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    2,4-D         JMPR 1974

    Explanation

          2,4-D was evaluated at the joint Meetings in 1970 (FAO/WHO, 1971)
    and 1971 (FAO/WHO, 1972). At the 1971 Meeting an ADI of 0-0.3 mg/kg
    was allocated and a tolerance of 0.02 ppm was established for barley
    oats, rye and wheat. Further work or information was desired on the
    metabolism and excretion of 2,4-D in animal species other than the rat
    and the occurrence of 2,4-D residues in crops following uses other
    than on cereals. Since the last evaluation (FAO/WHO, 1972b), further
    information on the use and residues of 2,4-D has become available and
    is summarized in the following monograph addendum.

    IDENTITY

    Purity

          No new information on the nature of the impurities occurring in
    commercial preparations of 2,4-D was available to the Meeting.
    However, the undated paper by Woolson et al. discussed in the 1971
    Monograph has been published (Woolson et al., 1972).

    EVALUATION FOR ACCEPTABLE DAILY INTAKE

          The compound was reconsidered by this Meeting on the basin of the
    published experimental data. Based upon this information there is no
    indication to change the previously allocated acceptable daily intake
    for man (0-0.3 mg/kg b.w.).

          A survey of "dioxin" content in 2,4-D has shown that the
    manufacturing process for 2,4-D in itself obviates the likelihood of
    the formation of "tetra, hexa and octadioxin" but it does not rule out
    the occurrence of other impurities.

    RESIDUES IN FOOD AND THEIR EVALUATION

    USE PATTERN

          The citrus industry has three registered uses for 2,4-D. One, as
    a pre-harvest orchard spray for oranges, grapefruit, and other citrus
    fruits to promote size and to prevent late season fruit drop. Two, as
    a post-harvest flooding or drip for lemons to retain the buttons
    (calyx) in a green condition and hence to increase the vitality of the
    fruit, thus retarding fungal growth during storage. Three, as a low
    pressure spray to the grove floor to control vine and broadleaf weeds.

          The use of 2,4-D as a growth regulator to promote size and to
    control fruit and leaf drop was reported by Hield et al. (1964) and
    Sarvoski and Stannard (1974) and is recommended for California citrus
    crops (California Agricultural Extension Service, 1974).

          The washing, dipping or flooding of lemons with 2,4-D where fruit
    is to be held in storage is a standard procedure and is effective in
    reducing Alternaria rot (De Wolfe et al., 1959). This use has
    reduced Alternaria from being one of the most serious fungus
    diseases of stored lemons to a minor cause of storage decay, and
    maintains the buttons in a sound green condition which helps the fruit
    retain its fresh appearance.

          2,4-D is registered for use on potatoes as a growth regulator in
    the U.S.A. It in claimed to reduce the yield of large, less desirable,
    tubers and increase that of the preferred medium size (Nelson and
    Nyland, 1963), and to intensify the skin colour on red varieties of
    potatoes (Nyland, 1956).

          Reports on the use of 2,4,D for the control of Eurasian water
    milfoil (Wojtalik et al., 1971) and water hyacinth (Schultz and
    Whitney, 1974) in aquatic environments have been published.

          Examples of some use patterns of 2,4-D are given in Table 1.

    RESIDUES RESULTING FROM SUPERVISED TRIALS

          Data are available on 2,4-D and 2,4-dichlorophenol residues in
    milk and cream from cows fed levels of 10 to 1000 mg/kg in their
    ration (Bjerke et al., 1972). Residues above the limit of
    determination (0.05 mg/kg) were found only at the highest dosing level
    (Table 2). Low residues of 2,4-D (0.6 mg/kg) were found in milk from
    cows grazing on pasture sprayed with 2,4-D (Klingman et al., 1966).
    Residue levels of 2,4-D in fish varied from <0.01 to 0.16 mg/kg in 60
    samples of fish taken from 3 different locations at various intervals
    following spraying of an aquatic environment (Shultz and Whitney,
    1974). Further details of all three experiments are given in the
    section "Fate of residues in animals."

