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    2,4-D         JMPR 1975

    Explanation

         The Joint Meeting in 1970 considered the use of 2,4-D as a
    selective herbicide on a range of cereal crops and data on the nature
    and fate of residues therein (FAO/WHO 1971). Recommendations were not
    made for maximum residue limits until an acceptable daily intake was
    established in 1971. (FAO/WHO 1972). In 1974 the use of 2,4-D as a
    pre- and post-harvest treatment for citrus fruit and as a growth
    regulator for potatoes was also evaluated and recommendations were
    made (FAO/WHO 1975).

         In view of the world-wide use of 2,4-D herbicides it was
    considered that the residue data, originating mainly in Europe,
    examined in 1970 possibly did not reflect the practices in countries
    with more extensive agriculture. Requests were therefore made for
    further data on the nature and level of 2,4-D residues in cereal
    crops.

         The Codex Committee on Pesticide Residues at its 8th Session in
    1975 expressed the view that the maximum residue limits proposed were
    too low (ALINORM 76/24 para 85). This monograph addendum confirms the
    necessity for a higher limit for 2,4-D residues in raw grain.

         The additional data which became available are summarized in this
    monograph addendum.

    EVALUATION FOR ACCEPTABLE DAILY INTAKE

    BIOCHEMICAL ASPECTS

    Absorption, distribution and excretion

         Following a single administration by gastric intubation to albino
    rats of sodium and diethylamine salts of 2,4-D at the doses of 555 and
    405 mg/kg respectively (the doses corresponding to the LD50 for these
    compounds) the highest blood concentrations were reached after 3
    hours. The half-retention periods were of 1 hour for the sodium salt
    and 72 hours for the diethylamine salt. Three hours after treatment
    the organs with the greatest distribution of the diethylamine salt
    were the liver and kidneys; the highest levels of this salt in tissues
    (heart, liver, kidney, muscles and brain) were observed 24 hours after
    administration and there was a substantial decrease after 10 days
    after the treatment.

         In the same study, albino rats were given by gastric intubation,
    daily doses corresponding to 1/20 and 1/5 respectively of the LD50 of
    2,4-D (23 and 91 mg/kg), sodium salt of 2,4-D (28 and 111 mg/kg),
    diethylamine salt of 2,4-D (20 and 80 mg/kg) and
    2-methyl-4-chlorophenoxypropionic acid (49 and 1975 mg/kg) for a
    period of 50 days.

         The tissue distribution (heart, liver, kidneys, muscles and
    brain) at the end of the testing period was found to be dose dependent
    (Buslovish et al., 1973)

    TOXICOLOGICAL STUDIES

    Special studies in teratogenicity

         Groups of 3-month old NMRI female mice randomly bred were
    injected subcutaneously with levels of 0, 50, 100 mg/kg bw of
    commercial mixture of 2,4-D and 2,4,5-T in dimethyl sulfoxide solution
    from day 6 through day 14 of gestation. The content of dioxins of the
    mixture was less than 1 ppm and the most common dioxin was
    2,3,7,8-tetrachlorodibenzo-p-dioxin. On day 18 of gestation, the
    animals were sacrificed, living foetuses removed, weighed and examined
    for gross malformation; microscopic examination for internal
    malformation was also carried out. At high dose foetal mortality was
    significantly increased, the frequency of cleft palate was increased,
    foetal weight considerably reduced, skeletal malformations were double
    with respect to the other group and controls, subcutaneous bleeding
    was three times as common, renal malformation such as dilated renal
    pelves, smaller and opaque medullae were slightly increased (Bage et
    al. 1973).

    OBSERVATIONS IN MAN

         Seventeen farm workers concerned with the transport of
    derivatives of chlorophenoxy acetic acid were examined by
    electroencephalography before and after massive exposure. EEG tracings
    before exposure showed slight abnormalities in 7 cases. After 4 to 28
    days of exposure disturbances of the cerebral bioelectrical activities
    were observed in 11 cases. In 5 of these cases overt pathological
    changes were observed in EEG tracings as revealed by the slowing down
    of the basal frequency and the presence of slow, high amplitude theta
    waves (Kontek et al., 1973).

