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    CHLOROBENZILATE                                 JMPR 1972

    Explanation

    Chlorobenzilate was briefly studied by the Joint Meeting in 1965
    (FAO/WHO, 1965) and a comprehensive evaluation was carried out in 1968
    (FAO/WHO, 1969). At this time information on several matters was
    requested and material now available concerning some of them is
    presented.

    Some additional information is included in the comprehensive review by
    Bartsch et al. (1971).

    RESIDUES IN FOOD AND THEIR EVALUATION

    Fulfilment of requirements

    In the following paragraphs, the numbers refer to the corresponding
    requirement as indicated by the 1968 Joint Meeting (FAO/WHO, 1969).

    1.   Composition of the technical product

         Analysis of technical chlorobenzilate is carried out by
         gas-liquid chromatography (6 ft   in column packed with 3%
         Carbowax 20M on Gas Chrom Q) with dibenzylsuccinate as an
         internal standard. For quantitative determination of the
         chlorobenzilate, a column temperature of 240C is used, but for
         qualitative separation and identification of the impurities,
         temperatures of 150C or 220C are used. Table 1 shows the nature
         and extent of the impurities observed in 13 batches of technical
         material over a two-year period. On the basis of retention times,
         relative to the parent compound, the unknown compounds are the
         same in all typical samples (Ciba-Geigy, 1972a). Methods for
         determining the active ingredient in technical material and
         formulated products have been described by Bartsch et al.
         (1971).

    2.   Nature of terminal residues

         In animals

         Knowles and Ahmad (1971) have investigated the comparative
         metabolism of chlorobenzilate, chloropropylate and GS-19851
         acaricides by rat hepatic enzymes. The limiting reaction was
         found to be the cleavage of the ester linkage by
         carboxylesterases which showed greater activity for
         chlorobenzilate than for the other two compounds. Degradation of
         all three materials was inhibited by di-isopropyl
         phosphorofluoridate and the main metabolite was 4-chlorobenzoic
         acid. Bourke et al. (1970) studied the distribution of residues

    TABLE 1  Nature and extent of impurities in technical chlorobenzilate

                                                                         

    Component                                    % Found (range)
                                                                         

    Chlorobenzilate                              93.8 - 97.3

    Ethyl 4-chlorobenzoate                       0.23 - 0.47

    4,4'-dichlorobenzophenone                    0.04 - 0.23

    Chloropropylate                              ND3 - 0.05

    2,4'-chlorobenzilate                         <0.01

    Ethyl ether of chlorobenzilate               0.14 - 0.59

    4,4'-dichlorobenzil  )1                      <0.01 - 0.11
    2,2'-dichlorobenzil  )

    "Unknown" RRT 0.492                          <0.01 - 0.03

       "      RRT 0.88                           0.01 - 0.15

       "      RRT 1.19                           0.06 - 0.10

       "      RRT 1.46                           0.04 - 0.09

       "      RRT 1.72                           0.10 - 0.38

       "      RRT 1.83                           ND - 0.09

    Total impurities measured                    0.90 - 1.61

    Total "unknowns" measured                    0.36 - 0.59
                                                                         

    1  Not separated by system used.
    2  RRT = retention time relative to chlorobenzilate (= 1.00).
    3  ND = not detected


    of these acaricides in rats. Adult male rats were fed 14C-labelled
    compounds and the following distribution was found: 42.78% in faeces,
    25.63% in urine, 15.47% in the gastrointestinal tract, 3.31% in liver
    and less than 1% in brain, heart, spleen or kidney. It appeared that
    chlorobenzilate was degraded to polar water-soluble metabolites more
    rapidly than was chloropropylate.

