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    PESTICIDE RESIDUES IN FOOD - 1979


    Sponsored jointly by FAO and WHO






    EVALUATIONS 1979





    Joint meeting of the
    FAO Panel of Experts on Pesticide Residues
    in Food and the Environment
    and the
    WHO Expert Group on Pesticide Residues
    Geneva, 3-12 December 1979



    CHLORPYRIFOS-METHYL

    Explanation

    In 1975 the Meeting evaluated information on chlorpyrifos-methyl.  An
    ADI was recommended, as well as MRLs for a number of commodities.
    Further information on residues in animal tissues, fat and eggs
    following feeding of residues in animal feeds was required before
    additional maximum residue limits could be recommended.

    The 11th Session of the CCPR noted that Germany (F.R.) possessed data
    supporting changes in the proposed MRLs and the addition of MRLs for
    other commodities (Alinorm 79/24-A, para. 133).

    New information was received on residues in animal tissues, fat and
    eggs from the feeding of rations fortified with chlorpyrifos-methyl at
    several levels; on residues in milk resulting from the feeding of
    feeds fortified at several levels, and on validated analytical methods
    for feeds and animal tissues, fat and eggs.  This information is
    assessed in the following monograph addendum.

    RESIDUES IN FOOD AND THEIR EVALUATION

    RESIDUES FROM SUPERVISED TRIALS

    These studies were designed to indicate residues of chlorpyrifos-
    methyl and its predominant metabolite, 3, 5, 6-trichloro-2-pyridinol,
    in animal tissues, fat, milk and eggs from the feeding of rations
    fortified at various levels.  Although the studies were not designed
    to indicate residues that actually result from good agricultural
    practices, they can be used to make reasonable estimates thereof.

    Cow's Milk and Cream

    Three cows were fed 0, 1, 3, 10, 30 and 100 mg/kg chlorpyrifos-methyl
    in the diet starting at the lowest level and increasing the dosage
    every 2 weeks (Kuper, 1978a).  Milk samples for each cow were obtained
    by combining equal portions of milk from two successive milkings.
    Residues were monitored throughout the feeding period and up to two
    weeks after removal of treated feed.  Equal portions of milk from each
    of the three cows were composited for cream samples.  Average residues
    of chlorpyrifosmethyl and 3,5,6-trichloro-2-pyridinol in milk and in
    cream from composited milk are summarized in Table 1.  Gross controls
    were <0.002 mg/kg chlorpyrifos or pyridinol metabolite equivalent.



        TABLE 1.  Average Residues of Chlorpyrifos-Methyl and 3,5,6-trichloro-2-pyridinol
    Found in Milk and in Composited Cream samples from Cows Fed Chlorpyrifos-Methyl

                                                                                              

                                            Average Residues Found, mg/kg1,4
    Chlorlpyrifos-Methyl     Chlorpyrifos-Methyl             3,5,6-Trichloro-2-Pyridinol
            Mg/kg               Milk           Cream               Milk            Cream
                                                                                              

               3                 -               -                 ND4
              10               <O.01                             <0.01
              30               <0.01            0.09              0.02             0.02
                                            (0.07-0.11)        (0.01-0.02)
             100                0.03            0.43              0.05             0.06
                            (0.02-0.04)     (0.31-0.50)        (0.03-0.07)      (0.06-0.07)
             1002              <0.01                             <0.01
                            (ND-<0.01)
             1003               ND              ND
                                                                                              

    1  Range is in parentheses if different than average value

    2  After 2 day withdrawal from feed

    3  After 4 day withdrawl from feed

    4  ND = less than twice the average of controls.  < indicates a response greater than
    twice the average of controls but less than the 0.01 mg/kg verified sensitivity of the
    method.  Values are adjusted for methodology recovery and controls.
    


    The data indicate that chlorpyrifos-methyl preferentially concentrates
    in the fat of milk whereas the pyridinol metabolite distributes
    equally in the aqueous and fat phases.  It also indicates that
    residues of 3,5,6-trichloro-2-pyridinol can equal or exceed residues
    of chlorpyrifos-methyl in milk.  Relative to milk, the concentration
    of chlorpyrifos-methyl in cream is proportional to the increase in the
    fat content.  At the 100 mg/kg feeding level, residues of
    chlorpyrifos-methyl and its pyridinol metabolite decrease to <0.01
    mg/kg after a 2-day withdrawal period.