         A man lost consciousness 2-3 hours after inhalation of vapours
    originating from a spraying pump containing a herbicide formulation
    (44% 2,4-D). Early manifestations included urinary incontinence and
    vomiting followed by myalgia, muscular hypertonia, slight fever,
    headache and constipation as well as intermittent nodal tachycardia as
    revealed by EEG. The cardiac arrhythmia and muscular hypertonia
    gradually subsided within 5 days of administration of quinidine
    hydrochloride (450 mg/day, 7 days). No abnormalities in the thyroid
    functions were observed. The patient was released from hospital in
    good health after 23 days (Paggiaro et al., 1974).

    Comments

         An ADI for man of 0 - 0.3 bw was allocated for this compound at
    the 1971 Joint Meeting.

         Since then further studies have become available on tissue
    distribution after gastric intubation to rats of sodium and
    diethylamine salts of 2,4-D. These studies supplement earlier studies
    in rodents.

         A teratology study in mice administered 2 4-D by subcutaneous
    injection showed no embryotoxic or teratogenic effect at 50 mg/kg body
    weight. Effects were found at 100 mg/kg.

         The results confirmed the no-effect level in earlier oral studies
    in rodents.

         Reports on apparent effects from occupational exposure to phenoxy
    herbicides were noted.

         The previously estimated ADI was reaffirmed.

    TOXICOLOGICAL EVALUATION

    Level causing no toxicological effect

         Rat: 625 ppm in the diet equivalent to 31 mg/kg bw

    Estimate of acceptable daily intake for man

         0 - 0.3 mg/kg bw

    RESIDUES IN FOOD AND THEIR EVALUATION

    USE PATTERN

         No information on new use patterns has become available and, in
    view of the fact that 2,4-D herbicides have been in world-wide use for
    more than 25 years it is assumed that there have been no now
    developments.

         Depending on the type of cereal crop, the weed spectrum, cultural
    practices and climatic factors, 2,4-D herbicides may be applied as
    salts, esters, amines or free acid formulations at rates ranging from
    250 g to 2 kg/ha (rarely up to 4 kg/ha). Application is usually made
    when weeds and cereal crop plants are small but in the case of maize
    (corn) and sorghum, treatment may be necessary when the crop is well
    advanced.

         With some varieties of wheat early treatment is essential to
    avoid phytotoxic damage which may occur once the seed-head has begun
    to develop. Usually wheat and oats are treated when the crop is 10-15
    cm tall and maize (corn) when 30-35 cm tall.

         However, when tall weeds growing up in the crop make harvesting
    difficult, if not impossible, 2,40 herbicides nay be applied 14-21
    days prior to harvest to destroy, defoliate or regulate the growth of
    the weeds so as to prevent interference with harvesting.

         During 1966 the US production of 2,4-D exceeded 31 000 000 kg
    (USA 1967). World consumption is estimated to exceed 50 000 000 kg.

    RESIDUES RESULTING FROM SUPERVISED TRIALS

    Cereal grains

         Data derived from supervised residue trials carried out in
    various regions of the USA have been provided by the US Department of
    Agriculture. These data were generated during the years 1960, 1961 and
    1965 and represent typical use patterns involving amine salt, butyl,
    iso-octyl, isopropyl and polypropylene glycol butyl ether ester
    formulations of 2,4-D. The residue analysis was carried out by the US
    FDA.

    Early post-emergence application

         Grain samples collected from plots sprayed 3-4 weeks after
    emergence with various formulations of 2,4-D were found to contain no
    residues of 2,4-D (limit of determination of 0.01-0.04 mg/kg) except
    in the case of oats, and then only when 4 kg/ha was applied. Wheat
    grain from crops receiving the same heavy application was free from
    residues.

    Pre-harvest application

         Trials carried out in Kansas during 1960 involved the application
    of butyl, isopropyl and iso-octyl esters of 2,4-D at 1 and 10 kg
    2,4-D/ha at intervals ranging from 5 to 23 days before harvest.

         The residue levels found are set out in Table 1.

         The data in Table 1 indicate that there is a noticeable
    accumulation of 2,4-D residues in the grain during the two weeks
    following application when treatment is made within 3 weeks of
    harvest. This is not unexpected in view of the systematic properties
    of the herbicide. However the amount of 2,4-D found in grain is not
    particularly great following normal rates of application, reaching
    levels above 0.5 mg/kg only at excessive rates of the order of
    10 kg/ha.

