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    PESTICIDE RESIDUES IN FOOD - 1984


    Sponsored jointly by FAO and WHO






    EVALUATIONS 1984




    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
    Rome, 24 September - 3 October 1984

    Food and Agriculture Organization of the United Nations
    Rome 1985

    METHOPRENE

    IDENTITY

    Chemical Name:    isopropyl (E,E)-(RS)-11-methoxy-3,7,11-
                                  trimethyldodeca-2,4-dienoate.

    Structural formula:

    CHEMICAL STRUCTURE 1

    Synonyms:         ALTOSID, APEX, DIACON, KABAT, DIANEX, PRECOR.
                        (All registered Trade Names) ZR-515; ENT-70460;
                        CAS No: 114-26-1

    Other information on identity and properties:

         Molecular formula        C19H34O3

         Molecular weight:        310

         Physical form:           Pale yellow liquid

         B.P.:                    100 at 0.05 mm Hg

         Vapour pressure:         2.37  10-5 mm Hg at 25C

                                  1.60  10-4 mm Hg at 40C

         Solubility:              all organic solvents-infinity
                                  water - 1.39 mg/l.

         Specific gravity:        0.9261 g/cc at 20C

         Octanol/water
         partition coefficient:   10,000

         Cis-2 : trans-2 ratio:   8:92

         Purity:                  methoprene          - 92-95%
                                  related isomers     - 5-2%
                                  solvent             - 1-2%
                                  unidentified        - 2-1%

         Stability:               extremely stable in sterile aqueous
                                  solutions over wide range. Readily
                                  biodegraded by common bacteria. Rapidly
                                  degraded in solution or in thin films by
                                  sunlight and UV light. Identity of
                                  degradation products known. (Quistad
                                  et al., 1975b).

         Formulations:            Several emulsifiable formulations for
                                  agricultural, stored product and public
                                  health use; solution formulation for
                                  treating cured tobacco; fogging solution
                                  for public health use; emulsifiable
                                  formulations for addition to feed
                                  supplements; slow release formulation
                                  for use in mosquito abatement;
                                  emulsifiable formulation for protection
                                  of houseplants.

    RESIDUES IN FOOD AND THEIR EVALUATION

    USE PATTERN

         Methoprene is an insect growth regulator (IGR), being a synthetic
    analogue of the insect juvenile hormone (Henrick et al., 1973).
    Unlike conventional insecticides, which act as direct poisons,
    methoprene acts by disrupting the morphological development of
    insects. After contact, inhalation or ingestion the insect grows and
    pupates normally, but the pupa dies in the pupal stage. As a result,
    methoprene is not generally used in pre-harvest situations when a
    quick kill of insect pests is required.

         Methoprene has found widespread use in mushroom culture to
    prevent the emergence of adult sciarid flies, where it is applied to
    compost or casing at the rate of about 100 g/100 sq.m. This
    application represents about 12% of methoprene use. A commonly used
    treatment is 175 ml of 650 g/l emulsifiable concentrate per 100 sq.m.,
    equivalent to 113 g a.i./100 sq.m. One or two applications are used.

         Special slow-release formulations are used to control mosquito
    breeding in floodwater sites, rice cultivations, storm drains, ponds,
    water treatment works etc. (Schaefer and Wilder, 1973; Mura and
    Takahashi, 1973).

         Methoprene is formulated into feed supplements for cattle to
    control adult hornfly breeding in cattle manure. The rate is
    equivalent to 0.016 mg/kg body weight daily. This, together with
    mosquito control represents about 50% of current use.

         Methoprene is marketed as a 65% emulsifiable concentrate
    formulation for use as a space spray for control of pests in food
    handling and storage establishments. It is applied as an aqueous spray
    or via mechanical foggers at a rate of 10 ml per 1,000 m2.

    Post-harvest treatments

         Methoprene has proved effective in the control of numerous pests
    of stored products such as peanuts where it is used at the rate of
    10g/tonne against most moth and beetle pests. It is being tested on
    cocoa beans and cereal grains as a 657g/l emulsifiable concentrate
    formulation. This is applied at a rate of 15ml/tonne of stored
    commodity, equivalent to 10g a.i./tonne, in either water or mineral
    oil.

         Trial work indicates that methoprene will control most insect
    pests affecting stored grain though it is significantly less effective
    against Sitophilus spp. which include three of the major pests of
    maize, rice and wheat. It may have to be combined with a conventional
    insecticide where these species are a problem.

         Methoprene has been used for more than five years for treating
    stored tobacco. The cured tobacco is treated, before packing, at the
    rate of 200g methoprene per tonne of tobacco. It is used for treating
    food-handling and tobacco-processing establishments to prevent insect
    breeding and development. A fogging formulation is used to control
    pre-adult fleas in premises.