    At the 10 mg/kg feeding level, the level of the highest proposed MRL
    on feed items that would contribute significantly to residues in
    animal products, residues in milk were 0.01 mg/kg (method sensitivity)
    for both chlorpyrifos-methyl and the pyridinol metabolite. This
    confirms the 0.01 ppm MRL previously recommended for milk (FAO/WHO,
    1976).

    Calves

    Cattle feed fortified at 0, 1, 3, 10, 30 and 100 mg/kg chlorpyrifos-
    methyl was fed ad libitum to three calves at each level for a period
    of 28 days with residue determinations for chlorpyrifos-methyl and
    3,5,6-trichloro-2-pyridinol up to 28 days after withdrawal (Kuper,
    1978b).  Residues in the fortified feed ranged from 70% of theoretical
    at the 30 mg/kg level to 130% at the 1 mg/kg level, averaging 92% of
    theoretical over all levels.  Data were not provided to show whether
    initial residue levels in the feed were maintained over the feeding
    period.  Assuming a half-life similar to that of stored grains (1-6
    months) possible 50% but probably no more than 10 - 30% of the
    chlorpyrifos-methyl would have dissipated.  Results of this feeding
    study are summarized in Table 2.  Controls were <0.001 mg/kg in all
    tissues for chlorpyrifos-methyl and <0.011 mg/kg (liver) for the
    pyridinol metabolite.

    At the 100 mg/kg feeding level maximum residues of chlorpyrifos-methyl
    at the end of 28 days were 0.05 mg/kg in kidney, 0.91 mg/kg in fat,
    and less than the validated method sensitivity for muscle and liver.
    Maximum residues of the pyridinol metabolite at this feeding level
    were 0.12, 2.17, 1.4, and 0.13 mg/kg respectively in muscle, liver,
    kidney and fat.  After 7 days withdrawal from treated feed, residues
    of chlorpyrifos-methyl and its pyridinol metabolite were less than the
    validated method sensitivities of 0.01 mg/kg and 0.05 mg/kg
    respectively.

    The chlorpyrifos-methyl therefore concentrates primarily in the fat
    and to a lesser extent in the kidney.  The pyridinol on the other
    hand, concentrates primarily in the liver and kidney.

    The highest MRL recommended for animal feed items which would be
    expected to contribute the most to potential residues of
    chlorpyrifos-methyl and 3,4,6-trichloro-2-pyridinol is 10 mg/kg in raw
    grains.  Maximum residues from feeding at the 10 mg/kg level were 0.03
    mg/kg chlorpyrifos-methyl in fat and 0.49 mg/kg pyridinol metabolite

    in liver.  Although the cattle diet would probably not be 100% raw
    grains, up to 80% could be.  Considering that initial feed
    fortification levels could have dissipated during the 28 day period,
    that some initial residues were less than 100% theoretical, that no
    data were available to demonstrate whether a 28 day feeding period was
    sufficient for tissue residues to peak (milk data gave some
    circumstantial indication that it would have), and control values up
    to 0.011 mg/kg for pyridinol, the residue data would indicate a need
    for a 0.05 mg/kg MRL for residues of chlorpyrifos-methyl in the meat,
    fat and meat by-products of cattle.

    Chickens and Eggs

    Mature White Leghorn laying hens were fed, ad libitum, feed
    fortified at 0, 1, 3, 10, 30 and 100 mg/kg chlorpyrifos-methyl for a
    period of 28 days (Kuper 1978c).  Residues in the fortified feed
    ranged from 70% of theoretical at the 30 mg/kg feeding level to 110%
    at the 1 mg/kg level (85% average over all levels).  No data were
    available to indicate whether initial residues in feed were maintained
    throughout the 28 day feeding period, or whether tissue residues
    peaked within that period.

    Muscle, fat, liver and egg tissues were analyzed for
    chlorpyrifos-methyl and 3,5,6-trichloro-2-pyridinol with methods
    validated on 0.01 and 0.05 mg/kg respectively.  Analyses were
    continued up to 14 days after withdrawal of treated feed.  Eggs were
    sampled on alternate days and composited per group.  Some individual
    eggs were analysed to demonstrate that the range of residues for
    individual eggs was essentially the same as for composited samples.
    Maximum apparent residues in chicken control were 0.004 mg/kg
    chlorpyrifos-methyl in muscle and 0.001 mg/kg pyridinol metabolite in
    liver and 0.011 mg/kg of the metabolite in eggs.