    TABLE 1.  Residues of 2,4-D in wheat following application of 2,4-D ester

                                                                        

                                Pre-harvest interval
    Ester       Rate. kg/ha             days             Residue, mg/kg
                                                                        

    butyl             1                   15                  0.07

                      1                   10                  0.05

                      1                    5                  0.06

                     10                   15                  2.45

                     10                   10                  1.50

                     10                    5                  0.60


    isopropyl         1                   17                  0.05

                      1                   11                  0.04

                      1                    5                  0.02

                     10                   17                  0.22

                     10                   11                  1.18

                     10                    5                  0.44


    Iso-octyl         1                   23                  0.09

                      1                   18                  0.40

                      1                   13                  0.27

                      1                    8                  0.05

                     10                   23                  0.73

                     10                   18                  1.06

                     10                   13                  0.84

                     10                    8                  0.51
                                                                        

         The tendency to accumulate in grain during the post-application
    period was confirmed by trials in Nebraska in 1960, in which the
    residues resulting from treatment 20 days before sampling were
    compared with those in the grain at application. The data presented in
    Table 2 show that the residue level is higher after 20 days and
    increases as the rate of application is increased.

         From the limited data it would appear that slightly more residues
    accumulate from the use of the water-soluble amine formulation than
    from the oil soluble ester form. Volatility of the latter could play a
    part.


    TABLE 2.  Residues of 2,4-D in grain on day of application and after
              20 days

                                                                         

                                 Pre-harvest interval
    Formulation    Rate, kg/ha          (days)             Residues, mg/kg
                                                                         

    ester               0.5                  0                  0.04

                          1                  0                  0.11

                          2                  0                  0.11

    amine                 1                  0                  0.05

    ester               0.5                 20                  0.09

                          1                 20                  0.14

                          2                 20                  0.34

    amine                 1                 20                  0.27
                                                                         


         Trials carried out in Kansas compared the residues of 2,4-D
    resulting from the use of the water-soluble dimethylamine salt with
    those from the volatile isopropyl ester and the less volatile
    iso-octyl ester. The results, which are presented in Table 3,
    generally support the observation that lower residues result from the
    use of the volatile ester than from the amine salt and the
    less-volatile ester. The difference is, however, not great.

    TABLE 3.  2,4-D Residues on Bison Wheat harvested at Hays, Kansas,
              at various intervals after treatment

                                                                            

              Residue, mg/kg after pre-harvest interval, days
                                                                            

                   7                   14                   21
                                                                            

    Dimethylamine salt

    0.0 lb/acre    0.05                0.01                 0.02

    0.5            0.16, 0.08, 0.10    0.19, 0.18           0.06, 0.06

    1.0            0.29, 0.06          0.31, 0.15           0.09, 0.12

    2.0            0.39                0.50                 0.29, 0.22

    4.0            1.24                1.46                 0.32

    Isopropyl ester

    0.0            0.07                0.02                 0.01

    0.5            0.05, 0.16, 0.11    0.03, 0.06, 0.03     0.04, 0.04, 0.05

    1.0            0.14, 0.22          0.15, 0.09           0.06, 0.04, 0.06

    2.0            0.21, 0.49          0.31, 0.31           0.13, 0.17

    4.0            0.78                1.80                 0.38

    Iso-octyl ester

    0.0            0.02, 0.02          0.0, 0.01            0.0, 0.03

    0.5            0.12, 0.12 0.10     0.15, 0.11           0.04, 0.06, 0.07

    1.0            0.18, 0.26, 0.36    0.35, 0.23, 0.23     0.09, 0.05, 0.16

    2.0            0.35, 0.45          0.63                 0.15, 0.22

    4.0            1.31                1.16                 0.53
                                                                            

         Cochrane and Russell (1975), using the extraction procedure of
    Yip (1962) found that when mixed n-butyl/isobutyl ester was applied at
    the rate of 430 g/ha to young wheat plants (three-leaf), the ester was
    immediately converted into free 2,4-D acid. Immediately after
    application 8.35 mg/kg of 2,4-D was found in the wheat plants and this
    level declined to 1.87 mg/kg on the third day after treatment.
    Thereafter decline continued at a rapid rate to less than 0.01 mg/kg
    after 44 days. No trace of 2,4-D could be found in grain or straw at
    harvest 80 days after treatment.