    RESIDUES RESULTING FROM SUPERVISED TRIALS

    Residues in stored agricultural commodities

    Peanuts.  During the period 1979-1981 the manufacturers, in
    conjunction with the US Department of Agriculture, undertook trials of
    methoprene for protecting peanuts against stored-product insect pests
    (Miller 1981). The methoprene was added as the peanuts were being
    placed in storage silos. Normally peanuts are stored for a period from
    a few months to a year.

         Treatment rates were 0, 4, 10, 25 and 100 mg/kg. Nuts (kernels)
    and hulls were analysed separately. Residues at the proposed use rate
    of 10 mg/kg on the day of treatment were below the limit of
    determination (0.05 mg/kg) in kernels with wrappers, 0.05-0.64 mg/kg
    in kernels without hulls (split hulls) and 14-20 mg/kg in hulls. After
    278 days storage the kernels with wrappers contained 0.13-0.38 mg/kg
    (mean of 11 samples 0.22 mg/kg), kernels without hulls (split hulls)
    0.38-2.1 mg/kg and hulls 8.3-14.0 mg/kg.

         Peanuts treated at exaggerated rates contained residues
    proportionally higher than those treated at the proposed use rate.

    Maize.  Eight studies were carried out on stored shelled maize
    (Miller, 1983a). Random samples from bulk lots treated at rates of 10,
    15, 20 and 23.5 mg/kg were drawn at intervals up to 13 months after
    treatment. At all application rates, residues showed no significant
    losses with time, even at 200 days after treatment. It is noteworthy
    that at no stage did the residues determined by analysis reflect the
    amount of active ingredient actually applied to the grain, and in most
    trials ranged between 40% and 70% of it. It is difficult to see
    whether this was due to failure to deposit the methoprene on grain,
    loss from the grain surface immediately after treatment, loss from the
    samples between collection and analysis, or failure to recover residue
    from the substrate.

         The analytical method included an internal standard which was
    added in the blender. Recovery of the internal standard was always
    high (80-110%), implying validity of the method and analytical
    procedures. However, there is no explanation of the apparent absence
    of methoprene. Nevertheless the residue levels found throughout the
    storage period were similar to, or higher than, those reported for the
    initial sample, indicating that little degradation had occurred during
    storage.

    Wheat.  Data from two studies (Miller, 1983b) were available. Stored
    wheat was treated with methoprene at 10 and 20 mg/kg and sampled 3,
    35, 95 and 150 days after treatment. Methoprene residues recovered by
    analysis were about one-half of the treatment rate but did not show
    any decline up to 150 days after treatment. Residues from the 10mg/kg
    treatment were 5.2 mg/kg after 150 days. An explanation for this
    discrepancy is needed.

         In a laboratory study at the University of California using GLC
    analysis, Mian and Mula (1983) found that when methoprene was applied
    at rates of 1, 5 and 10 mg/kg to wheat (13.5% moisture content) stored
    at 27  1C, losses were 61, 66 and 62% respectively over a period of
    12 months. The results of analyses carried out at six times within the
    12-month period are given in Table 1.

         These data seem more reliable than those reported by Miller
    (1983b), since the main initial recovery of insect growth regulator was
    99% of that applied. Thus it can be accepted that the loss over 12
    months is of the order of 60% of the amount applied.

         Rowlands (1976), determined the rate of breakdown at 20C during
    10 weeks storage in the dark, using radiolabelled methoprene with
    three samples of wheat: fresh wheat of 19.1% moisture content, old
    wheat of 12% moisture content and old wheat of 18% moisture content.
    Table 2 (adapted from Rowlands, 1976) indicates that the breakdown was
    quite rapid at the high moisture levels (half-life of three weeks at
    18-19% moisture) and was about half as fast on grain with 12% moisture
    (half-life six weeks).

    Table 1.  Residues of methoprene in treated wheat grain at 
              various post-treatment intervals 1

                                                                
                                Residue, mg/kg
    Interval                                                    
                            Application rate, mg/kg
                    1              5                10
                                                                

    0               0.99a          4.97a            9.92a
    1 wk            0.99a          4.60a            9.39a
                    (0)            (7)              (5)
    1 mo            0.94a          3.93a            6.62b
                    (5)            (21)             (33)
    4 mo            0.82a          3.49ab           5.53bc
                    (17)           (30)             (44)
    8 mo            0.80a          1.78bc           4.07c
                    (19)           (64)             (59)
    12 mo           0.38b          1.71c            3.73c
                    (61)           (66)             (62)
                                                                

    1  Mean of four analyses. Means followed by the same letters
       in a column are no significantly different from one another
       (Duncan's multiple range test. P = 0.05). Values in 
       parentheses below each mean represent percent loss in 
       residues at indicated post-treatment interval.


