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    PESTICIDE RESIDUES IN FOOD - 1980


    Sponsored jointly by FAO and WHO






    EVALUATIONS 1980





    Joint meeting of the
    FAO Panel of Experts on Pesticide Residues
    in Food and the Environment
    and the
    WHO Expert Group on Pesticide Residues
    Rome, 6-15 October 1980




    GUAZATINE

    Explanation

    Guazatine was evaluated by the FAO/WHO Joint Meeting in 1978
    (FAO/WHO, 1979a) at which time it was required that, by 1980,
    information be provided on the level, distribution, and fate of
    guazatine residues when the material is applied to major citrus
    fruit varieties in commercial practice, on the level and fate of
    guazatine residues in meat and milk following the feeding of citrus
    pulp containing residues of guazatine to cattle, and on an
    analytical method suitable for regulatory purposes.

    Information on the distribution and levels of guazatine residues in
    citrus and rock melons was received from Australia and on residue
    levels in citrus from Sweden.  Methods of residue analysis were
    received from both sources and are evaluated.


    RESIDUES IN FOOD

    USE PATTERN

    Post-harvest treatments

    Citrus.  After cropping, the fruits ideally are treated immediately
    on a conveyor packing line system.  They are lightly scrubbed,
    partially-dried, flooded (spray drenched) with 500 mg/l guazatine
    solution, partially dried, coated with a thin wax film, and finally
    dried. Treated fruits are then ready either for transport to market
    or for cold temperature storage preceding later marketing or
    export.  If the fruits cannot be treated on the packing line
    immediately, they are bulk dipped in 250 mg/l guazatine solution
    for protection against rot while they are stored temporarily (not
    more than 24 hours) at ambient temperature (Rippon, 1980).

    Rock melons.  At present the cropped fruits are bulk-dipped in a
    guazatine-benomyl mixture: 500-500 mg/l, before being dried and
    then transported to market or stored for later transport.  If
    conditions are favourable for rot-development the guazatine-dip
    concentration is doubled (Rippon, 1980).

    RESIDUES RESULTING FROM SUPERVISED TRIALS

    Citrus treatments in Australia included a bulk dip for holding
    purposes, a flood plus wax packing line treatment, and a combined
    holding and packing line treatment.  Treated citrus were subjected
    to ambient (20C) and cold (7.5C) storage to simulate local and
    export conditions).  Recommended citrus treatments are a 250 mg/l
    bulk dip and a 500 mg/l flood application.

    Rock melon treatment consisted of dipping in guazatine alone
    (250-2000 mg/l) or in combination with benomyl (250-750 mg/l).  A
    500 or 1000 mg/l guazatine dip in combination with 500 mg/l benomyl
    is recommended, depending on seasonal conditions.

    Results shown in Tables 1, 2, 3, and 7 were obtained from
    preliminary citrus and rock melon trials.  The data indicated
    residues in whole citrus up to 1.5 mg/kg, and in whole rock melon
    up to 5 mg/kg.

    In subsequent rock melon trials (Tables 8 and 9), the more complete
    data obtained confirmed that whole fruit residue of up to 4.5 mg/kg
    could result from likely treatments, irrespective of the presence
    of benomyl.  The low residues found in the edible pulp (up to 0.3
    mg/kg) may be due to contamination during sample preparation.  The
    presence of benomyl in mixed dips did not interfere in the
    guazatine analysis.

    In subsequent citrus trials (Tables 4-6), the more complete data
    indicated higher average residues than obtained in the initial
    trials, ranging from about 0.9 mg/kg for the dip, up to 2.5 mg/kg
    for the flood and combined treatments.  All trials indicated that
    most of the citrus residue remains on the surface of the fruit
    during 28 days storage after treatment, with appreciable decrease
    by 56 days.  This decrease could be either due to migration of
    guazatine into the fruit or simply reflect strong binding of the
    guazatine to the fruit surface.  Pulp residues were always very
    low, ranging from 0.1 to 0.4 mg/kg.

    Lemon residues were always somewhat higher than for orange or
    tangor. This could be due to the shape and surface characteristics
    of lemon, and differences in the age and maturity of the fruits
    (Australia, 1980).

    Citrus Preliminary trials

    Trial 1

        TABLE 1.  Guazatine Residues in Citrus Fruit Following Post-harvest Treatment

                                                                                         
    Guazatine      Fruit Variety       Storage      Guazatine Residue (mg/kg) in
    Treatment                           temp.               stored fruit
                                                   0 Days    7 Days    14 Days   21 Days
                                                                                         

                   Valencia orange      20C       0.42      0.42         Not Analysed
    520 mg/l       Eureka lemon         20C       0.56      0.48          "      "
    Bulk dip       Ellendale tangor     20C       0.43      0.48          "      "

                   Valencia orange      20C       0.53      0.53         Not Analysed
    520 mg/l       Eureka lemon         20C       1.01      0.93          "      "
    Bulk dip       Ellendale tangor     20C       0.92      0.99          "      "
    + Wash,        Valencia orange      7.5C      0.53      0.64        0.58     0.60
    695 mg/l       Eureka lemon         7.5C      1.01      0.84        1.01     1.05
    Flood/wax      Ellendale tangor     7.5C      0.92      0.50        0.45     0.30