         Studies made at the University of California (Harvey, 1970) of
    the residues resulting from the application of 2,4-D amine to barley,
    oats and wheat at various growth stages have revealed that the
    residues in grain at harvest vary according to the growth stage at
    time of treatment. These data indicate that even though the residue in
    the whole plant exceeded 10 mg/kg 7 days after application, there was
    a steady decline in the residue level, so that less than 3 mg/kg
    remains in the straw and less than 0.3 mg/kg in the grain at harvest.
    The results of these studies are summarized in Table 4.

         Klingman et al (1966) found 58 mg/kg 2,4-D acid one half-hour
    after applying 2.25 kg butyl ester/ha and this decreased to 5 mg/kg on
    the fifth day.

         Lokke (1975) developed improved analytical techniques for the
    determination of 2,4-D and amino acid conjugates in cereal grains and
    with these found that the application of 4 kg (4 times the normal
    rate) of 2,4-D per ha to barley at the three-leaf stage resulted in
    residues of only 0.04-0.06 mg/kg in the grain at harvest. By
    conventional methods of extraction the same grain was estimated to
    have less than 0.02 mg/kg of 2,4-D. Treatment made at the same
    excessive rate after the spikes formed on barley left residues of
    0.35-0.45 mg/kg when measured by the method of Lokke (1975) or Yip
    (1971). By direct extraction methods, only 0.04 mg of 2,4-D could be
    recovered per kg of barley. Lokke reported that treatment at the same
    rate after the green grains were fully formed resulted in residues of
    4.0-4.3 mg/kg. This is 8-10 times higher than the residue found in the
    same sample by conventional extraction procedures.

         In Sweden and other Scandinavian countries where the harvesting
    of berries and mushrooms regularly takes place in forests, the use of
    2,4-D and 2,4,5-T herbicides has become a regular feature of forest
    management to regulate the growth of underbrush.

         Erne and Hartman (1973) made an extensive study of the residues
    of 2,4,5-T in mushrooms and both 2,4-D and 2,4,5-T in berries
    harvested in forests following the aerial and ground application of
    brush control herbicides. The results of their findings with respect
    to 2,4-D are summarized in Table 5.


    
    TABLE 4.  2,4-D residues in whole plant, grain and straw following treatment at different stages of growth

                                                                                                                            

                                                      Residues found at different growth stages, mg/kg
                                                                  (Intervals from treatment/days)

               Application   Stage of                    Late                                 Mature     Mature     Mature
    Crop       rate kg/ha    treatment    Tillering   Tillering   Boot    Harvest    Dried    (plant)    (grain)    (straw)
                                                                                                                            

    Oats           0.75      Tillering        4.4         2.7      1.1      0.57        -          -       0.05        1.8
                   1.5           "           12.3         5.2      2.8      0.77        -          -       0.07        2.6
                                              (7)        (14)     (28)      (56)                          (110)      (110)
    Barley         0.75      Late               -        0.84        -      0.37     0.18          -       0.03       0.48
    1.5                      tillering          -         9.5        -       1.2     0.28          -       0.02       0.85
                                                          (7)               (15)     (28)                  (63)       (63)
    Barley         0.75      Late boot          -           -        -       6.5      2.8        2.6       0.14        2.3
                   1.5           "              -           -        -      13.8      7.7        6.5        0.3        5.5
                                                                             (7)     (14)       (28)       (49)       (49)
    Wheat          0.75      Tillering          -         5.5      2.3         -     0.31          -       0.02       0.11
                   1.5           "              -        17.5      8.2         -     0.45          -       0.02       0.11
                                                          (7)     (14)               (28)                  (65)       (65)
    Wheat          0.75      Boot stage         -           -        -         -     4.8         0.6       0.02       0.54
                   1.5           "              -           -        -         -     13.5        1.5       0.02       1.10
                                                                                      (7)       (28)       (50)       (50)
    Barley         0.75      Tillering        3.3           -      1.0         -     0.02          -       0.02       0.02
                   1.5           "           11.8           -      2.4         -     0.35          -       0.02       0.05
                                              (7)                 (14)               (28)                  (58)       (58)
    Barley         0.75      Boot stage         -           -        -       4.5      3.1          -       0.02       0.03
                   1.5           "              -           -        -        14      7.7          -       0.03       0.02
                                                                             (7)     (14)                  (43)       (43)
                                                                                                                            