    Table 2.  Breakdown of methoprene on stored wheat grains at 20C*

                                                                
                             Residue, mg/kg found in grain
    Storage time                                                
    in weeks            Fresh wheat    Old wheat      Old wheat
                          (19%)          (12%)          (18%)
                                                                
    0                      5.6            9.8            9.6
    1                      7.2            9.2            7.7
    2                      5.5            7.9            6.3
    3                      4.4            7.6            5.0
    4                      3.4            7.0            4.3
    5                      2.2            6.2            3.2
    6                      1.4            5.1            2.1
    7                      0.5            4.2            1.4
    8                      0.2            3.2            0.5
    9                      0.1            2.4            0.4
    10                     nil            1.5            0.2
                                                                

    *  Results are means from triplicate 25g samples, treated at 
       10 mg/kg (approx.)

    Cocoa beans.  In two trials carried out in Brazil, methoprene was
    applied to cocoa beans at rates of 1, 2.5, 5.0, 10.0 and 15 mg/kg. The
    beans were sampled on the day of treatment and after 50 days storage
    at 27C. Less than half of the applied methoprene was recovered by
    analysis but the analytical results on samples taken after 50 days in
    storage were not significantly different from those on the initial
    samples (Miller, 1983c).

    Residues in mushrooms

         Data from 7 studies were available (Miller 1978), reflecting both
    compost and casing treatment at 0.2 to 4.6 times the recommended
    application rates. Samples were collected at 0, 9 and 35 days after
    application. When less than 2.5 times the recommended combined
    treatment rates were used, the residues were undetectable
    (<0.05 mg/kg). At 2.5 and 4.6 times the recommended application rate,
    residues ranged from 0.17 mg/kg to 150 mg/kg. The high values were due
    to contamination with compost or peat moss "casing" which was shown to
    contain methoprene at hundreds of mg/kg. Subsequent samples were
    rinsed with cold water to remove the contamination. Most samples
    examined in these later experiments contained no methoprene or the
    residues were less than 0.1 mg/kg (Miller, 1978).

         Dust samples from food-handling establishments were analysed in
    six studies (Miller, 1984). Isolated areas were treated with
    0.6g a.i./1000 sq.m. (the food being covered according to label
    direction before application). Samples were collected in petri dishes
    located in appropriate areas of the treated spaces 0, 30, 60 and 90
    days after treatment. Residues expressed as mg/kg dust were high owing
    to the small amount of dust collected in the dishes. Consequently, the
    total quantity of methoprene collected is a more realistic value to
    consider when evaluating the results.

         Immediately after application methoprene residues were high,
    ranging from 26 micrograms to 191 mg. However within 30 days they
    declined significantly and ranged from 0.5 to 24 mg. Although there is
    some variation in the results, within 60 days the majority of the
    residues were below the level of detection (0.5 micrograms).

    Residues in forage and field crops

         Methoprene is marketed for the control of floodwater mosquitoes
    as a 10% slow-release encapsulated aqueous solution (Schaefer and
    Wilder, 1973). Since treated areas include pastures, rice fields and
    marshlands, residues might occur on certain food crops. Consequently,
    Zoecon conducted a series of studies on rice, legumes and forage crops
    to analyse for possible methoprene residues which could enter the food
    chain through ingestion by livestock.

         In a study on rice, methoprene was applied prior to "heading"
    (Miller, 1974a). The crop was then grown to harvest. Samples were
    collected at 45, 62 and 71 days after application, resulting in a
    built-in pre-harvest interval of two months. No detectable residues
    (<0.05 mg/kg) were found in the rice grains, hulls or straw of any of
    the samples.

         In nine studies on pasture legumes and forage crops (Miller,
    1974b) pasture land was treated at rates of 9, 11, 23 and 45 g/ha. The
    highest treatment rate is four times the highest recommended rate.
    Samples were collected 0, 1, 2, 3, 5, 7, 9, 11 and 365 days after
    application. Residue levels indicated that methoprene is rapidly
    degraded. By day 3, the residues from the highest treatment ranged
    from 0.12 to 0.30 mg/kg.

    FATE OF RESIDUES

    In animals

         Numerous studies have been conducted to determine the fate of
    methoprene in livestock because of the commercial use in animal feeds
    to suppress the breeding of flies in manure.

         Quistad et al. (1974b, 1975a) administered [5-14C] methoprene
    to a steer which was slaughtered two weeks later. Samples of fat,
    muscle, liver, lung, blood and bile were analysed for radioactive
    residues. No primary methoprene metabolites could be characterised but
    the majority of the tissue radioactivity was positively identified as
    cholesterol. A total of 72% of the bile radioactivity was contributed
    by cholesterol, cholic acid and deoxycholic acid. Radioactivity from
    metabolised methoprene was associated with protein and cholesteryl
    esters of fatty acids.