                   Valencia orange      20C       0.40      0.51         Not Analysed
    695 mg/l       Eureka lemon         20C       0.42      0.91          "      "
    Flood/wax      Ellendale tangor     20C       0.67      0.83          "      "
                   Valencia orange      7.5C      0.40      0.67        0.51     0.61
                   Eureka lemon         7.5C      0.42      0.76        0.89     0.72
                   Ellendale tangor     7.5C      o.67      1.03        0.60     0.45
                                                                                         
    
    TABLE 2.  Guazatine Residues in Peel, Pulp and Whole Citrus Fruit after
    21 days storage at 7.5C following Post-harvest Treatment

                                                                           
    Guazatine treatment   Fruit            Sample             Guazatine
                          variety                          residues (mg/kg)
                                                                           

    520 mg/l Bulk Dip,    Valencia         Peel               3.0
    + Wash + 695          Orange           pulp               0.2
    mg/l Flood/Wax                         Whole Fruit        0.8

                          Eureka           Peel               1.6
                          Lemon            pulp               0.4
                                           Whole Fruit        0.86

                          Ellendale        Peel               3.5
                          Tangor           pulp               0.55
                                           Whole Fruit        1.2
                                                                           

        TABLE 3. Guazatine Residues in Citrus Fruit following Post-harvest Treatment

                                                                                             
    RESIDUES (mg/kg)
    Guazatine          Fruit        Storage     0 Days   4 Days   7 Days   14 Days  28 Days
    Treatment          Variety      Temp.       Storage  Storage  Storage  Storage  Storage
                                                                                             

    250 mg/l           Valencia
                       Orange       20C         0.54     0.52      NA1     NA       NA
    Bulk Dip           Eureka
                       Lemon        20C         0.63     0.62      NA       NA       NA
                       Ellendale
                       tangor       20C         0.63     0.63      NA       NA       NA

    500 mg/l           Valencia     20C         0.50     NA        0.43     0.42     NA
    Flood/Wax          orange       7.5C        0.50     NA        0.38     0.40     0.50
                       Eureka       20C         0.63     NA        0.52     0.51     NA
                       lemon        7.5C        0.63     NA        0.46     0.44     0.63
                       Ellendale    20C         0.62     NA        0.63     0.60     NA
                       tangor       7.5C        0.62     NA        0.46     0.42     0.38

    250 mg/l           Valencia     20C         0.42     NA        0.45     0.44     NA
    Bulk Dip +         orange       7.5C        0.42     NA        0.43     0.42     0.49
    Wash               Eureka       20C         0.53     NA        0.65     0.64     NA
    500 mg/l           lemon        7.5C        0.55     NA        0.50     0.51     0.40
    Flood Wax          Ellendale    20C         0.64     NA        0.63     0.63     NA
                       tangor       7.5C        0.64     NA        0.61     0.63     0.60
                                                                                             

    1 NA = not analysed
    
    Citrus advanced trials

    Trial 1

    TABLE 4.  Guazatine Residues in Citrus Fruit Following 250 mg/l
    Post-harvest Dip

                                                                       
    Fruit          Distribution          Residues (mg/kg)
    Variety        of Residues       0 Days1   4 Days1     Mean2
                                                         (whole fruit)
                                                                       

    Valencia        Surface           0.54       0.52      0.86
    Orange          Whole             0.91       0.8
                    Pulp              0.10       NA
                    Peel              2.50       NA

    Eureka          Surface           0.63       0.62      0.99
    Lemon           Whole             1.03       0.95
                    Pulp              0.10       NA
                    Peel              1.60       RA

    Ellendale       Surface           0.63       0.63      0.94
    tangor          Whole             0.94       0.94
                    Pulp              0.10       NA
                    Peel              3.60       NA
                                                                       

    1 Each result mean of 4 replicates
    2 Mean of 8 analyses

        Trial 2

    TABLE 5.  Guazatine Residues (mg/kg) in Citrus Fruit Following 500 mg/l
    Flood and Storage at either 20C or 7.5C

                                                                                                
    Fruit           Distribution    0 Days   7 Days  14 Days  28 Days   56 Days   Mean  s.d.
    Variety         of Residues                                                  mg/l whole fruit
    Temp. at
    storage
                                                                                                

    Valencia        Surface         0.50     0.43    0.42
    Orange          Whole Fruit     1.48     1.30    1.20
    20C            Pulp            0.1        -       -
                    Peel            4.10       -       -

    7.5C           Surface                  0.38    0.40      0.50      0.11
                    Whole Fruit              1.5     1.2       1.3       1.6
                    Pulp                      -       -                  0.1     1.44  0.251
                    Peel                      -       -        4.0        -

    Eureka          Surface         0.63     0.52    0.51
    Lemon           Whole Fruit     2.1      1.83    1.70
    20C            Pulp            0.1        -       -
                    Peel            3.6        -       -