    

        TABLE 5.  Residues of 2,4-D in berries resulting from the aerial
              spraying of forests

                                                                                    

                                              Residue, mg/kg, at interval, days,
                                              after spraying. (No. of samples in
                                              parenthesis)
                                                                                    
                    Rate of Application
    Commodity             kg/ha               2-5           10-14         24-32
                                                                                    

    Raspberries           1.375               0.4-0.9       0.3-0.6       <0.03-0.3
                                              (3)           (3)           (3)
    Lingonberries         0.980               2.6-7.7       0.8-7.0       1.2-3.6
         "                                    (3)           (8)           (9)
         "                1.290               0.8-3.5       0.4-1.0       1.2-2.5
                                              (3)           (3)           (3)
         "                1.075               -             -             0.7-2.7
                                                                          (3)
         "                0.980               -             0.3           0.6-0.8
                                                            (1)           (2)
         "                nil                 <0.03         <0.03         <0.03
         "                1.075               1.5-4.0       1.2-2.0       0.7-2.0
                                              (3)           (3)           (3)
         "                1.015               2.0           0.9-1.5       -
                                              (3)           (3)
    Bilberries            1.290               2.0-2.4       0.5-1.3       0.9-1.8
                                              (3)           (3)           (3)
         "                1.075               1.4-2.9       1.3-2.5       -
                                              (4)           (3)
         "                nil                 <0.03         <0.03-0.2
                                              (1)           (2)
                                                                                    
    

    FATE OF RESIDUES

    In plants

         An enormous amount of research has been carried out over more
    than 25 years on the mode of action of 2,4-D, the reason for its
    selective action and the mechanisms of degradation in plants.

         Loos (1969) has published a 50 page review including references
    to 166 papers on the degradation of phenoxyalkanoic acids of which
    2,4-D is by far the most important. Only a few of the basic principles
    can be touched upon in this monograph.

         In the experiments referred to in the previous section (p. 179),
    Klingman et al (1966) found that virtually all the butyl and 75% of
    the 2-ethylhexyl esters of 2,4-D had been hydrolized to the acid
    within 30 minutes after application to forage. When 2.25 kg butyl
    ester per ha was applied, initial residues of 58 mg/kg decreased to 5
    mg/kg in five days. This work confirmed studies with radio-labelled
    2,4-D (Crafts, 1960) and others in which residues were determined by
    bioassay (Morré and Rogers, 1960) or gas chromatography (Hagin and
    Linscott, 1965).

         Similar trials by Cochrane and Russell (1975), in which the butyl
    ester was applied to wheat, were described in the previous section
    (p.179). No trace of butyl ester was found after the application of
    430 g/ha, but residues of 8.35 mg/kg of the free acid were found
    immediately after application and decreased rapidly.

         There appear to be three principal mechanisms for the metabolism
    of 2,4-D by plants, viz. degradation of the acetic acid side chain,
    hydroxylation of the aromatic ring and conjugation with plant
    constituents.

         Degradation of the side chain has been observed in many plants
    but in only a few species or varieties does it appear to play a major
    role in the breakdown of herbicidal activity. Loss of the side chain
    from a phenoxyacetic acid without further metabolic changes to the
    molecule would yield the corresponding phenol. Luckwill and
    Lloyd-Jones (1960) and Chkanikov et al. (1965) have demonstrated the
    production of 2,4-dichlorophenol in strawberries, beans, sunflowers,
    corn and barley. Simultaneously the side-chain carbon atoms are
    released as CO2 in some plants.

         Fawcett et al. (1959) showed that wheat and pea tissue
    hydroxylated 2,4-D in the 4-position. Similar reactions were observed
    in oats, barley and maize (corn), but not in various legumes, by
    Wilcox et al. (1963).

         The identity of the principal 2,4-D metabolite in bean, which was
    believed by Holley (1952) and Crosby (1964) to be a
    hydroxyphenoxyacetic acid, was studied by Thomas et al. (1964) who
    confirmed the major phenolic acid as
    2,5-dichloro-4-hydroxyphenoxyacetic acid and the minor phenolic acid
    as 2,3-dichloro-4-hydroxyphenoxyacetic acid. The 4-hydroxylation of
    2,4-D in bean was thus made possible by a chlorine shift from the 4
    to the 5 or 3 position. These two metabolites accumulate in bean as
    the glucosides.