         In plants

         Chlorobenzilate, chloropropylate and GS-19851 (isopropyl
         4,4'-dibromobenzilate) labelled with 14C in both aliphatic
         carbon atoms of the benzilic acid moiety were applied to leaves
         of soybean plants grown in glasshouse pots. Each leaflet was
         treated with 25 000 cpm of acaricide in 10 ml of 95% ethanol with
         a microsyringe. Specimens were analysed after 0, 4, 8, 12 and 16
         days. Only limited transportation of the acaricides to other
         tissues of the plant was observed. Recovered radioactivity on Day
         0 was 84.7% (chlorobenzilate), 94.8% (GS-19851) and 96.5%
         (chloropropylate). On day 16 these values were 10.5%, 17.7% and
         10.3%, respectively. Autoradiography of thin-layer chromatograms
         revealed that the major component (greater than 95%) was the
         parent compound in all cases (Hassan and Knowles 1969).

    3.   Residue data from countries other than U.S.A.

         Table 2 lists data on residues resulting from applications to
         fruit according to good agricultural practice in countries other
         than U.S.A. (Ciba-Geigy, 1972b). Table 3 gives residues observed
         following treatment of tea (Bortsch et al., 1971).

    4.   Data on disappearance of residues in soils

         No further data available.

    5.   Data on residues in wine

         No data available.

    6.   Occurrence of residues in milk

         Data have been received concerning the residues of
         chlorobenzilate occurring in milk due to feeding cows with known
         dosages of the compound (Ciba-Geigy, 1972 c). Two sets of three
         cows in the middle of lactation were fed with either 50 mg or 200
         mg of chlorobenzilate per day; milk samples were taken 7, 3 and 1
         day before feeding was started and 0, 1, 2, 4, 7, 10, 21, 28, 29,
         30, 32, 35 and 42 days after starting the dosed feeds. Two cows
         were also used as untreated controls. The feeding levels
         corresponded to 5 ppm and 20 ppm of chlorobenzilate in citrus
         pulp cattle feed, assuming a maximum intake of 10 kg feed per cow
         per day. Residues of chlorobenzilate in the milk from cows on the
         50 mg per day dosage were in the range <0.025 to 0.03 ppm
         throughout the treatment period. Cows on the 200 mg per day
         dosage yielded milk containing from <0.025 to 0.06 ppm of
         chlorobenzilate. Residues of the two potential metabolites,
         4,4'-dichlorobenzophenone and 4,4'-dichlorobenzilic acid were
         below 0.02 ppm in all milk samples examined.

    TABLE 2  Chlorobenzilate residues in fruit

                                                                             

    Crop and          Application         Days after    Residue in plant
    country        conc.(% a.i.)   No.    application   parts (ppm)
                                                                             

    Apples                                              fruits

    Switzerland    0.0375          1      0             2.1
                                          3             1.8
                                          7             1.5
                                          10            1.5
                                          14            1.1
                                          21            0.75
                                          28            0.64
                                          35            0.55
                                          101           0.13
                   control                -             >0.01

    Pears

    Switzerland    0.0375          1      65            0.13
                   control                -             <0.05

    Oranges                                             pulp      peel

    Spain          0.05            1      150           <0.03     2.1
                   control                -             <0.03     <0.03
    Cyprus         0.02            1      167           <0.03     1.75
                   control                -             <0.03     <0.03
    Israel         0.03            1      165           <0.03     <0.03
                   control                -             <0.03     <0.03
    South Africa   0.025           1      14            <0.01     1.6, 1.7
                                          28            <0.01     1.6, 1.6
                                          42            <0.01     2.0, 1.8
                   control                -             <0.01     <0.01

    Tomatoes       dosage                               fruits

    Israel         0.375 kg        2      21            <0.1
                   a.i. per ha            71            <0.1
                                          141           <0.1
                   control                -             <0.1
                                                                             

    1  Days after second application.

    TABLE 3  Chlorobenzilate residues in tea (Bartsch et al., 1971)

                                                                             

                 Applications     Days after        Chlorobenzilate
    Country      conc.     No.    application            (ppm)
                                                                             

                                                 dried          fermented
                                                 leaves         leaves

    Indonesia    50 g      1      1              17.0, 13.0     1.4
                 a.i./ha          4              12.0, 10.0     1.7
                                  7              1.5, 1.4       <0.20
                                  9              ---            0.63