          Washington navel orange trees were sprayed with 20 mg/kg (acid
    equivalent) 2,4-D (isopropyl ester) as practised for growth regulation
    and preharvest fruit drop. Orange samples were taken before, 1 day
    after and 7 days after spraying. Residues of 2,4-D averaged <0.1
    mg/kg before, 0.1 mg/kg 1 day after, and <0.1 mg/kg 7 days after
    treatment (Erickson and Hield, 1962).

          Grapefruit and pineapple orange plots were sprayed with 2,4-D
    (butoxyethyl ester) for weed control. Orange and grapefruit samples
    were picked 5 mouths after treatment. Residues of 2,4-D were below the
    limit of detection (<0.01 mg/kg) in all samples of pulp and peel from
    washed and unwashed fruit analyzed (Phillips, 1969).



        TABLE 1.

    Some examples of use patterns of 2,4-D.

                                                                                                           

                                                                      Dosage levels            Pre-harvest
    Crop             Use                     Material Used            (acid equivalent)        interval
                                                                                                           

    Citrus           pre-harvest fruit       2,4-D (isopropyl         4-24 mg/kg (spray)       7 days
                     drop and growth         ester, ethyl ester,
                     regulator               amine salt)

    Lemons           increase storage        2,4-D (isopropyl         500 mg/kg                -
                     life, retard            ester)
                     fungal growth

    Citrus           control vine and        2,4-D (butoxyethyl       2.2 kg/ha                5 months
                     broadleaf weeds         ester)

    Potatoes         growth regulator        2,4-D (propylene         70-140 g/ha              1 month
                                             glycol butyl
                                             ether ester)

    Aquatic          control Eurasian        2,4-D (dimethylamine     22 and 44 kg/ha          -
    environment      water milfoil           salt)

    Aquatic          control water           2,4-D                    4.48 kg/ha               -
    environment      hyacinth                (dodecyltetradecyl
                                             amine salts)

                                                                                                           

    TABLE 2.

    Residues of 2,4-D and 2,4-dichlorophenol in milk and cream from cows fed a ration containing
    1000 mg/kg of 2,4-D (Bjerke et al, 1972

                                                                                                       

    Days                  2,4-D (mg/kg)                             2,4-dichlorophenol (mg/kg)
    on diet       Milk, cow no.              Cream,               Milk, cow no.            Cream
                22        7        12        composite        22        7        12        composite
                                                                                                       

     3          0.05     0.06     <0.05      -                <0.05     0.06      0.05     -

    10          <0-05    0.08     <0-05      -                <0.05     0.06     <0.05     -

    17          0.05     0.11     0.05       0.12             <0.05     0.05     <0.05     <0-05

    18          0.05     0.12     <0-05      <0.05            <0.05     0.06     <0-05     <0.05

    19          0-05     0.09     <0.05      0.05             <0-05     0.06     <0-05     <0-05

    20          0.06     0.12     <0-05      0.06             0.08      0.06     <0.05     <0-05

    21          <0-05    0.07     <0.05      <0.05            <0.05     0.05     <0.05     <0-05

                                                                                                       
    


          Residue data for lemons treated with 2,4-D formulations to
    increase storage life and retard fungal growth were available from the
    U.S.A. (Erickson et al., 1963) and Australia (Johns, 1974). The U.S.A.
    data, summarized in Table 3, show that 2,4-D residue levels ranged
    from 0.25 to 0.56 mg/kg 1 day after treatment with different
    formulations at 500 mg/kg. These residues were persistent up to 42
    days. The storage conditions for the fruit were not given. In the
    Australian trial lemon fruit were treated with 2,4-D (sodium salt) at
    several rates and modes of application. The data shown in Table 4
    indicate that relatively low levels of 2,4-D occurred at the time of
    treatment at rates up to 500 Mg/kg. The residue levels had declined to
    <0.01 mg/kg at the end of 8 weeks storage. Only in the case of double
    treatment (two 500 mg/kg applications) were residues, 0.6 mg/kg,
    detected after 10 weeks storage.

          Potatoes treated with 2,4-D as a growth regulator (Bristol and
    Nelson, 1974) contained 2,4-D and 2,4-dichlorophenol residues of
    approximately 0.1 and 0.005 mg/kg respectively. The data are
    summarized in Table 5. Phenolic metabolites other than
    2,4-dichlorophenol were not detected. 2,4-D was stable in potatoes
    under conditions of frozen storage but residues in whole tubers stored
    at 38°C decreased with a half-life of approximately 12 weeks. See also
    "Fate of residues in plants," and "in storage."