         A metabolic balance study was conducted on a 338 kg Jersey cow
    dosed with 207 mg of [5-14C] methoprene by capsule (Chamberlain
    et al 1975b). This is 40 times the proposed dose for fly control.
    The study proceeded for 7 days after dosing. It was found that 16.4%
    of the radioactivity was excreted as CO2 between 0 and 170 hours,
    with peak excretion after a little under 24 hours. A total of 30.3% of
    the applied dose was eliminated via the faeces within about 50 hours
    and 19.7% via the urine in 30 hours. By the end of the experiment 7.6%
    of the radiolabel had been excreted via the milk. Radioactivity was
    measured in 30 tissue samples after the cow was sacrificed 7 days
    after dosing. The tissues contained a total of 20% of the applied
    dose, intestine and contents contributing 5.96% and fat 4.59%. The
    organs of metabolism, lung, liver and kidney, contributed a further
    1.68%. The presence of radiocarbon in the tissues was attributed to
    steroidal derivatives in which labelled acetate from the methoprene
    was anabolically incorporated into natural body constituents.

         The results of the above two studies and another on a guinea pig
    were compared by Chamberlain et al. (1975a). It was reported that a
    rather large percentage of the radiolabel was incorporated in the
    tissues and respired by the animals. A small proportion was
    incorporated into free primary metabolites, a greater amount into
    glucuronides and a considerable proportion within polar components,
    possibly complex conjugates or biochemicals. Methoprene was not found
    in the urine, but accounted for approximately 40% of the radiolabel in
    the faeces. This explains the activity of the administered methoprene
    against fly larvae developing in dung pats. The formation of
    conjugates and complex metabolites was more extensive in the steer
    than the guinea pig. Comprehensive information was provided on the
    radiolabel profiles in tissues, organs and other components of the
    animals.

         On the basis of detailed biochemical analysis of tissues and
    organs from the above three studies Quistad et al. (1975c)
    elucidated the metabolism by the cow to natural products in milk and
    blood. Only a trace of methoprene (0.015/l) and no detectable primary
    metabolites occurred in milk.

         Quistad et al. (1976a) studied the metabolism of methoprene in
    leghorn chickens give single oral doses of [5-14C] methoprene.
    Residual radioactivity was found in tissues and eggs. Although a high
    initial dose (59 mg/kg)resulted in residues of methoprene in muscle
    (0.01 mg/kg), fat (2.13 mg/kg) and egg yolk (8.03 mg/kg), these
    represented only 39% and 2% of the total radiolabel in fat and egg
    yolk respectively. Radiolabelled natural products were by far the main
    14C residues in tissues and eggs, particularly at the lower dose of
    0.6 mg/kg where cholesterol and normal fatty acids contributed 8% and
    71% of the total radiolabel in egg yolk.

         When chickens were given feed treated with methoprene at 0, 25,
    50 and 100 ppm for 14-63 days, residues in poultry meat and eggs were
    less than 0.1 mg/kg (Zoecon, 1973).

         Quistad et al. (1976b) studied methoprene metabolism in
    bluegill fish in a dynamic flow-through system and a model aquatic
    ecosystem. The fish in the flow-through system acquired moderate
    residues of largely unmetabolised methoprene when continuously exposed
    to about 30 times the anticipated environmental levels of methoprene,
    but these were 93-95% eliminated within two weeks when the fish were
    transferred to flowing uncontaminated water. In the model aquatic
    ecosystem, the fish accumulated 14C, but the radioactivity was almost
    exclusively in radiolabelled natural products, including cholesterol,
    free fatty acids, glycerides and protein. Less than 0.1% of the total
    radioactivity could be attributed to methoprene or its primary
    metabolites.

    In plants

         Methoprene is rapidly degraded in plants with a half-life of
    about 2 days in alfalfa and about 0.5 days in rice. The major
    metabolic pathways include ester hydrolysis, O-demethylation and
    oxidative scissions of the 4-ene double bond, producing primary
    metabolites which are present in relatively low yields (Figure 1)
    (Quistad et al. 1974a). These low amounts suggest that subsequent
    degradation or conjugation occurs, and in fact the recovery of polar
    metabolites as well as the loss of radioactivity through
    volatilization are evidence of additional metabolism. In addition,
    unextractable residues suggest the extensive degradation of methoprene
    and incorporation into natural cellular products. The metabolic fate
    of methoprene in alfalfa and rice is shown in Figure 1. Metabolism
    resulted in five primary non-polar products isolated from the plant
    (ZR-669, ZR-725, ZR-724, ZR-1945 and ZR-1602, see Figure 1). The
    combined yield of these products was 1.4% of the applied dose after 7
    days from alfalfa and 2.1% after 3 days from rice. The most abundant
    non-polar metabolite was ZR-1564 (13% from rice, 2% from alfalfa)
    which was isolated from vapours and not found within the plant. The
    main aglycones after enzymic cleavage of alfalfa conjugates were
    ZR-725 (2.2%), ZR-1945 (0.8%), and ZR-724 (7.4%). The major aglycone
    from rice was ZR-1945 (1.2%) with a small amount of ZR-1602 (0.3%).
    After 30 days, only 1% of the applied dose remained in alfalfa as
    unmetabolised methoprene and 0.4% remained in rice after 15 days. The
    initial rapid loss of methoprene and radiolabel from plants was
    attributed to evaporation of methoprene and 7-methoxycitronellal
    (ZR-1564) on the evidence from supporting studies of methoprene
    evaporation from glass plates and of compounds volatilized from leaf
    surfaces.

