, but then
    decreased steadily to only 2% at 70 days. The bound radioactivity
    never exceeded 2% of the originally applied radioactivity. The major
    components in the organosoluble extracts of pond sediment were
    isofenphos and IOA. Minor components identified in the sediment were
    IPS, SA, cyclic-isofenphos and cyclic-IOA. The overall rate of
    isofenphos dissipation was exponential. The half-life of isofenphos
    was 13 days. After 70 days, only 2% of the initially applied
    isofenphos itself remained in the pond.

    In activated sludge

         Ring µL-(14C)-isofenphos was studied in a laboratory model of an
    activated sewage sludge system (Spare 1979). Activated sludge,
    synthetic sewage and (14C)-isofenphos were aerated for 23 h cycle.
    During the remaining hour/cycle, the mixture was allowed to settle and
    a portion of the supernatant was replaced with fresh synthetic sewage

    and an increased amount of (14C)-isofenphos. The concentration of
    isofenphos during the first cycle was 0.1 mg/kg and was increased
    through 10 cycles to 100 mg/kg.

         After the 100 mg/kg cycle concluded, three cycles were carried
    out with no addition of test compound. Isofenphos had no detrimental
    effect on the bacteria, yeasts or actinmycetes in the system, but at
    isofenphos concentrations of 60, 80 and 100 mg/l, protozoa were absent
    or few in the treated flasks. The quantity of radioactivity in the
    settled solids increased steadily during the course of the study from
    10% to 50% of the added radioactivity. (14C)-isofenphos was not
    degraded appreciably by the organisms in the activated sludge system.
    Greater than 96% of the added (14C)-isofenphos was recovered intact
    from the supernatant and solid fractions.

    Photodegradation

    In aqueous solution

         A study of the photodecomposition of ring-uL-(14C)-isofenphos in
    aqueous solution was carried out in a photoreactor containing a high
    pressure, quartz, mercury-vapour, 200-watt, Hanovia immersion lamp as
    the light source (Strankowski 1977e). Decomposition of isofenphos was
    slow in the pH 7 solution with only 30% decomposition in 30 days of
    continuous exposure to the high intensity light. The isofenphos half-
    life was calculated to be 51 days. In an acetone sensitized solution,
    the photodecomposition was increased, and the half-life of isofenphos
    was reduced to approximately 14 h.

         Isofenphos was the only component found in the dark control
    solutions from either the unsensitized or sensitized studies. It was
    also identified in the photolysis solutions; approximately 75% and 3%
    of the total radioactivity remained as isofenphos at the end of the
    unsensitized and sensitized studies, respectively.

         The major photoproduct, which represented nearly 21% of the total
    radioactivity in the sensitized test, was identified by Poje (1979) as
    3,3-dimethyl isoindoline-l-one by mass spectroscopy and nuclear
    magnetic resonance spectroscopy. This product contains no phosphorous.
    The remaining photoproducts, none of which accounted for more than 5%
    of the total radioactivity, included IOA, DAI, SA and catechol.

    On soil

         In a study of the photodegradation of (14C)-isofenphos on soil,
    isofenphos had a half-life of 2.6 days when applied to a thin layer of
    soil and subjected to light from a 200 watt Hanovia mercury lamp
    (Weissenburger and Pollock 1979). Six photoproducts were detected
    on the soil. IOA (33% of the applied radioactivity) was the principal

    photo-product. Small quantities (approx. 5%) of DNI, DNIOA, des-N-
    isopropyl cyclic isofenphos, IPS and phenol were also detected. During
    26 days of exposure to the light, 25% of the applied radioactivity was
    bound to the soil and 33% was lost through volatility. A trapping
    study, carried out for 3 days, showed that the volatile compounds
    included isofenphos (91%), DNI (3%), IPS (1%) and phenol (5%). All of
    the applied activity was accounted for in the trapping study.