    TABLE 3.  2,4-D residues in lemon fruit following treatment with various
              2,4-D formulations.

                                                                            

                                        Residue, mg/kg, in whole fruit
                                       at interval (days) after treatment
    Formulation1                       1         7         14        42
                                                                            

    2,4-D (emulsifiable acid)          0.42      0.33      0.35      0.22

    2,4-D (isopropyl ester)            0.56      0.27      0.31      0.22

    2,4-D (sodium salt)                0.30      0.39      0.50      0.33

    2,4-D (diethanolamine salt)        0.29      -         0.29      -

    2,4-D (triethanolamine salt)       0.25      -         0.39      -
                                                                            

    1  All treatments were applied as a wax emulsion at 500 mg/kg acid
       equivalent.


    
    TABLE 4.  2,4-D residues in lemon fruit following treatment with 2,4-D (sodium salt)

                                                                                                        

                                                      Residues in whole fruit (mg/kg),
                                                    at interval (weeks) after treatment

        Treatment            No. of             6 weeks (11°C)      8 weeks (11°C)      20 weeks (11°C)
    (2,4-D sodium salt)      Samples     0     +2 weeks (20°C)     +2 weeks (20°C)     + 2 weeks (20°C)
                                                                                                        

    100 mg/kg, wax foam        4        0.5          <0.01                <0.01              <0.01

    100 mg/kg, wax dip         4        0.5          <0.01                <0.01              <0.01

    200 mg/kg, war foam        4        0.6          <0.01                <0.01              <0.01

    300 mg/kg, war foam        4        0.8          <0.01                <0.01              <0.01

    500 mg/kg, wax foam        4        1.1          <0.01                <0.01              <0.01

    500 mg/kg, wax dip         4        1.3          <0.01                <0.01              <0.01

    500 mg/kg,
    pre-processing dip +
    500 mg/kg wax foam         1         -             -                   0.6                 -
                                                                                                        

    TABLE 5.  2,4-D residues in potatoes (Bristol and Nelson 1974)

                                                                                                            

                                                                          Residue, mg/kg
              Treatment
                rate        Variety         No. of               2,4-D                2,4-dichlorophenol
    Year      (g/ha)1      of potato        samples       Range         Average     Range           Average
                                                                                                            

    1972      0            Red Pontiac      10            <0.02         <0.02       <0.002          <0.002

    1972      140          Red Pontiac      10            0.06-0.10     0.08        <0.002-0.003    <0.002

    1972      280          Red Pontiac      10            0.07-0.14     0.10        <0.002-0.004    <0.002

    1973      0            Red Pontiac      4             <0.02         <0.02       <0.002          <0.002

    1973      140          Red Pontiac      4             0.09-0.14     0.11        0.003-0.005     0.004

    1973      0            Red Norland      4             <0.02         <0.02       <0.002          <0.002

    1973      140          Red Norland      4             0.07-0.11     0.09        0.006-0.011     0.008

    1973      0            Norland          4             <0.02         <0.02       <0.002          <0.002

    1973      140          Norland          4             0.07-0.11     0.09        0.004-0.009     0.006
                                                                                                            

    1  Spray applications were made in two equal treatments two weeks apart.
    

    FATE OF RESIDUES

    General comments

          Information was available from experiments with 2,4-D-14C that
    showed that 4-hydroxy metabolites were found in plants. These were
    present as glycosides as well as the free aglycones. Low levels of
    2,4-D residues are excreted in the milk of dairy cows fed high levels
    of 2,4-D in their ration. Information is lacking on the levels and
    fate of residues in tissues, soils and crops grown on contaminated
    soils. Some of the experiments described below are also mentioned in
    the section "Residues resulting from supervised trials."

    In animals

          Milk from cows grazing on pasture sprayed at the rate of
    2.2 kg/ha with either the isopropyl, 2-ethylhexyl, or butyl esters of
    2,4-D contained residues of 2,4-D ranging from 0.01 to 0.06 mg/kg
    during the first 2 days after spraying and declined to 0.01 mg/kg or
    lower by day 3 (Klingman et al., 1966). Analysis of forage samples
    from these trials showed 2,4-D residue levels after spraying of 59 and
    48 mg/kg for the butyl and 2-ethylhexyl ester treatments,
    respectively. The residues had declined to 5 and 15 mg/kg on day 7.