         10-3H-methoprene painted on the leaf surface or injected into
    the stem of the dwarf Lima bean plant was found not to be
    translocated. When injected into the stem, the parent compound was
    partially metabolised to ZR-724, ZR-725 and a polar conjugate,
    possibly the glycoside. Topical application resulted in more rapid
    metabolism to the same metabolites after 24 hours incubation
    (Schooley, 1974).

         Rowlands (1976) studied the uptake and metabolism of methoprene
    and two other IGRs by stored wheat grains. He applied the
    radiolabelled compounds topically to individual grains and to small
    samples of wheat in glass jars. The wheat contained 19.5% moisture and
    was kept at 20C in the dark. The methoprene penetrated rapidly from
    both vapour and topical treatments, but the quantities sorbed differed
    between the two routes. Two days after treatment most of the intact
    methoprene was found in the aleurone layers, much less in the germ and
    virtually none in the endo-sperm proper or outer seedcoat. The rate of
    degradation is indicated in Table 2 above. Degradation is promoted by
    grain moisture, wheat with 19% moisture giving a half-life of 2-3
    weeks, 18% moisture 3-4 weeks and 12% moisture 6-7 weeks.

         Mian and Mullar (1983) determined the residues of methoprene on
    wheat over a 12-month storage period. Details are given above
    ("Residues resulting from supervised trials").

    In soil

         Schooley et al. (1975b) studied the degradation of methoprene
    in soil as a function of time under various conditions. On aerobic
    sandy loam methoprene showed an initial half-life of about 10 days at
    a surface treatment rate of 1 kg/ha; decomposition was much slower on
    autoclaved soil. Only small amounts of non-polar metabolites were
    isolated, including the hydroxy ester resulting from O-demethylation
    (0.7% of the applied dose). Over 50% of the applied dose was converted
    to CO2. Radioactivity from [5-14C] methoprene was incorporated into
    humic acid, fulvic acid, and humin fractions of sandy loams. These
    studies indicate rapid and extensive breakdown of methoprene in soils.

         Leaching experiments with radiolabelled methoprene were performed
    to determine the mobility of the chemical in different soils. The four
    agricultural soils used were sand, sandy loam, silt loam and clay
    loam. Experiments with soil columns and soil thin-layer plates
    revealed that no significant leaching of methoprene occurred with the
    four soils tests (Zoecon, 1984b).

         Quistad and Staiger (1974a) determined the amount and nature of
    bound residues in soil. They found that in sandy loam soil treated
    with [5-14C] methoprene humic acids (3%) and fulvic acids (8.4%) were
    most abundant after 30 days. The total recovery of the label from
    treated soil was essentially quantitative.

         Quistad and Staiger (1974b) determined the partition between
    water and soil using a 0.01 mg/l solution of [5-14C] methoprene
    agitated with sandy loam soil. After thorough mixing for 15 minutes,
    87% of the applied dose was bound to the soil.

         Staiger et al. (1974a) grew wheat plants in soil that had been
    treated 6 months previously to determine the uptake by a second crop
    grown in methoprene-contaminated soil. After treatment of the soil at
    the rate of 1 kg/ha, less than 0.006% of the administered label was
    taken up in wheat six months after dosing. This uptake is about 
    one-tenth of that observed when soil was treated with radiolabelled
    methoprene at the time seeds were planted.

         Staiger et al. (1974b) determined the uptake of methoprene by
    wheat plants when wheat seeds were planted in sandy loam and
    radiolabelled methoprene was incorporated at the rate of 1 kg/ha at
    the seed level and in the soil at a depth of 9 cm below the seed. The
    wheat was harvested 11 days after planting and assayed after total
    combustion. Uptake was found to be 0.09% of the applied radioactivity
    when the IGR was inserted at the level of the seed and 0.04% of the
    radioactivity when it was incorporated 9 cm below the seeds. It appears
    that very little methoprene or its metabolites are translocated into
    wheat from soil.