    In storage

         Stability of isofenphos and its metabolites were studied during
    frozen storage (-28°C to -18°C) (Mobay 1980a). Concentration of
    isofenphos and IOA did not change significantly in maize kernel,
    brassica leafy vegetables, green forage, green beans (pods and vines)
    and onions kept deep frozen for 400 to 800 days. However, 24% of
    isofenphos decomposed in sugarbeet during 420 days of storage. DNI and
    DNIOA residues decreased by 20% add 32% respectively in alfalfa stored
    at -23 to -18°C.

         Poultry tissues, eggs and giblets, fortified at 1 mg/kg with
    isofenphos and its metabolite, were stored frozen for 255, 90 and 60
    days respectively without significant changes in concentrations of
    residues.

         Cattle tissues and milk were fortified at 1.0 mg/kg with
    isofenphos and metabolites and placed in frozen storage. Fat and
    muscle tissues showed no significant decomposition of the compounds
    after 24 to 28 days. Milk showed no significant decomposition of the
    compounds after 76 days in frozen storage. In kidney and liver,
    isofephos, isofenphos oxygen analogue and des-N-isopropyl isofenphos
    were stable for 28 to 35 days. The liver and kidney samples were
    extracted within 4 and 8 days, respectively. There was no
    decomposition of des-N-isopropyl isofenphos oxygen analogue in kidney
    after 10 days of frozen storage; however, it did not disappear
    completely in liver after 5 days of frozen storage.

    METHODS OF RESIDUE ANALYSIS

         A gas chromatographic procedure for the determination of residues
    of isofenphos and its oxygen analogue in various crops, soil and water
    has been described by Wagner (1976). Plant samples of high water
    content and soils are extracted with acetone. The acetone extract is
    diluted with water and the residues are partitioned into chloroform or
    dichloromethane. Rape, maize and water are extracted with acetonitrile
    and chloroform respectively. The concentrated extracts are cleaned on
    neutral alumina or on active carbon columns. The residues in the
    concentrated extracts are separated on DC-200, QF-1 or OV-17 columns
    and detected with a thermionic detector. Recovery data from

    experiments run on a large variety of crops by adding known amounts of
    isofenphos and its oxygen analogue at the blending step were generally
    in the 77% to 105% range. The limit of determination is 0.005 mg/kg
    for each compound in plant and soil samples and 0.002 mg/kg in water.

         Gas chromatographic methods for the determination of residues of
    isofenphos, isofenphos oxygen analogue, des-N-isopropyl isofenphos and
    des-N-isopropyl isofenphos oxygen analogue in maize cob, forage, husk
    and kernel and in onions are described by Stanley (1977b, 1979a) and
    in bovine and poultry tissues, milk and eggs by Shaw II (1977a). For
    all four compounds, recoveries were 65 to 70%. The limit of
    determination for each compound is 0.01 mg/kg, except for milk and
    eggs (0.001 mg/kg). Multiplication factors for converting metabolite
    residues to isofenphos equivalents are 1.05 for IOA, 1.139 for DNI and
    1.204 for DNIOA.

         Interference studies for the determination of residues of
    isofenphos and its metabolites as maize and onions were done by
    Stanley (1977c, 1979b) and in milk, animal tissues and eggs by Shaw II
    (1977b, 1979). All possible interferences by 20 or 27 organophosphate
    pesticides registered for use on maize and onions or investigated in
    animal products respectively were separated from isofenphos and its
    metabolites by GLC on the standard (10% DC-200 + 2% OV-225 on
    Chromosorb WHP) or on the confirmatory (5% OV 210 on Supelcoport or 5%
    OV 275 on Chromosorb WHP) columns or by clean-up on the silica gel
    column used in the residue method. The silica gel column was applied
    as follows: 372 g silica gel is deactivated with 28 cm3 water, 10 g
    is filled into the column under hexane and settled, and a 1-cm layer
    of granular Na2SO4 is placed on top of the column.

         Hexane is drained to the top of the sodium sulphate. The
    concentrated extract is transferred in the column with 5 to 10 cm3
    portions of hexane and finally the column is washed with hexane (total
    amount-50 cm3). The portions of hexane is drained through the column
    at a rate of 5 to 10 cm3 until 50 cm3 is collected. Eluents (freshly
    prepared) and fractions are: (1) 185 cm3 hexane-dichloromethane (1:1)
    (isofenphos); (2) 150 cm3 dichloromethane (DNI); (3) 65 cm3
    dichloromethane-methanol (99:1) (discarded); (4) 135 cm3
    dichloromethane-methanol (99:1) (IOA).