          Cows were fed rations containing 2,4-D at 10, 30, 100, 300 and
    1000 mg/kg levels for 2 or 3 weeks at each level (Bjerke et al.,
    1972). Residues were determined in samples of milk and cream from
    these cows during the feeding period and for 7 days following
    withdrawal at the highest feeding level. No residues of 2,4-D or
    2,4-dichlorophenol greater than 0.05 mg/kg (limit of determination)
    were found in milk or cream at the 300 mg/kg or lower feeding levels.
    Residues of 2,4-D and 2,4-dichlorophenol determined in milk and cream
    from cows fed at the 1000 mg/kg level are shown in Table 2. The
    highest residues found in any sample were 0.12 and 0.06 mg/kg of the
    acid and phenol respectively. No residues greater than 0.05 mg/kg were
    found in any of the samples taken during the withdrawal period. An
    acid hydrolysis extraction procedure was used to extract any phenoxy
    acid or phenol that might have been bound physically or chemically to
    natural constituents of the milk.

          2,4-D residues were determined in 5 different species of fish
    (total of 60 samples) taken from 3 different locations at various
    intervals following spraying of an aquatic environment with 2,4-D
    (dodecyl-tetradecyl amine salts) at a rate of 4.48 kg/ha (Scholtz and
    Whitney, 1974). Residue levels varied from <0.01 (limit of detection)
    to 0.16 mg/kg. Of the 60 samples analyzed, 3 contained residues of
    2,4-D greater than 0.1 mg/kg and 8 had residue levels between 0.01 and
    0.1 mg/kg.

    In Plants

          Residues arising in treated citrus are discussed above.
    ("Residues resulting from supervised trials.")

          Low residues of 2,4-D and 2,4-dichlorophenol (approximately 0.1
    and 0.005 mg/kg respectively) were found in potatoes following
    treatment with 2,4-D for growth regulation (Bristol and Nelson, 1974.)
    See Table 5. The potato samples were analyzed by two different
    procedures: a total residue analysis which involved hydrolysis with
    sulphuric acid before extraction, and a free residue procedure without
    the initial hydrolysis. Differences between total and free residue
    levels of 2,4-D were small but significant, indicating the presence of
    small amounts of conjugated and/or bound residues. Phenolic
    metabolites of 2,4-D other than 2,4-dichlorophenol were not detected
    in any of the potato samples. The small differences in residue levels
    between the different varieties of potato sampled in the 1973 test
    were not significant.

          Experiments with carboxyl - MC labelled 2,4-D acid indicated that
    part of the 2,4-D reacted with some plant constituents of lemons to
    produce an ester-like complex (Erickson et al., 1963).

          In bean plants treated with 2,4-D-1-14C the major metabolite was
    2,5-dichloro-4-hydroxyphenoxyacetic acid, while
    2,3-dichloro-4-hydroxyphenoxyacetic acid was a minor metabolite
    (Hamilton et al., 1971). These metabolites accumulated as glycosides
    but were also present as the free aglycones. The glycosides appeared
    to be relatively stable in bean plant tissue since they were still
    the predominant residue 22 days after treatment. Similar metabolites
    were found in wheat, barley, oats and soybeans but not in buckwheat
    or maize. In further experiments with soybean cotyledon callus tissue
    cultures Feung et al, (1971) confirmed that the major water-soluble
    metabolites were similar to those in bean plants. In addition a major
    ether-soluble metabolite was identified as the 2,4-D glutamic acid
    conjugate. Chkanikov et al., 1971) used 2,4-D-2-14C to study the
    water-soluble metabolites of maize and bean plants. They confirmed
    that the major metabolites in bean plants were glucosides of
    hydroxylated 2,4-D and that the metabolism in bean plants differs from
    that in maize.