    In water

         Sterile aqueous solutions of methoprene (0.5 mg/l), buffered at
    various pH values, were found to be extremely stable to hydrolysis
    over four weeks at 20C in the dark. No degradation was seen for the
    duration of the experiment in sterile water buffered at pH 7 and 9,
    and similar stability was observed in pH 5 buffer for 21 days. However
    the pH 5 buffer became non-sterile between the 21- and 30-day
    observations, and 59% degradation occurred between days 21 and 30.
    (Schooley and Bergot, 1975a).

         Schaefer and Dupras (1973) studied the persistence of methoprene
    in water. They found that residues were greatly affected by sunlight
    and temperature. The methoprene tended to remain on the water surface
    and its distribution was affected by wind. The half-life in field
    water was only about two hours. A slow-release formulation showed
    almost no detectable residues after 24 hours, although biological
    activity persisted for several days.

         Schooley and Bergot (1974) measured the dissipation of methoprene
    in pond water and sewage at dose rates of 0.001 and 0.01 mg/l. A
    half-life of 30-45 hours was observed in pond water, with a 60-70 hour
    half-life in sewage. This significantly longer half-life in sewage may
    have been due to the fact that the primary sewage sample had been
    standing in a closed bottle for 30 days in the laboratory, while the
    pond water had been collected fresh on the day of the experiment.

         On standing for thirteen days in unsterilised pond water
    containing suspended sediment in full summer sunlight at a
    concentration of 0.66 mg/l, methoprene was metabolised and/or
    photodegraded mainly to one product, ZR-1945 methoxycitronellic acid
    (Schooley et al. 1975a). This is one of many products formed by the
    combined action of oxygen and sunlight on thin films of methoprene on
    glass plates. ZR-724, ZR-725 and ZR-669 (See Figure 1) were either not
    present or present in very low concentrations.

         A 3-day study was conducted on methoprene in non-sterile pond
    water at ambient temperature in direct sunlight (Schooley and Bergot,
    1975b). Three primary products, ZR-724, ZR-725, ZR-669 were
    identified. Each compound, as well as the recovered methoprene, was
    found to be an approximately 1:1 mixture of cis-2 and trans-2
    isomers, in contrast to the 94% trans-2 methoprene which was
    applied. This conversion has been shown in other studies to be ready
    photoisomerization rather than an enzymic process.

         The biodegradation of (2E)-[10-3H] methoprene was studied in
    pond water containing unknown micro-organisms (Schooley et al
    1975a). The methoprene showed a half-life of approximately 30 hrs at
    0.001 mg/l and 40 hr at 0.01 mg/l. Incubation of the same preparation
    for 3 days at 0.42 mg/l generated three primary metabolites, the
    result of ester hydrolysis and/or O-demethylation. These metabolites
    and the recovered methoprene were photoequilibrium mixtures of 2-ene

    isomers. In another incubation experiment with (2E)-[5-14C]
    methoprene at 0.66 mg/l in a pond water sample with presumably
    different microflora, a completely different metabolic profile was
    observed. The main and only identifiable metabolite, resulting from
    oxidative scission of the 4-ene double bond, was 7-methoxycitronellic
    acid (29% of the applied dose).

    In processing and cooking

    Peanuts.  Peanuts from one of the trials Miller (1981) (see
    "Residues resulting from supervised trials") with residues ranging
    from 0.18 to 1.21 mg/kg at the time of treatment, were processed into
    peanut butter and peanut oil. The peanut butter was reported to
    contain less than 0.05 mg/kg of methoprene, indistinguishable from
    untreated controls. The peanut oil, however, contained 0.25 mg/kg
    (0.20-0.29) of methoprene. These results do not appear to be
    consistent.

         A trial was carried out in Australia (Zoecon 1984c) in which
    methoprene was applied to wheat at the rate of 10 mg/kg. After storage
    for two weeks the wheat was milled through an experimental Buhler mill
    to provide bran, pollard and white flour. A separate milling produced
    wholemeal. The residues found, and the proportion of each fraction in
    the first milling, are shown in Table 3.

         A separate trial carried out at Kansas State University (Zoecon,
    1984d) involved treatment of two lots of wheat with methoprene at the
    rate of 10 mg/kg followed, 9 days later, by milling through a Ross
    Walking Flour Mill. A separate milling through a pin mill produced
    wholemeal.

         The white and wholemeal flours were used to prepare bread. The
    results of analysis are shown in Table 4.

    METHODS OF RESIDUE ANALYSIS

         Residues of methoprene have been determined in waters, soils,
    plants, stored grain and corn, milk, eggs, fish, shellfish, poultry
    and cattle tissues, blood, urine and faeces. Residues in all samples
    are extracted by high-speed blending with acetonitrile followed by
    vacuum filtration. Fatty extracts are subjected to cold-temperature
    precipitation and filtration to remove fat. Petroleum ether
    partitioning, Florisil and neutral alumina chromatography are used for
    clean-up. The concentrated eluates are analyzed by GLC on columns of
    differing polarity, using flame ionization detectors. Confirmation of
    identity by additional GLC and mass fragmentography (MF). The lower
    limits of determination are: soils, blood and urine, 0.001 mg/kg;
    forage grasses, forage legumes and rice foliage, 0.005 mg/kg; milk,
    eggs, stored grain and corn kernels, fish, shellfish, poultry and
    cattle tissues and faeces, 0.01 mg/kg. Limits of determination and
    recoveries were validated by analysis of laboratory and field samples
    fortified with 14C-methoprene (Miller et al., 1975).