    Average residues, corrected for controls and method recovery are
    summarized in Table 3 which shows increasing residues with increasing
    ingestion levels.  At the 100 mg/kg feeding level, maximum
    chlorpyrifos-methyl residues at 0.1 mg/kg in chicken fat and 0.03
    mg/kg in eggs, again demonstrating its affinity for fat tissues
    relative to other tissues.  Maximum residues of the pyridinol
    metabolite at this feeding level are 0.12 mg/kg in liver and 0.08
    mg/kg in eggs, which is relatively low compared to similar feeding
    levels in calves.  Even so, residues of the metabolite in chicken
    tissues may equal, or exceed, those of chlorpyrifos-methyl.  Residues
    of chlorpyrifos-methyl and 3,5,6-trichloro-2-pyridinol in chicken and
    egg tissues are generally less than validated method sensitivities
    after a 7 day withdrawal from treated feed.  Although not shown in
    Table 3, both chlorpyrifos-methyl and its pyridinol metabolite showed
    a greater affinity for egg yolk than the white.



        TABLE 2.  Average Residues of Chlorpyrifos-Methyl and 3,5,6-Trichloro-2-Pyridinol found in tissues of Calves fed Chlorpyrifos-Methyl
    for 28 days

                                                                                                                                             
    Feed Rate
    Mg/Kg                       Average Residues in Tissues, Mg/Kg1
    in Feed                        Chlorpyrifos-Methyl                                         3,5,6-Trichloro-2-Pyridinol
                                                                                                                                         
                   Muscle         Liver         Kidney         Fat                 Muscle         Liver           Kidney          Fat
                                                                                                                                             

      1               -             -              -           ND2                    -            0.07            <0.05              -
                                                            (ND-<0.01)                         (0.06-0.07)
      3               -             -              -           0.02                   -            0.15             0.07              -
                                                            (<0.01-0.02)                       (0.13-0.19)       (0.07-0.08)
     10               -             -              -           0.03                   -            0.44             0.21            <0.05
                                                            (0.02-0.03)                        (0.39-0.49)       (0.13-0.35)
     30               ND           ND           <0.01          0.09                 <0.05          1.17             0.63             0.06
                  (ND-<0.01)    (ND-<0.01)                  (0.04-0.16)           (ND-<0.05)   (0.96-1.54)       (0.55-0.78)      (0.05-0.06)
    100             <0.01          ND            0.03          0.77                  0.08          2.16             1.22             0.88
                  (ND-<0.01)                  (0.02-0.05)   (0.65-0.91)           (0.05-0.12)  (2.15-2.17)       (0.93-1.4)       (<0.05-0.13)
    1003              ND           ND             ND            ND                    ND          <0.05            <0.05            <0.05
                                                            (ND-<0.01)
    1004              -         -             -                 ND                    -             ND               ND               -
                                                            (0.00-<0.01)
                                                                                                                                             

    1  Range for three calves in parentheses if different than average.  All valves adjusted for controls and recovery of methods.

    2  ND = less than twice the average of controls.

    3  After 7 day withdrawal from treated feed.

    4  After 14 day withdrawal from treated feed.

    TABLE 3.  Average Residues of Chlorpyrifos-Methyl and 3,5,6-Trichloro-2-Pyridinol found in Tissues of Chickens fed Chlorpyrifos-Methyl 
    for 28 Days

                                                                                                                                             
    Level
    In Feed                     Average Residues in Tissues, Mg/Kg1
    Mg/Kg                          Chlorpyrifos-Methyl                                         3,5,6-Trichloro-2-Pyridinol
                                                                                                                                         
                   Muscle         Liver         Kidney         Fat                 Muscle         Liver            Kidney            Fat
                                                                                                                                             