    In storage

          Chopped potato samples were fortified with 0.05, 0.1 and 0.2
    mg/kg levels of 2,4-D before being put into frozen storage (Bristol
    and Nelson, 1974). Analysis of samples removed from storage at times
    varying from 12 to 73 weeks gave an average recovery of 87.6 ± 2.3%
    for 26 of these samples compared with 87.9 ± 3.0% before storage,
    demonstrating that 2,4-D was stable under these conditions. In another
    study residue levels of 2,4-D decreased with a half-life of
    approximately 12 weeks, while those of 2,4-dichlorophenol remained low
    throughout the period, in whole potatoes stored at 38°C. The data are
    shown in Table 6.

    
    TABLE 6.  Effect of storage at 38°C on 2,4-D residues in potatoes
              (Bristol and Nelson, 1974)

                                                                                      

                                        Residue, mg/kg.
    Storage
    period,            Control Sample                     Treated Samples1
    weeks          2,4-D     2,4-dichlorophenol       2,4-D        2,4-dichlorophenol
                                                                                      

    0              <0.02     <0.002                 0.10, 0.14     0.005, 0.005

    16             <0.02     <0.002                 0.04, 0.05     0.004, 0.005

    20             <0.02     <0.002                 0.04, 0.03     0.006, 0.005

    25             <0.02     <0.002                 0.03, 0.02     0.004, 0.005

    30             <0.02     <0.002                <0.02, <0.02    0.003, 0.004

    35             <0.02     <0.002                <0.02, <0.02    0.005, 0.004
                                                                                      

    1 Individual residue data are given for each of two treated samples.
    
    METHODS OF RESIDUE ANALYSIS

          The chromatographic methods of analysis of 2,4-D residues have
    been reviewed by Cochrane and Purkayastha (1973). This review covers
    extraction, clean-up, derivation and GLC conditions for residue
    determinations in water, soil, plant material, animal tissue and fish.

          Methods for the analysis of 2,4-D residues in animal and plant
    tissue, soil and water have been made available since the 1970
    evaluation and these are summarized in Table 7. Only 2 methods (Bjerke
    et al., 1974; Bristol and Nelson 1974) included the 2,4-dichlorophenol
    metabolite. The extraction procedures range from extraction with
    acetone for plant tissue to alkali and acid hydrolysis for mussel
    tissues. It is known that 2,4-D conjugates with plant constituents
    (Hamilton et al., 1971) and most of these procedures probably do not
    hydrolyse the conjugates. Development and validation of extraction
    procedures to determine total 2,4-D residues including 2,4-D acid,
    2,4-D-dichlorophenol and conjugates of each are needed.


        TABLE 7.  Methods for the determination of 2,4-D residues

                                                                                                                                               

                                                                                                 Sensitivity        Recovery
    Substrate     Extraction        Clean-up              Esterification        Determination1   (mg/kg)            (%)               Reference
                                                                                                                                               

    Fish          Hexane/           Acid-hexane/          2-chloroethanol/      GLC-ED           0.001              70                Renberg,
                  acetone           ether partition;      sulphuric acid;                                                             1974
                                    Sephadex              diazomethane
                                    QAE ion exchange

    Fish          Methanol/         Acid/                 diazomethane          GLC-E Cond.      0.01               90 ± 2.5          Schultz
                  phosphoric        alkalichloroform                                                                                  and
                  acid              partition                                                                                         Whitney,
                                                                                                                                      1974

    Fish,         Alkali and acid   Acid/buffer-ether/    boron tri-fluoride/   GLC-EC           0.1                -                 Wojtalik
    mussels       hydrolysis of     petroleum ether       methanol                                                                    et al.,
                  ethanol extract   partition;                                                                                        1971
                  of fish alkali    Florisil
                  and acid          column
                  hydrolysis of
                  mussel tissue.

    Meat,         Ethanol/          Acid/                 diazomethane          GLC-EC;          0.01               74-98             Yip,
    fish,         sulphuric         alkalichloroform                            GLC-MC                                                1971
    poultry,      acid/chloroform   partition;
    dairy                           Florisil
    products                        column after
                                    esterification

    Milk,         Acid              Alumino               diazomethane          GLC-EC;                                               Bjerke
    cream         hydrolysis,       column, acid/         (2,4-D only)          GLC-MC           0.05               90-100            at al.,
                  ether             alkali-benzene                                                                  (2,4-D);          1972
                                    partition for                                                                   84-100
                                    2,4-dichlorophenol;                                                             (2,4-
                                    acid-ether                                                                      dichlorophenol)
                                    partition for
                                    2,4-D

    TABLE 7.  (Cont'd.)