         The esterification of glucose with phenoxyacetic acids has been
    observed. Klambt (1961) reported the formation of the glucose ester of
    2,4-D in wheat, and Thomas et al. (1964) reported that oats converted
    2,4-D to its beta-D-glucose ester.

         Aspartic acid combines with 2,4-D to form
    2,4-dichlorphenoxyacetyl-aspartic acid in wheat (Klambt, 1961) and
    probably in other plants.

         Feung et al. (1971, 1972, 1973a, b) demonstrated that 2,4-D is
    rapidly conjugated by means of an amide bond with a number of amino
    acids. Seven amino acid conjugates have been isolated and identified
    (glutamic and aspartic acids, alanine, valine leucine, phenylalanine,
    tryptophane) and these have been shown to have 2,4-D-like herbicidal
    activity (Feung et al., 1974). Such conjugation cannot be considered
    as a detoxification mechanism but perhaps as an activation step. Feung
    et al., 1974, have postulated that the conjugated form of 2,4-D could
    be exclusively the physiologically active form.

         Feung et al. (1975) demonstrated that the two ring-hydroxylated
    metabolites (4-OH-2, 5-dichloro and 4-OH-2, 3-dichloro) which together
    represent 97% of the soluble metabolites do not possess any
    growth-stimulating activity and exist as aglycones. In later studies,
    corn, the only monocotyledon tested, was shown to produce also
    3-hydroxy-2, 4-dichlorophenoxyacetic acid (3-OH-2, 4-D) and
    4-hydroxy-2-chlorophenoxyacetic acid which were not found in other
    plants tested. The authors were prompted to suggest that corn possess
    a significant alternative pathway of metabolism that may account for
    its resistance to herbicidal quantities of 2,4-D.

         On the other hand Hagin et al. (1970) have shown that three
    grasses metabolize 2,4-D to 2,4-dichlorophenoxypropionic acid, which
    is biologically inactive and this is assumed to be a major
    detoxification mechanism of these grasses.

    In animals

         Jensen and Miller (1975) report studies in which chickens were
    fed rations containing 10 or 100 mg/kg 2,4-D for 30 days. Tissue
    samples and eggs from these birds were analysed for residues of 2,4-D
    and 2,4-dichlorophenol using a method which had a validated
    sensitivity of 0.05 mg/kg for either compound. No dichlorophenol was
    detected in any sample. The average 2,4-D residues found are given in
    Table 6.

    TABLE 6.  2,4-D residues in chickens fed 2,4-D in the diet

                                                                

                              2,4-D residues (mg/kg)
    2,4-D in diet
       (mg/kg)        muscle/skin      fat        liver     eggs
                                                                

       10               <0.025       <0.025     <0.05     <0.025

       100              <0.2         <0.05      0.5       0.1
                                                                

         There is no reasonable expectation of residues in poultry tissues
    and eggs from ingestion of feeds containing up to 10 mg 2,4-D/kg. This
    level is far above the amount of 2,4-D (<0.2 mg/kg) that might be
    expected from treatment of crops used for chicken feed.

    EVIDENCE OF RESIDUES IN FOOD IN COMMERCE OR AT CONSUMPTION

         The only new information on the incidence and level of 2,4-D
    residues in food in commerce came from Hungary. In 1974 56 samples of
    barley, oats, rye and wheat from lots which were known to have been
    treated with 2,4-D were analysed by food inspection laboratories. None
    of the samples contained residues above the limit of determination
    (0.005 mg/kg). Whether these laboratories used methods capable of
    measuring conjugated residues is not known.

    METHODS OF RESIDUE ANALYSIS

         Several studies, reviewed by Loos (1969) and Feung et al.
    (1973a,b) have confirmed the conjugation of chlorophenoxy acids,
    including 2,4-D, with plant constituents. The conjugates are suggested
    to consist mainly of combinations of chlorophenoxy acids and amino
    acids. In spite of this these conjugates are not hydrolysed during
    most of the published analytical methods for chlorophenoxy acids. Chow
    et al. (1971) studied the original procedure of Yip and Nay (1966)
    which involves extraction (without hydrolysis) with 50% diethyl
    ether/hexane in the presence of sulphuric acid. By heating wheat plant
    tissue under alkaline conditions they found that less than 25% of the
    residue could be accounted for by analysing only the organic phase as
    in the Yip and Nay (1966) method.