    Table 3.  Methoprene residues in wheat fractions Zoecon, 1984c)

                                                                  

    Fraction           % wt./wt/ of each      Methoprene (mg/kg)
                       fraction (excluding
                       wholemeal)
                                                                  

    Wheat                                            8.9
    Wholemeal flour                                  6.7
    White flour              74                      1.8
    Bran                     23                     11.4
    Pollard                   3                     17.7

    An internal standard was used in the analysis.
                                                                  


    Table 4  Methoprene residues in wheat fractions (Zoecon, 1984d)

                                                                        

    Fraction           % mt/wt of each fraction   Methoprene (mg/kg)
                       (excluding wholemeal)
                                                                       
    
                       Trial     Trial            Trial      Trial
                                                                        

    Wheat (whole)                                 5.0         5.7(Note 1)

    White flour        70.2       70.1            2.8         2.8

    Wholemeal flour                               4.1         5.5

    Bran               14.1       14.4            8.7        10.2

    Shorts  )          12.7       12.4            9.6         9.6
             pollard
    Red dog )           2.4        2.5            9.5        11.0

    Germ                0.6        0.6            8.7        10.7

    White bread (33% moisture)                         0.41

    Wholemeal bread (33% moisture)                     0.88
                                                                        

    Note 1  It appears that a significant proportion of the original
            deposit (10 mg/kg) was lost, possibly by evaporation, between
            application and analysis. Spiked samples analysed at the same
            time yielded the results expected.

         Mian and Mulla (1983) describe a method derived from that of
    Dunham and Miller (1978) which they applied successfully to studying
    the persistence of methoprene in stored wheat. This involved Florisil
    and Alumina chromatography with petroleum ether followed by GLC/FID on
    a column of 3% OV-101 on 100-120 mesh Chromosorb W (acid-washed,
    dimethyldichlorosilane treated), conditioned at 350C. The column and
    injector temperatures were 190C for retention times of 8 min for
    methoprene and 12 min for n-docosane used as an internal standard. The
    detector temperature was 350C. The limit of determination was
    reported to be 0.01 mg/kg and the recovery of 10, 5 and 1 mg/kg in
    grain extracts ranged between 80 and 100%.

    RESIDUES IN FOOD IN COMMERCE OR AT CONSUMPTION

         No information was available.

    NATIONAL MAXIMUM RESIDUE LIMITS

         The meeting was advised that the following national maximum
    residue limits had been established in the USA.

         Meat of cattle, goats, hogs, sheep,
         poultry and horses                           0.1 mg/kg

         Fat of cattle, goats, hogs, sheep,
         poultry and horses                           0.3 mg/kg

         Meat by-products of cattle, goats, hogs,
         sheep, poultry and horses                    0.1 mg/kg

         Milk fat                                     0.05 mg/kg

         Eggs                                         0.05 mg/kg

         Peanuts                                      2.0 mg/kg

         Mushrooms                                    1.0 mg/kg

         A petition has been presented for tolerances of 10 mg/kg on
    barley, buckwheat, corn, millet, oats, rice, rye, sorghum, sunflower
    and wheat. In addition a substantial increase has been proposed in the
    tolerances for indirect residues, derived from animal feeds, in meat,
    fat and meat by-products of cattle, goats, hogs, sheep, poultry and
    horses and in eggs and milk fat.

         The meeting was not aware of similar action by any other national
    authority.

    APPRAISAL

         Methoprene is an insect growth regulator. It is an analogue of
    the insect juvenile hormone. After contact, inhalation or ingestion it
    interferes with the normal process of insect development preventing
    the emergency of adults from pupae or larvae.

         So far there have been no practical pre-harvest uses of
    methoprene on crop plants and it appears unlikely that such uses will
    develop. There is an important application for the control of sciarid
    flies in mushroom culture and as a feed supplement for cattle to
    prevent the breeding of hornflies in cattle manure. Methoprene is
    applied to water to prevent the breeding of mosquitoes.

         The most important application currently under development is the
    use of methoprene to prevent the breeding of stored-product insect
    pests in tobacco, peanuts, cocoa beans and a wide range of stored
    commodities. There are good prospects for use in stored grain to
    inhibit the development of stored-product insects. Extensive studies
    on these uses are at an advanced stage in several countries.