                 100 g     1      1              9.1, 18.0      5.60
                 a.i./ha          4              21.0, 16.0     5.50
                                  7              7.6, 6.1       4.10
                                  9              ---            5.30

    India                                        manufactured dried leaves
                 312 g     1      7                       45.6
                 a.i./ha                               brewed tea
                                                 wet leaves     brew
                                                    37.4        0.06
                                                                             


    7.   Methods of residue analysis

         Chlorobenzilate residues have been determined by gas
         chromatography following extraction from various foods using
         petroleum ether (FAO/WHO, 1969), methanol (Delley et al., 1964)
         or mixtures of iso-propanol and benzene (Benfield and Richardson,
         1965). Alumina or Florisil columns have been used (U.S. Food and
         Drug Administration, 1968) for clean-up with various eluants. The
         basic alumina column clean-up was preferred because of its more
         consistent activity. Oily samples such as nuts, seeds and citrus
         peel required an additional cleanup stage (partitioning with
         acetonitrile) before column chromatography. The microcoulometric
         detector was used with a column of 1 m X 4 m 5% G.E. XE60 on
         Anakrom ABS 50-60 mesh or 2 ft x  in 30% Carbowax 20M on 60/80
         Gaschrom Q (FAO/WHO, 1969). A 6 ft column packed with 3% XE60 on
         80/100 Gaschrom Q and an electron capture detector has been found
         to be the more efficient system (U.S. Food and Drug
         Administration, 1968). Interferences from DDT and TDE were
         eliminated by the clean-up procedure. These materials were eluted
         in the benzene fraction using the Florisil column and in the
         hexane fraction with the alumina column. Delley et al. (1964)
         used glass columns packed with 2.5% Reoplex 400 on Anakrom ABS
         maintained at 170C with a hydrogen flame detector, which gave a

         detection limit of 0.1 g. These gas chromatographic procedures
         are suitable for use for regulatory purposes.

         Details of gas chromatographic procedures for determining
         residues of chlorobenzilate and its metabolites in milk have been
         described by Formica et al. (1972). The milk sample is mixed
         with anhydrous sodium sulphate and extracted with benzene in a
         Soxhlet apparatus. The benzene extract is evaporated to dryness,
         redissolved in hexane and partitioned against acetonitrile to
         separate chlorobenzilate and 4,4'-dichlorobenzophenone from milk
         fats. The acetonitrile phase is concentrated and further cleaned
         by thin-layer chromatography on silica gel developed with
         benzene. Chlorobenzilate and 4,4'-dichlorobenzophenone are
         finally determined by gas chromatography, using microcoulometric
         and electron capture detection, respectively.

         To test for 4,4'-dichlorobenzilic acid, acetone is added to the
         milk and after filtration and evaporation of the acetone, ammonia
         is added to the aqueous extract. The fats are separated by
         partitioning with hexane, and the remaining water extract is
         acidified with hydrochloric acid. The 4,4'-dichlorobenzilic acid
         is extracted with ether and oxidized with acid potassium
         dichromate to 4,4'-dichlorobenzophenone which is then determined
         by gas chromatography with electron capture detection. By the
         methods stated, the limits of detection are quoted as
         chlorobenzilate, 0.025 ppm (5 mg); 4,4'-dichlorobenzophenone,
         0.02 ppm (0.1 mg); 4,4'-dichlorobenzilic acid, 0.02 ppm (0.1 mg).
         The Coulson electrolytic conductivity detector system, normally
         used for nitrogen detection has been used to determine
         halogenated compounds such as chlorobenzilate. The quartz
         reduction tube was packed with a platinum gauze and the glass
         column with 5% SE30 on Chromosorb W.S. (Coulson, 1966). Bartsch
         et al. (1971) give a detailed review of residue methods.