    10               -            <0.01           -             ND                    -             -              <0.05            <0.05
                                (ND-<0.01)                  (ND-<0.01)                                           (ND-<0.05)
    30               ND            0.01           ND          <0.01                 <0.05         <0.05            <0.05            <0.05
                                (<0.01-0.02)                (<0.01-0.02)                                         (<0.05-0.06)
    100             0.01           0.08           ND           0.02                 <0.05         <0.05             0.06             0.06
                                (0.05-0.1)                  (0.01-0.03)           (ND-<0.05)                     (0.05-0.12)      (<0.05-0.08)
    100              ND6          ND6             ND6          0.023                <0.056        <0.056           <0.056          
    <0.053
                                                                                                                 (<0.05-0.06)
                                                              <0.014                                               <0.057
                                                                                                                 (ND-0.05)
                                                                                                                                             

    1  Range in parentheses if different than average.

    2  ND = less than twice average for controls.

    3  After two day withdrawal from treated feed.

    4  After four day withdrawal from treated feed.

    5  After six day withdrawal from treated feed.

    6  After seven day withdrawal from treated feed.

    7  After 14 day withdrawal from treated feed.
    


    At the 10 mg/kg feeding level, the highest recommended MRL for a feed
    item which would contribute significantly to chicken feed, maximum
    residues were less than validated method sensitivity for both
    chlorpyrifos-methyl and its pyridinol metabolite in all chicken
    tissues and eggs.  This is true even at the 30 mg/kg feeding level for
    average tissues.

    The available information would support an MRL of 0.05 mg/kg combined
    residues of chlorpyrifos-methyl and 3,5,6-trichloro-2-pyridinol in the
    meat, fat and meat by-products of chickens and in eggs.

    FATE OF RESIDUES

    No additional data were submitted for review.

    METHODS OF RESIDUE ANALYSIS

    Validated methods were received for the determination of
    chlorpyrifos-methyl and 3,5,6-trichloro-2-pyridinol in animal tissues
    and eggs.  These appear similar to methods previously submitted with
    some variations for specific tissues.  These methods are also similar
    to methods for chlorpyrifos and its pyridinol hydrolytic product as
    available in the U.S. Food and Drug Administration's Pesticide
    Analytical Manual, Volume II.

    Method ACR 77.6 is used for the determination of chlorpyrifos-methyl
    in bovine and chicken tissues and in eggs (Kuper, 1977).  Residues are
    extracted with acetone, except in the case of fat, in which case
    hexane is used.  The acetone from the filtrate is evaporated. 
    Residues are taken up in hexane.  All hexane extracts are partitioned
    with acetonitrile, followed-by cleanup on a de-activated silica gel
    column.  Quantitation is by gas chromatography using a flame
    photometric detector.  Average recoveries range from 78  6 to 95  6
    percent over the 0.01-1 mg/kg fortification levels.  The method is
    validated at 0.01 mg/kg for the specified tissues.

    A modification of ACR 77.6 (ACR 77.6.S1) is used for the determination
    of chlorpyrifos-methyl in milk and cream (Kuper, 1978d).  Methanol and
    sodium chloride is added to warmed milk or cream before shaking with
    hexane.  After centrifugation and repeat extractions, the procedure is
    continued as in ACR 77.6 with the acetonitrile partitioning of the
    hexane extract.  Over the 0.01 to 1.0 mg/kg fortification level
    average recoveries in milk and cream are 87  2 and 84  4 percent
    respectively.  The method is validated for milk and cream at 0.01
    mg/kg.

    Method ACR 78.9 is used for the determination of
    3,5,6-trichloro-2-pyridinol in bovine muscle, liver, kidney and fat
    (Kuper, 1978e).  Residues are extracted with methanol and
    chromatographed on acidic alumina, eluting with hydrochloric acid. 
    The eluate is partitioned with benzene.  In the case of liver and
    kidney, the benzene phase is partitioned with sodium bicarbonate
    solution.  The aqueous phase is acidified and partitioned with

    benzene.  Residues of all tissues are derivatized with N,O-bis
    (trimethylsilyl) acetamide for determination by electron capture gas
    chromatography.  Average recoveries at the 0.05 - 2.0 mg/kg
    fortification levels range from 83.5 to 92  4 percent.  The method is
    validated to 0.05 mg/kg for bovine tissues.