         Shaw II (1977b, 1979) applied different conditions: 12 g silica
    gel and 6 g Na2SO4; column is washed with 40 cm3 hexane, 100 cm3
    hexane-benzene (8:2) 75 cm3 hexane-benzene (6:4); elution: (1)
    fraction (isofenphos) 25 cm3 hexane-benzene (6:4) + 100 cm3 hexane-
    benzene (4:6) + 100 cm3 hexane-benzene (2:8) + 50 cm benzene; (2)
    fraction (IOA + DNI) 50 cm3 benzene + 300 cm3 benzene-acetone (8:2).

         A gas chromatographic method for the determination of isofenphos
    and isofenphos oxygen analogue in soil and water, using an alkaline
    thermionic emission detector, is described by Shaw II (1974).

    Recoveries of isofenphos and its oxygen analogue were 93 to 101%; the
    sensitivity of the method is approximately 0.005 mg/kg for soil and
    0.002 mg/kg for water samples.

         The determination of isofenphos and its oxygen analogue residues
    in aged soil by alkaline thermionic emission gas chromatography was
    investigated by Shaw II (1977c). The soil was extracted in a Soxhlet
    apparatus for 16 to 24h with chloroform/methanol (7:3). The extract
    was concentrated, partitioned with hexane/acetonitrile (1:1) and
    partially purified with three hexane extractions of the residue in a
    methanol/water solution. Isofenphos recoveries were >79% and
    isofenphos oxygen analogue recoveries were >95% from soils fortified
    at 0.05 mg/kg. The sensitivity of this method is 0.01 mg/kg.

         Isofenphos and IOA were determined by Brown and Williams (1976)
    in cabbage, potato, rapeseed and soil. The rapeseed was extracted with
    acetonitrile and cabbage, potato and soil with ethylacetate. The
    concentrated extracts were cleaned on a column consisting of the
    mixtures of Florisil, silica gel, alumina and Nuchar C. The cleaned-up
    extracts were separated on DEGS or OV275 liquid phases and detected
    with FPD. The recoveries at 0.01 to 1 mg/kg levels were in the range
    of 84 to 98%. The limits of determination were 0.003 mg/kg and 0.005
    mg/kg for isofenphos and IOA respectively in potato, rapeseed and
    soil, while they were five times higher in cabbage.

         The methods described by Wagner (1976) or by Brown and Williams
    (1976), in combination with the confirmatory columns and GLC
    separation (Stanley 1977b, 1979a), are recommended for regulatory
    purposes.

    NATIONAL MAXIMUM RESIDUE LIMITS REPORTED TO THE MEETING

         Isofenphos is registered in the following countries: Austria,
    Bulgaria, Chile, Denmark, Federal Republic of Germany, German
    Democratic Republic, Indonesia, Israel, Italy, Morocco, Mexico,
    Norway, The Netherlands, South Africa, Spain, Sweden and the USA.

         Maximum residue limits and preharvest intervals were reported
    from some of the countries and are summarized in Table 14.

    EVALUATIONS

    COMMENTS AND APPRAISAL

         In the mammalian species tested, the oral LD50 ranged from about
    20 mg/kg bw in rats to approximately 150 mg/kg bw in rabbits. In the
    rat, the oxygen analogue of isofenphos was shown to be slightly, but
    not substantially, more toxic orally than isofenphos, both acutely and
    subacutely.