         Lokke (1975) following the lead of Chow et al. (1971) developed a
    procedure involving hydrolysis of the starch under acidic conditions
    followed by treatment with diastase and further hydrolysis of amino
    acid conjugates through the action of proteolytic enzymes. These steps
    resulted in a considerable increase in the yield of 2,4-D residues
    from barley treated at various stages of growth with 2,4-D. The
    proteolytic enzyme (papain or ficin) was shown to be responsible for
    releasing about 50% of the 2,4-D found.

         In parallel experiments Lokke found that a straightforward
    extraction with 65% acetonitrile (Yip, 1971) gave comparable recovery.
    In the light of the efficient liberation of residues through
    hydrolysis by proteolytic enzymes, it is remarkable that the simple
    extraction with acetonitrile is of similar efficiency. Lokke went on
    to show, however, that neither the extraction procedure nor the
    enzymic hydrolysis can be considered completely exhaustive. It is not
    yet clear what amounts of 2,4-D conjugates remain after either of
    these procedures have been applied to grain.

         Glas (1975) has used aqueous sodium hydroxide to extract 2,4-D
    from grain and straw before purification on an alumina microcolumn for
    GLC quantitation using an electron capture detector.

    NATIONAL TOLERANCES REPORTED TO THE MEETING

         Particular attention was drawn to the following tolerances which
    have been established in national legislation (Table 7).

    TABLE 7. National tolerances reported to the meeting

                                                                        

    Country     Commodity                                 Tolerance mg/kg
                                                                        

    USA         forage of barley, oats, rye and wheat           20

                grain of barley, oats, rye and wheat            0.5

                flour of barley, oats, rye and wheat            0.5

                other milled fractions of barley, oats,
                 rye and wheat                                  2

                potable water                                   0.1
                                                                        

    APPRAISAL

         In response to the requirements of the 1970 Meeting, data from
    supervised trials on grain crops were received, together with a number
    of papers published on the degradation of 2,4-D in plants and soils
    and on the nature and measurement of conjugates in plants. Some
    information was also received on 2,4-D residues in food sampled in
    commerce.

         Most of the supervised trials were conducted in the 1960/65
    period and there is now evidence that the extraction methods then in
    use only recovered a portion of the free 2,4-D and none of the residue
    conjugated with amino acids. More recent experimental evidence
    indicates that there may be more significant residues present in grain
    at harvest following approved use of 2,4-D herbicides than was
    revealed in most of the data generated previously. On the basis of
    this evidence a higher maximum residue limit is recommended.

         Following the application of 2,4-D herbicides for brush control
    in forests in Sweden an extensive published study indicates that
    significant amounts of 2,4-D residue will be found in raspberries,
    lingonberries and bilberries harvested from the treated forests. The
    Meeting felt justified in extending limits based on this study to
    other vaccinium berries and blackberries. Maximum residue limits for
    2,4-D in these berries are therefore recommended.

         Since 2,4,5-T is usually combined with 2,4-D in brush-killing
    herbicides used in forests and in view of the evidence that residues
    of 2,4,5-T can occur in berries and mushrooms, it would appear to be
    necessary to recommend maximum residue limits for 2,4,5-T in these
    commodities also.

    RECOMMENDATIONS

         Previous recommendations for maximum residue limits are amended
    (raw grain) and new recommendations are made (berries) as follows.

    MAXIMUM RESIDUE LIMITS

                                                               

    Commodity                                    Limit, mg/kg
                                                               

         Milk and milk products                   0.05*

         Meat and eggs                            0.05*

         Raw grain                                0.2

         Vaccinium berries
         e.g. lingonberries, bilberries           5

         Blackberries, raspberries                5
                                                               

    * At or about the limit of determination

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    Buslovich, S. Yu., Voinova, I. V. and Milchina, G. (1973) Distribution
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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 4)
       D, 2,4- (Pesticide residues in food: 1980 evaluations)