    A modified version of ACR 78.9 (ACR 78.9.S1) is used for the
    determination of 3,5,6-trichloro-2-pyridinol in the muscle, fat, liver
    and eggs of chickens (Kuper, 1978f).  The procedure is basically the
    same as for bovine liver and kidney except a 3% OV-17 gas
    chromatography column is used in lieu of 6% DC 200.  Average
    recoveries in chicken tissues and eggs over the 0.05 - 1.0 mg/kg
    fortification levels range from 88  3 to 93  4 percent.  The method
    is validated at 0.05 mg/kg for chicken tissues and eggs.

    A modified version of 78.9 (78.9 S2) is used for the determination of
    3,5,6-trichloro-2-pyridinol in milk and cream (see Kuper, 1978d, p.
    5).  The procedure is basically the same as used for bovine liver and
    kidney except the initial extraction is with benzene  lieu of
    methanol.  Recoveries over the concentration range of 0.01-1.0 mg/kg
    average 93  3 percent for milk and 88  4 percent for cream.  The
    method is validated at 0.01 mg/kg for milk and cream.

    APPRAISAL

    Following the evaluation in 1975, the Meeting required information on
    residues in animal tissues, fat and eggs following the feeding of
    chlorpyrifos-methyl residues in animal feeds before additional maximum
    residue limits could be recommended.  Other studies were listed as
    desirable.  Data on residues in animal tissues, fat and eggs,
    additional data for milk, and validated analytical methodologies have
    been provided and are evaluated.

    Calves, dairy cows and hens were fed diets containing
    chlorpyrifos-methyl at concentrations ranging from 0 - 100 ppm.
    Tissues, milk, fat and eggs were analyzed for chlorpyrifos-methyl and
    3,5,6-trichloro-2-pyridinol.  Analytical methods similar to those
    previously evaluated were validated in all tissues down to 0.01 mg/kg
    chlorpyrifos-methyl and 0.05 mg/kg 3,5,6-trichloro-2-pyridinol.  In
    the case of milk both chlorpyrifos-methyl and its pyridinol hydrolytic
    product were validated at 0.01 mg/kg.

    In calves, residues of chlorpyrifos-methyl were maximal in the fat at
    the end of 28 days feeding.  Except for trace residues in the kidney
    at exaggerated feeding levels, residues were less than method
    sensitivity in muscle and liver.  Maximum residues of the pyridinol
    were in muscle and liver.  7 days after withdrawal of the diet
    containing 100 mg/kg chlorpyrifos-methyl, residues of
    chlorpyrifos-methyl and the pyridinol were below validated method
    sensitivity.

    In hens, maximum residues of chlorpyrifos-methyl were found in fat and
    maximum of 3,5,6-trichloro-2-pyridinol in the liver.  While the

    pyridinol concentrates in the liver of hens as it does in the liver of
    calves, the concentration of residues in hens is significantly lower
    than in the liver of calves receiving similar concentrations in feed.

    In eggs, residues of the 3,5,6-trichloro-2-pyridinol are 2-3 times
    those of chlorpyrifos-methyl.  Egg yolk showed a greater affinity than
    the white for both chlorpyrifos-methyl and the pyridinol.  Except for
    one sample, residues of chlorpyrifos-methyl and the pyridinol in hen
    tissues and eggs were less than method sensitivities 7 days after
    withdrawal from the diet containing 100 mg/kg chlorpyrifos-methyl.

    Chlorpyrifos-methyl was found to concentrate in the fat of milk
    whereas the 3,5,6-trichloro-2-pyridinol distributed equally in the
    aqueous and fat phases.  Residues of chlorpyrifos-methyl in cream
    increased over that of milk proportionally to the increased fat
    content.  Residues of the pyridinol on the other hand remain
    relatively constant.  The study confirms conclusions on milk residues
    reached by the 1975 Meeting.

    None of the animal tissue, milk or egg samples were analyzed for the
    oxygen analogue of chlorpyrifos-methyl or possible conjugated
    residues.  As the 1975 Meeting concluded, data indicate little
    likelihood of the oxygen analogue occurring.  However there appears to
    be little experimental evidence to demonstrate the absence of
    conjugated residues in animal tissues.  It has been demonstrated that
    the pyridinol is conjugated in the urine as the glucuronide.  More,
    definitive experimental evidence to demonstrate the presence or
    absence of conjugated moieties in animal tissues is desirable.