        TABLE 14.  National MRLs reported to the Meeting
                                                                                        

                                                      Preharvest
    Country             Crop/Commodity                intervals                MRL
                                                      (days)                   (mg/kg)
                                                                                        

    Fed. Rep. of        Leafy and stem
      Germany             vegetables                                           0.1
                        Rapeseed                                               0.05

    Italy               Pear                          42                       0.1
                        Fruit                                                  0.1
                        Vegetables                                             0.1
                        Sugarbeet                                              0.1

    Netherlands         Cauliflower                   56                       0.1
                        Cabbages (including
                          Brussels sprouts)           56                       0.1
                        Onions                                                 0.1
                        Celery                                                 0.05
                        Celeriac                                               0.05

    Norway              Root, tuber and
                        bulb vegetables               90
                        Brassica leafy vegetables     Apply not later
                                                        than transplanting

    South Africa        Citrus fruit                  180                      0.2

    Spain               Cabbage                       Apply before sowing
                        Onions                        21
                        Garlic                        21
                        All crops (except
                          root vegetables)            Apply before sowing
                        and potato)

    USA                 Maize                         75
                        Corn, forage                                           1.0
                        Corn, fodder                                           1.0
                        Corn, fresh (including
                        sweet)                                                 0.1
                        Corn, grain                                            0.1
                        Meat, fat, meat by-
                        products of cattle,
                        sheep and poultry
                        Milk                                                   0.02
                        Eggs                                                   0.02
                                                                                        
    
         Metabolism studies in rats, pigs and cows, treated orally with
    ring-µL(14C)-isofenphos indicated that the compound is rapidly
    eliminated in both faeces and urine, with the latter being the
    predominant route of excretion. It is metabolized mainly by oxidative
    desulphuration, dearylation, hydrolysis, deamination and conjugation.
    There is no indication that isofenphos and/or its metabolites
    accumulate in mammalian tissue. The excretion pattern and proposed
    metabolic pathways for isofenphos appear to be similar in the three
    mammalian species studied.

         A 3-generation reproduction study in rats (with 2 litters in the
    first generation (F0) but only one litter each in the second (F1)
    and third (F2) generations showed a marginal effect in the F0
    generation at the second mating on the pregnancy rate, even at 1 ppm,
    the lowest tested level. No indication of teratogenic effect was noted
    in a rabbit teratology study at dosages as high as 5 mg/kg bw.
    Teratology studies in rats exposed orally, dermally or via inhalation
    were all negative.

         In vitro microbial assays, including reversion-test and rec-assay
    and a dominant lethal study in mice, failed to give any evidency of
    mutagenicity. Long-term studies in mice and rats revealed no
    carcinogenic activity. An acute delayed neurotoxicity study in hens,
    which could be considered as a screen, was negative. The acute oral
    toxicity of isofenphos was found to be potentiated by malathion but
    not by a number of other organophosphorus insecticides tested.

         Plasma cholinesterase inhibition was observed as the most
    sensitive indicator of toxicity in the short-term rat studies and the
    108-week mouse and 2-year rat feeding studies. A dietary level of
    1 ppm was a marginal no-effect level in the rat and a no-effect level
    in the mouse with respect to plasma cholinesterase. In the dog, a
    90-day study suggested that dietary levels of 10 ppm and above caused
    significant plasma cholinesterase depression and an increase in liver
    weight, although the latter was not accompanied by histopathological
    alteration of the tissue. A 2-year feeding study in the same species
    demonstrated 2 ppm to be the no-effect level, based on plasma
    cholinesterase. The information provided on observations in humans was
    very scanty.

         Acceptable data was available to permit the establishment of no-
    effect levels in three mammalian species. Owing to the unavailability
    of an appropriate neurotoxicity study in hens, only a temporary ADI,
    was allocated.

         Isofenphos, O-ethyl O-2-isopropoxycarbonylphenyl
    isopropylphosphoramidothioate, is an organophosphorus insecticide
    applied mainly in soil at a rate of 1.5 to 2.5 kg a.i./ha or 1 to 5 kg
    a.i./ha for row or overall surface treatment, respectively.