                                                                                                                                               

                                                                                                 Sensitivity        Recovery
    Substrate     Extraction        Clean-up              Esterification        Determination1   (mg/kg)            (%)               Reference
                                                                                                                                               

    Plant         Acetone           Acid/                 methanol/             GLC-MC           0.01               72-96             Munro,
    tissues                         buffer-chloroform     sulphuric                                                                   1972
                                    partition; sweep      acid
                                    co-distillation
                                    after esterification

    Oranges,      Acetone           Acid/                 dimethyl              GLC-EC           0.01               73-95             Phillips,
    grapefruit                      buffer-chloroform     sulphate                                                                    1969
                                    partition

    Cereals,      35%               Acid/alkali           diazomethane          GLC-EC;          0.01               84-100            Yip
    fruits,       aqueous           chloroform            (2,4-D)               GLC-MC                                                1971
    vegetables    acetonitrile      partition;
                  or                Florisil column
                  acetonitrile      after
                                    esterification

    Potatoes      Alkali            Acid/                 diazomethane          GLC-E Cond.      0.02 (2,4-D);      87.9 ± 3.0        Bristol
                  extraction;       alkali-benzene        (2,4-D)                                                   (2,4-D);          and
                  acid              partition;                                                   0.002 (2,4-        84.5 ± 5.7        Nelson,
                  hydrolysis        alumina column;                                              dichlorophenol)    (2,4-             1974
                                    acid/alkali-benzene                                                             dichlorophenol
                                    partition for
                                    2,4-dichlorophenol;
                                    acid-ether
                                    partition for 2,4-D

    Barley        Acid and          Partition             diazomethane          GLC-EC           0.02               75-82             Lokke,
                  enzymic           into ether/                                                                                       1974
                  hydrolysis        hexane;
                                    alumina
                                    column

    TABLE 7.  (Cont'd.)

                                                                                                                                               

                                                                                                 Sensitivity        Recovery
    Substrate     Extraction        Clean-up              Esterification        Determination1   (mg/kg)            (%)               Reference
                                                                                                                                               

    Soil          Alkali            Sephadex              2-chloroethanol/      GLC-EC           0.001              70-74             Renberg,
                  extraction        QAE ion               sulphuric acid;                                                             1974
                                    exchange              diazomethane
                                    column

    Soil          Diethyl           Acid/alkali-ether     boron trichloride/    GLC              0.01               84-103            Woodham
                  ether/            partition             2-chloroethanol                                                             et al.,
                  sulphuric                                                                                                           1971
                  acid

    Soil          (a) Ethanol/      Acid/alkali-ether     diazomethane;         GLC-EC           0.03-0.05          84-100            Purkayastha,
                  sulphuric         partition             diazobutane                                               (methyl           1974
                  acid/di-ethyl                                                                                     ester);
                  ether

                  (b) Alkali/                                                                                       76-92
                  chloroform                                                                                        (butyl
                                                                                                                    ester)

    Sediment      Acetone/          Acid/                 Boron trifluoride/    GLC-EC           0.1                -                 Wojtalik
                  petroleum         buffer-chloroform     methanol                                                                    at al.,
                  ether/                                                                                                              1971
                  sulphuric acid/
                  ethyl ether

    Water         -                 Sephadex QAE          2-chloroethanol/      GLC-EC           0.0001             97                Renberg,
                                    ion exchange          sulphuric acid;                                                             1974
                                    column                diazomethane

    Water         Diethyl           Alkali/               diazomethane;         GLC-EC           0.05               82-94             Purkayastha,
                  ether/            buffer-diethyl        diazobutane                                                                 1974
                  sulphuric         ether
                  acid

    TABLE 7.  (Cont'd.)