    No analyses were conducted for animal tissue, milk or egg residues of
    O-methyl-O-hydrogen-O(3,5,6-trichloro-2-pyridyl) phosphorothionate
    which has been found in appreciable quantities in the urine of ewes
    and rats (1975 Evaluations).  This information is desirable.

    The data indicate that in animal tissues, milk and eggs maximum
    residues of 3,5,6-trichloro-2-pyridinol usually exceed maximum
    residues of chlorpyrifos-methyl although not necessarily in the same
    tissues.

    Even the limited data on 3,5,6-trichloro-2-pyridinol residues in
    plants (1975 Evaluation, p. 126) indicate that residues of the
    pyridinol can exceed those of chlorpyrifos-methyl after 1-2 weeks from
    application.

    The data on residues in animal tissues, fat, and eggs are adequate to
    fulfill data requirements from the 1975 Evaluations and to support
    recommendations for additional animal products.

    RECOMMENDATIONS

    On the basis of additional information before the Meeting, the
    previously recommended MRL for milk is confirmed and the following
    MRLs are recommended.  The recommended figures refer to residues of
    chlorpyrifos-methyl.

    Commodity                               Limit, mg/kg
                                                         

    Carcase meat of cattle, cattle fat,         0.05
    cattle by-products

    Meat of chickens, chicken fat,              0.05
    chicken by-products
                                                         

    FURTHER WORK OR INFORMATION

    Desirable

    1.  Analysis of milk, tissues, fat and eggs from animals fed feed with
    chlorpyrifos-methyl fortified rations for possible residues of
    O-methyl-O-3,5,6-trichloro-2-pyridyl phosphorothionate.

    2.  Residue data for the carcase meat, fat and meat by-products of
    pigs from feeding of chlorpyrifos-methyl fortified rations.

    3.  Additional information or work listed as desirable by the 1975
    Meeting.

    REFERENCES

    Kuper, A.W.  Residues of Chlorpyrifos-Methyl and
    3,5,6-Trichloro-2-Pyridinol in Milk and Cream from Cows fed
    Chlorpyrifos-Methyl. (1978a) Unpublished Report GH-C 1161, Dow
    Chemical U.S.A., Midland, Michigan. December 27, 1978.

    Kuper, A.W.  Residues of Chlorpyrifos-Methyl and
    3,5,6-Trichloro-2-Pyridinol in Tissues from Calves fed with
    Chlorpyrifos-Methyl. (1978b) Unpublished Report GH-C 1118, Dow
    Chemical U.S.A., Midland, Michigan. July 5 1978.

    Kuper, A.W.  Residues of Chlorpyrifos-Methyl and
    3,5,6-Trichloro-2-Pyridinol in Tissues and Eggs from Chickens fed
    Chlorpyrifos-Methyl.  (1978c) Unpublished Report GH-C 1155, Dow
    Chemical U.S.A., Midland, Michigan. November 15, 1978.

    Kuper, A.W.  Determination of Residues of Chlorpyrifos-Methyl in
    Bovine Tissues. (1977) Unpublished Method ACR 77.6, Dow Chemical
    U.S.A., Midland, Michigan, April 11, 1977.

    Kuper, A.W.  Determination of Residues of Chlorpyrifos-Methyl in Milk
    and Cream. Modification ACR 77.6.S1 of Method ACR 77.6, Dow Chemical
    U.S.A., Midland, Michigan. November 9, 1978.  (1978d).

    Kuper, A.W.  Determination of Residues of 3,5,6-Trichloro-2-Pyridinol
    in Bovine Tissues.  (1978e) Unpublished Method ACR 78.9, Dow Chemical
    U.S.A., Midland, Michigan. May 12, 1978.

    Kuper, A.W.  Determination of Residues of 3,5,6-Trichloro-2-Pyridinol
    in Chicken Tissues. Modification ACR 78.9.S1 of Method ACR 78.9, Dow
    Chemical, U.S.A., Midland, Michigan. July 13, 1978. (1978f).
    


    See Also:
       Toxicological Abbreviations
       Chlorpyrifos-methyl (WHO Pesticide Residues Series 5)
       Chlorpyrifos-methyl (Pesticide residues in food: 1991 evaluations Part II Toxicology)
       Chlorpyrifos-methyl (Pesticide residues in food: 1992 evaluations Part II Toxicology)