         Supervised trials were carried out on various crops at different
    locations in the United States, Canada and Europe. Isofenphos and its
    main metabolites, isofenphos oxygen analogue (IOA), des-N-isopropyl
    isofenphos (DNI) and des-N-isopropyl isofenphos oxygen analogue
    (DNIOA), were analysed. It was found that the residue consisted mainly
    of isofenphos and IOA. The residues measured in brassica leafy
    vegetables, rapeseed and root and tuber vegetables were below
    0.1 mg/kg and usually at or about the limit of determination after the
    recommended pre-harvest intervals. No residues were detected in maize
    kernels, either in the milk or dry stages, while residues up to
    2 mg/kg were found in green forage 3 to 4 weeks after application. The
    total residue detected in mature onion bulbs was always below 1 mg/kg.

         Rotational crops can also take up residues from previous soil
    treatments. The residues, consisting mainly of isofenphos and IOA,
    were at or about the limit of determination in cereal grains, leafy
    and root vegetables and edible oil crops grown in soil treated with
    isofenphos 9 or more months earlier, The residue level might exceed
    1 mg/kg in green wheat forage but it is under 0.1 mg/kg in the straw
    at the time of harvest.

         Isofenphos is degraded in soil, plants and animals, but its route
    of degradation in soil and plants is somewhat different from that in
    animals. Oxidation seems to be the first reaction in soil and plants.
    In animals, hydrolysis and oxidation both appear to be of major
    importance. The degradation of isofenphos was not rapid in any of the
    systems studied. Soil micro-organisms seem capable of splitting the
    aromatic ring to produce CO2 but only after the phosphoramidothioate
    rouping has been removed from the molecule. In plants, oxidation is
    followed by depropylation, hydrolysis and conjugation. IOA and
    isofenphos are the primary residues but DNIOA also appears in smaller
    amounts. The approximate ratio of isofenphos, IOA and DNIOA in mature
    maize stalks was 1:8:1. 2,5-Dihydroxybenzoic acid (DHBA), salicylic
    acid (SA) and deaminated isofenphos oxygen analogue (DAIOA) appear in
    traces.

         In animals, isofenphos undergoes hydrolysis, oxidation,
    N-depropylation, deamination and conjugation. Excretion is rapid
    in orally dosed animals. Pigs, cows and hens excreted 81%, 79% and
    72% of the applied dose in the urine or excreta within the first
    24h after treatment. Urine contained isopropyl salicylate (IPA),
    O-hydroxyhippuric acid (HHA) SA and DAIOA in non-conjugated and/or
    conjugated forms; 4 to 5% of the administered dose is eliminated in
    the faeces over a 72 to 80h period.

         During a period of multiple dosing for 6 consecutive days at a
    rate of 15 mg/kg/day, rats were able to eliminate residues of
    isofenphos fast enough to maintain a relatively constant level of
    radioactivity in the tissues. One day after cessation of the
    isofenphos treatment a 10-fold decrease in radioactive residue
    concentration was detected in the muscle, fat and liver. Dairy cows

    and laying hens were kept on feed containing residues of isofenphos,
    IOA and DNIOA in the ratio of 1:8:1 at various levels between 2 and
    20 mg/kg feed and 5 and 150 mg/kg respectively for 28 days. Residues
    in all the bovine tissues, except liver and kidney at the 20 mg/kg
    level, were <0.01 mg/kg. The liver and kidney samples collected from
    the 20 mg/kg group contained residues of isofenphos ranging from
    <0.01 to 0.02 mg/kg and 0.01 to 0.04 mg/kg respectively. Residues of
    isofenphos equivalents in milk from all samples, except the 20 mg/kg
    group, were below 0.001 mg/kg. The milk samples collected on the 28th
    day from the high level group contained residues ranging from 0.001 to
    0.01 mg/kg.

         Isofenphos residues in poultry tissues and eggs from the control
    and 5 mg/kg groups were below the limits of determination of the
    analytical method (0.01 mg/kg for tissues, 0.001 mg/kg for eggs). The
    hens fed at the 15 mg/kg level had no detectable residues in the
    tissues and had residues ranging from <0.001 to 0.002 mg/kg in the
    eggs. In the 50 mg/kg group, muscle and fat samples had no measurable
    residues, while giblets and skin samples had residues ranging from
    0.002 to 0.004 mg/kg. Residues were detected in all tissues, except
    muscle, and ranged from <0.01 to 0.05 mg/kg and in eggs from 0.003 to
    0.017 mg/kg at the exaggerated 150 mg/kg dietary level. Control values
    were <0.01 mg/kg in tissues and <0.001 mg/kg in eggs. No detectable
    residues can be expected in products derived from animals fed with
    forage that has been treated with isofenphos at recommended
    application rates.