                                                                                                                                               

                                                                                                 Sensitivity        Recovery
    Substrate     Extraction        Clean-up              Esterification        Determination1   (mg/kg)            (%)               Reference
                                                                                                                                               

    Water         Chloroform/       -                     diazomethane          GLC-E Cond.      0.001              97.5 ± 2.5        Schultz
                  sulphuric                                                                                                           and
                  acid                                                                                                                Whitney,
                                                                                                                                      1974

    Water         Alkali            Acid/sodium           Boron trifluoride/    GLC-EC           0.001              -                 Wojtalik
                  hydrolysis;       sulphate-             methanol                                                                    et al.,
                  chloroform/       chloroform                                                                                        1971
                  phosphoric        partition
                  acid
                                                                                                                                               

    1  GLC-EC = GLC - electron capture detection

       GLC-MC = GLC - microcoulometric detection

       GLC-E Cond = GLC - electrolytic conductivity
    

    TABLE 8.  Comparison of three extraction procedures for determining
              2,4-D in barley (Lokke, 1974)

                                                                              

                  2,4-D, mg/kg, in field-treated barley
    Extraction                                             Recovery of 2,4-D
    Procedure     Sample 1    Sample 2     Sample 3        Added to barley, %
                                                                              

    Lokke,
    1974          0.06        0.45         4.3             75-82

    Yip,
    1971          0.04        0.35         4.0             68-102

    Ether/
    hexane        <0.01       0.04         0.51            72-95

                                                                              

          Most of the clean up procedures require an acid/alkali-organic
    solvent partition step. The methods that include the
    2,4-dichlorophenol employ an alumina column step to separate the
    phenol, which is determined separately from 2,4-D. Esterification of
    the acid is usually with diazomethane but other reagents such as boron
    trifluoride/methanol, 2-chloroethanol/sulphuric acid, 
    methanol/sulphuric acid and diazobutane have also been used. A
    Florisil column (Yip, 1971) and sweep co-distillation (Munro, 1972)
    have been used for further clean-up of the esterified extract.

          GLC with the electron capture, microcoulometric or electrolytic
    conductivity detectors were used as the determinative step.
    Sensitivities claimed ranged from 0.1 to 0.0001 mg/kg. Yip (1971)
    reported lower background responses and generally higher recoveries
    with the microcoulometric detector than with the electron capture
    detector. The increased specificity would make either the
    microcoulometric or electrolytic conductivity the detector of choice.
    Recoveries of >70% were reported for all methods.

          Lokke, 1974 compared three methods for the determination of 2,4-D
    in barley grain. Barley was field-treated with 2,4-D to obtain samples
    with weathered residues. The extraction procedures used were acid and
    enzymic hydrolysis (Lokke, 1974, Table 7), 35% aqueous acetonitrile
    (Yip, 1971, Table 7) and extraction with ether/hexane. The results
    from the study are summarized in Table 8 and show that the extraction
    procedures of Lokke, 1974 and Yip, 1971 give similar results but the
    ether/hexane extraction procedure gave residues lower by a factor of
    10. The three procedures gave essentially the same recovery values
    from fortified samples. These investigations demonstrate the
    importance of the extraction procedure. Lokke, 1974 reported that
    further analysis of the filter cakes from both the Lokke and Yip
    extraction procedures indicated still unhydrolyzed 2,4-D conjugates.

          Although the clean-up, esterification and determinative steps for
    2,4-D residues appear to be satisfactory, extraction Procedures
    require development and validation, as mentioned above.

    NATIONAL TOLERANCES REPORTED TO THE MEETING

          Some examples of national tolerances were reported to the Meeting
    and are listed in Table 9.

    TABLE 9.  Examples of national tolerances reported to the meeting

                                                                            
                                                                Tolerance
    Country        Commodity                                    mg/kg
                                                                            

    Australia      Apples, apricots, beets, carrots,
                   citrus, corn, cucumbers, grapes,
                   melons, onions, peaches, pears,
                   peppers, plums, potatoes, tomatoes           5

    Belgium        Apples, apricots, boots, carrots,
                   citrus, corn, cucumbers, grapes,
                   melons, onions, peaches, pears,
                   peppers, plums, potatoes, tomatoes           0.05

    Canada         Asparagus                                    5

                   Barley, corn, flax, oats, rye,               Negligible
                   strawberries, wheat                          Residue

    Fed. Rep.      Apples, apricots, beets, carrots,
    of Germany     citrus, corn, cucumbers, grapes,
                   melons, onions, peaches, pears,
                   peppers, plums, potatoes, tomatoes           0.1