         Methoprene formulations are stable under normal storage
    conditions but are rapidly degraded by light. Sterile aqueous
    solutions are stable over a wide pH range but non-sterile solutions
    are rapidly biodegraded.

         When used for the control of insects in stored products
    methoprene is applied alone at the rate of 10 mg/kg or in conjunction
    with conventional insecticides at much lower rates (1-2 mg/kg).
    Although the residues studies available at this stage are not
    extensive it would appear that there is relatively little degradation
    on dry stored commodities over many months. Degradation is promoted by
    moisture and probably also by temperature.

         In numerous field studies on peanuts, maize, cocoa beans and
    wheat, investigators have failed to recover more than half of the
    methoprene applied. This difficulty was not encountered in one small
    scale laboratory study at the University of California. This suggests
    a pronounced tendency for the methoprene to evaporate with the water
    used to apply the insect growth regulator or a fundamental weakness in
    the trial technique. This feature is currently receiving further
    study.

         The meeting received studies from Australia and the USA designed
    to determine the distribution of methoprene in grain and the effect of
    processing and cooking on the residues. These enabled the meeting to
    propose temporary maximum residue limits.

         One study with the labelled compound applied to individual wheat
    grains indicated rapid penetration. A study on peanuts indicated that
    by far the greater proportion of the deposit remained on the hulls and
    wrappers and was not taken up by the kernel. However, allowance must
    be made for the fact that a proportion of the kernels lose their
    shells. When the peanut kernels were processed into peanut butter the
    methoprene disappeared but significant amounts were detected in peanut
    oil. These studies should be repeated.

         When methoprene is applied to mushroom compost or casing at rates
    which will control sciarid flies there are no detectable residues in
    mushrooms. However, it is necessary to wash the peat moss casing from
    the mushrooms before analysis to prevent anomalous results.

         Methoprene would be suitable for the treatment of surfaces and
    structures to prevent insect pests breeding in food storage and
    preparation areas. Several studies indicated that such treatments
    would not lead to significant residues in foodstuffs.

         Studies have been conducted with cattle, poultry and fish to
    elucidate the metabolic processes and determine whether residues occur
    when methoprene is administered to livestock directly for fly control,
    or in their feed from cereal grain and milling products, or when the
    compound is used for mosquito control. All available information
    points to the rapid degradation of the parent compound and
    incorporation of fragments into natural body components but the
    meeting, recognizing that it would not be practical to withdraw
    treated feed before slaughter, recommended temporary MRLs for
    methoprene residues in foods of animal origin to cover the feeding of
    treated grain and its milling products.

         Methoprene is rapidly metabolised in plants, yielding products
    that are further degraded to normal plant building materials. When
    applied to soil, most of the dose is converted rapidly to CO2. There
    is no significant leaching of methoprene from treated soil and no
    uptake by plants. There appears to be no likelihood of methoprene
    being taken up by rice, forage or other crops growing in water treated
    for mosquito control.

         Methoprene residues may be determined by extraction with
    acetonitrile followed by clean-up on Florisil and alumina columns and
    quantitation by GLC with a flame ionization detector. The method
    appears suitable for regulatory purposes. The limit of determination
    ranges from 0.001 mg/kg to 0.01 mg/kg.

         The meeting was aware that MRLs had been established for a range
    of commodities in the USA and that others were under consideration.

    RECOMMENDATIONS

         The meeting recognized that there were, as yet, only a limited
    number of registrations for the use of methoprene but that potentially
    important developments might not proceed in the absence of appropriate
    maximum residue limits. To assist these further developments and to
    cater for existing uses, the following temporary MRLs are recommended
    to apply to residues of methoprene resulting from the direct
    application of methoprene or the feeding of methoprene-treated
    commodities. They apply to methoprene only.

                                                                

    Commodity                      MRL (mg/kg)
                                                                

    Cereal grains                  10
    Bran                           20
    Flour (wholemeal)              10
    Flour (white)                  5
    Carcase meat                   0.2 (in the carcase fat)
    Meat byproducts                0.1
    Eggs                           0.05
    Milk                           0.002
    Peanuts                        2
    Mushrooms                      1
                                                                


    FURTHER WORK OR INFORMATION

    Required  (before maximum residue limits can be confirmed):

    1.   Information from studies (now in progress) in which methoprene is
         used for the control of pests of stored grain.

    2.   Studies to define the rate of degradation of methoprene in stored
         products under a range of conditions.

    3.   Further information on the fate of methoprene residues in cereal
         grains and other stored products as a result of processing and
         cooking.

    4.   Information from current trials with stored dried lentils, beans,
         dried fruit, cocoa beans, coffee beans, spices and oilseeds to
         define the treatment necessary, the rate of degradation and the
         fate of residues in processing and cooking.

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    See Also:
       Toxicological Abbreviations
       Methoprene (Pesticide residues in food: 1984 evaluations)