         Catfish exposed continuously to isofenphos for 28 days in water
    containing 10 mg/l isofenphos accumulated residues of 0.75 mg/kg,
    consisting entirely of intact isofenphos, within the first 4 days of
    exposure. However 87% of this was eliminated within the first day
    after transfer of the catfish to uncontaminated water.

         Isofenphos is readily absorbed by soil. It is only slowly leached
    in all the soils tested, but its degradation products move somewhat
    more rapidly. In soil persistence studies with isofenphos incorporated
    into the soil at the single-application field-use rate, the residue of
    isofenphos itself declined steadily in all soils tested, and the total
    organophosphorus residue decreased to half the initial concentration
    within 45 to 200 days.

         Hydrolysis of isofenphos occurs at acidic and alkaline pH, but it
    is stable near neutral pH.

         In an activated sludge system, isofenphos has no detrimental
    effect on the sludge organisms, and the parent compound was not
    degraded.

         Photodegradation of isofenphos in aqueous solution is slow and
    yields principally a re-arrangement product, 3,3-dimethylisoindoline-
    l-one. On soil, photodegradation is rapid and the main product is IOA.

         Residue analytical methods for the determination of isofenphos
    and IOA, the major residue component, and for DNI and DNIOA are
    available. Recoveries are in the range of 77 to 105%.

         The limits of determination for isofenphos and IOA in plant
    samples, animal tissues, and milk and eggs are 0.005 - 0.01, 0.01 and
    0.001 mg/kg respectively, in water 0.002 and in soil 0.01 mg/kg.

    Level causing no toxicological effect

    Mouse : 1 ppm in the diet equivalent to 0.15 mg/kg bw/day
    Rat   : 1 ppm in the diet equivalent to 0.05 mg/kg bw/day
    Dog   : 2 ppm in the diet equivalent to 0.05 mg/kg bw/day

    Estimate of temporary acceptable daily intake for man

         0 - 0.0005 mg/kg bw

    RECOMMENDATIONS OF RESIDUE LIMITS

         The Meeting estimated the maximum residue levels likely to occur
    in various commodities and concluded that they were suitable for
    establishing temporary MRLs.

         The limits refer to the sum of isofenphos and its oxygen
    analogue.

                                                                        
                                              Preharvest intervals (days)
    Commodity                  Limit (mg/kg)  on which recommendations
                                              are based
                                                                        
    Celeriac                   0.02 *                     150
    Swedes                     0.02 *                      90
    Turnips                    0.02 *                      90
    Brassica leafy vegetables  0.1                         60
    Celery                     0.02 *                      90
    Maize                      0.02 *                      90
    Maize, fodder              0.5                         90
    (dry)
    Sweet corn                 0.02 *                      90
    Sweet corn fodder (dry)    0.5
    Rapeseed                   0.02 *                      90
    Carcass meat               0.02 *
    Animal fats                0.02 *
    Meat by-products           0.02 *
    Cattle milk                0.01 *
    Meat of chicken            0.02 *
    Chicken by-products        0.02 *
                                                                        
    *  Level at or about the limit of determination.

    FURTHER WORK OR INFORMATION

    Required (by 1983)

         An appropriate neurotoxicity study in hens.

    Desirable

    1.   2-generation (2 litters/generation) reproduction study.

    2.   In vitro biochemical studies on purified isofenphos with respect
         to anticholesterase activity.

    3.   Further observations in humans.

    4.   Additional data from supervised trials on potatoes and onions,
         including information on soil residues and soil moisture content.

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    See Also:
       Toxicological Abbreviations
       Isofenphos (Pesticide residues in food: 1982 evaluations)
       Isofenphos (Pesticide residues in food: 1984 evaluations)
       Isofenphos (Pesticide residues in food: 1986 evaluations Part II Toxicology)