    Netherlands    Apples, apricots, beets, carrots,
                   citrus, corn, cucumbers, grapes,
                   melons, onions, peaches, pears,
                   plums, potatoes, tomatoes                    0.05

    Switzerland    Apples, apricots, citrus, grapes,
                   melons, peaches, pears, plums                0.05

    U.S.A.         Forage of barley, oats, rye,
                   and wheat                                    20

                   Apples, asparagus, citrus, pears,
                   quinces                                      5

                   Barley, oats, rye, wheat                     0.5

    TABLE 9.  (Cont'd.)
                                                                            

                                                                Tolerance
    Country        Commodity                                    mg/kg
                                                                            

                   Potatoes                                     0.2

                   Cucurbits, forage grasses,
                   forage legumes, fruiting
                   vegetables, leafy vegetables,
                   nuts, root crop vegetables, seed
                   and pod vegetables, small fruits,
                   stone fruits, and the individual
                   raw agricultural commodities,
                   avocados, cotton-seed, hops, and
                   strawberries                                 0.1
                                                                            

    APPRAISAL

          Information was presented to the Meeting on several uses of 2,4-D
    on citrus: for weed control, as a pre-harvest growth regulator and for
    post-harvest treatment of lemons and mandarins to retain buttons and
    hence prolong storage life and assist in control of fungus. It is also
    used on potatoes as a growth regulator, and in aquatic environments
    for weed control.

          2,4-D translocates into plants from the soil and from topical
    application. Metabolites of 2,4-D accumulate as glycosides and are
    also present as the free aglycones. In bean plants,
    2,5-dichloro-4-hydroxyphenoxyacetic acid and
    2,3-dichloro-4-hydroxyphenoxyacetic acid were identified as major
    and minor metabolites respectively. Similar metabolites were found in
    wheat, barley, oats and soybeans but not in buckwheat or maize. Small
    amounts of conjugated and/or bound residues of 2,4-D were found in
    potatoes and lemons. Low levels of 2,4-dichlorophenol were detected
    in potatoes and in milk from cows.

          Residue data indicated that low residues occurred from the
    pre-harvest use of 2,4-D on citrus and potatoes. The post-harvest use
    on lemons resulted in residues of <2 mg/kg.

          Low residue levels of 2,4-D occurred in the milk of cows fed
    rations containing high levels of 2,4-D (1000 mg/kg) in their diet.
    Residues decreased to below detectable levels on withdrawal of 2,4-D
    from the diet.

          Experiments with potatoes demonstrated that 2,4-D was stable for
    periods exceeding 1 year under conditions of frozen storage. 2,4-D
    residues in whole potatoes stored at 38°C decreased with a half-life
    of approximately 12 weeks.

          Gas-chromatographic methods utilizing electron capture,
    micro-coulometric and/or electrolytic conductivity detection should
    be adaptable for regulatory purposes. A hydrolysis procedure prior
    to extraction is required to liberate conjugated and/or bound
    residues. Additional clean-up steps before and after esterification
    appear to be of advantage for some samples.

          National tolerances are in effect in a number of countries.

    RECOMMENDATIONS

          In addition to the tolerances of 0.02 mg/kg for barley, oats, rye
    and wheat recommended at the 1971 Joint Meeting (FAO/WHO, 1972), the
    following tolerances are recommended.

    TOLERANCES

                                Tolerance
          Commodity              mg/kg

          Citrus                2
          Potatoes              0.21
          Milk                  0.052

    1 The tolerance is based on a pre-harvest interval of 30 days

    2 At or about the limit of determination

    FURTHER WORK OR INFORMATION

    Desirable

    1.    Information on the storage and fate of 2,4-D in food animals.

    2.    Information on the fate of 2,4-D in soils and residue data on
          crops grown on contaminated soil.

    3.    Development of extraction procedures to determine total 2,4-D
          residues including 2,4-D acids, 2,4-dichlorophenol and their
          conjugates.

    REFERENCES

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    Bristol, D. and Nelson, D.C. (1974). Residue levels of
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    See Also:
       Toxicological Abbreviations
       D, 2,4- (AGP:1970/M/12/1)
       D, 2,4- (WHO Pesticide Residues Series 1)
       D, 2,4- (WHO Pesticide Residues Series 5)
       D, 2,4- (Pesticide residues in food: 1980 evaluations)