    APPRAISAL

    Information has been supplied which fulfills five of the seven
    requirements listed by the 1968 Joint Meeting. No information was
    available regarding disappearance from soils or carry-over of residues
    into wine as a result of treating grapes. The following tolerances are
    recommended:

    RECOMMENDATIONS

    TOLERANCES
                                                           ppm
         Apples, pears, grapes                             2
         Citrus fruits                                     1
         Melons, cantaloupes                               1
         Almonds and walnuts (shelled), tomatoes           0.2
         Milk (from the feeding of treated forage)         0.05*

         * at or about the limit of determination

    FURTHER WORK OR INFORMATION

    DESIRABLE

    1.   Data on the possible carry-over of residues into wine as a result
         of the treatment of grapes.

    2.   Further data on the disappearance of residues in soils.

    3.   Further data on residues occurring from usage on tea.

    REFERENCES

    Bartsch, E., Eberle, D., Ramsteiner, K., Tomann, A. and Spindler, M.
    (1971) The carbinole acaricides: chlorobenzilate and chloropropylate.
    Residue Reviews 39: 1-93.

    Benfield, C.A. and Richardson, D. (1965) Report No. CP/64/Anal/21.
    Fisons Pest Control Ltd., Chesterfield Park Research Station.

    Bourke, J.B., Broderick, E.J. and Stoewsand, G.S. (1970) Elimination
    rate and tissue residues of chloropropylate and chlorobenzilate in
    rats. Bull. Environ. Contam. Toxicol., 5: 509-514.

    Ciba-Geigy. (1972a) Composition of chlorobenzilate technical. 
    (unpublished)

    Ciba-Geigy. (1972b) Residue data on chlorobenzilate. Report 1960, RAR
    40/71, RVA 53/72, RVA 54/72, RVA 55/72 and RVA 58/72. (unpublished)

    Ciba-Geigy. (1972c) Residues of chlorobenzilate and two of its
    metabolites in milk of Swiss cows. Report RVA 63/72. (unpublished)

    Coulson, D.M. (1966) Selective detection of nitrogen compounds in
    electrolytic conductivity gas chromatography. J. Gas Chromatog., 4:
    285-287.

    Delley, R., Friedrich, K. and Geiser, A. (1964) Report of Geigy method
    ROS No. 2526. (unpublished)

    FAO/WHO. (1965) Evaluation of the toxicity of pesticide residues in
    food. FAO PL/1965/10/1: WHO/Food Add./27.65.

    FAO/WHO. (1969) 1968 Evaluations of some pesticide residues in food.
    FAO/PL: 1968/M/9/1; WHO/Food Add./69.35.

    Formica, G., Gabrielova, M. and Magnin, B. (1972) Gas chromatographic
    residue determinations of chlorobenzilate and two of its metabolites
    in milk. Ciba-Geigy report REM 13/72. (unpublished)

    Hassan, T.K. and Knowles, C.O. (1969) Behaviour of three 14C-labelled
    benzilate acaricides when applied topically to soybean leaves. J.
    Econ. Entomol., 62: 618-619.

    Knowles, C.O. and Ahmad, S. (1971) Comparative metabolism of
    chlorobenzilate, chloropropylate and bromopropylate acaricides by rat
    hepatic enzymes. Can. J. Physiol. Pharmacol., 49: 590-597.

    U.S. Food and Drug Administration. (1968) The determination of
    chlorobenzilate and chloropropylate in plant materials. Pesticide
    Analytical Manual. Vol.II, Section 120 p. 218.
    


    See Also:
       Toxicological Abbreviations
       Chlorobenzilate (ICSC)
       Chlorobenzilate (FAO Meeting Report PL/1965/10/1)
       Chlorobenzilate (FAO/PL:1968/M/9/1)
       Chlorobenzilate (WHO Pesticide Residues Series 5)
       Chlorobenzilate (Pesticide residues in food: 1977 evaluations)
       Chlorobenzilate (Pesticide residues in food: 1980 evaluations)
       Chlorobenzilate (IARC Summary & Evaluation, Volume 5, 1974)
       Chlorobenzilate (IARC Summary & Evaluation, Volume 30, 1983)