    7.5C           Surface                  0.46    0.44     0.63      0.13
                    Whole Fruit              1.84    2.2      1.85      2.1
                    Pulp                       -      -                 0.1       1.95  0.291
                    Peel                       -      -                 2.6

    Ellendale       Surface         0.62     0.63    0.60
    Tangor          Whole Fruit     1.75     1.4     1.5
    20C            Pulp            0.3       -       -
                    Peel            2.8       -       -

    7.5C           Surface                  0.46    0.42     0.38      0.12
                    Whole Fruit              1.36    1.4      1.34      1.4
                    Pulp                                                0.3       1.45  0.171
                    Peel                                                2.6
                                                                                                

    1 Mean of 28 analyses
    
        Trial 3

    TABLE 6.  Guazatine Residues (mg/kg) in Citrus Fruit Following Post-harvest
    Bulk Dip (250mg/l) and Flood Treatment (500 mg/l)

                                                                                                
    Fruit           Distribution    0 Days   7 Days  14 Days  28 Days   56 Days   Mean  s.d.
    Variety         of Residues                                                  mg/l whole fruit
    Temp. at
    storage
                                                                                                

    Valencia        Surface         0.42     0.45    0.44
    Orange          Whole Fruit     1.2      1.3     1.4
    20C            Pulp            0.1       -       -
                    Peel            2.4       -       -

    7.5C           Surface          -       0.43    0.42     0.49      0.06      1.33  0.261
                    Whole Fruit      -       1.2     1.6      1.4       1.2
                    Pulp             -       -       -         -        0.2
                    Peel             -       -       -         -        2.2

    Eureka Lemons   Surface         0.53     0.65    0.64
    20C            Whole Fruit     1.6      1.83    1.75
                    Pulp            0.2       -       -
                    Peel            1.9       -       -

    7.5C           Surface                  0.50    0.51     0.37      0.11      1.83 + 371
                    Whole fruit              1.8     1.8      1.8       2.2
                    Pulp                      -       -       0.2
                    Peel                      -       -       2.1

    Ellendale       Surface         0.64     0.63    0.63
    Tangor          Whole Fruit     1.6      1.52    1.6
    20C            Pulp            0.31      -       -
                    Peel            2.1       -       -

    7.5C           Surface                  0.61    0.63     0.60      0.08      1.52  .31
                    Whole Fruit              1.4     1.6      1.6       1.3
                    Pulp                                                0.4
                    Peel                                                2.2
                                                                                                

    1 Mean of 28 analyses
    
    Rock melon preliminary trial

    TABLE 7. Guazatine Residues in Whole Rock Melon Following Post-harvest
    Dipping

    Initial Deposits
                                                                        
    Guazatine           Actual Conc.      Guazatine residues (mg/kg) in
    treatment (mg/l)    of dip (mg/l)     whole fruit except seeds
                                                                        

          500                480                     2.7
         1000               1090                     3.4
         2000               2420                     4.9
                                                                        


        Rock melon advanced trials

    TABLE 8.  (Trial 1) Guazatine Residues in Rock Melon Following Post-harvest
    Bulk Dipping in Guazatine and Guazatine/Benomyl Dips

                                                                                       
    Treatment               Distribution             Guazatine Residues (mg/kg)
    (mg/l)                                          0 Days1     7 Days1   Mean  s.d.2
                                                                          Whole fruit
                                                                                       
    500                     Pulp                      Nil        Nil
    Guazatine               Peel                      2.7        2.9
                            Whole Fruit               1.12       1.30     1.21  0.27

    1000                    Pulp                      0.29       0.15
    Guazatine               Peel                      6.50       6.50
                            Whole Fruit               3.60       4.2      3.90  0.52

    500 Guazatine +         Pulp                      Nil        Nil
    500 Benomyl             Peel                      2.87       3.20
                            Whole Fruit               2.00       1.30     1.65  0.46

    1000 Guazatine +        Pulp                      0.35       0.30
    500 Benomyl             Peel                      5.85       6.6
                            Whole Fruit               3.52       4.3      3.96  0.49
                                                                                       

    1 each result mean of 4 replicates
    2 mean of 8 analyses
    
    TABLE 9.  (Trial 2) Guazatine Residues in Rock Melon Following
    Post-harvest Bulk Dipping in Various Mixtures of Guazatine and Benomyl
                                                       

    Treatment             Guazatine residues (mg/kg)1
    (mg/l)              Pulp1    Peel1    Whole Fruit1
                                                       
    Guazatine Nil        ND       ND          ND
    Benomyl 250

    Guazatine Nil        ND       ND          ND
    Benomyl 500

    Guazatine Nil        ND       ND          ND
    Benomyl 750

    Guazatine 250        ND       1.2         0.6
    Benomyl Nil

    Guazatine 250        ND       1.3         0.6
    Benomyl 250

    Guazatine 250        0.3      1.6         0.9
    Benomyl 500

    Guazatine 250        0.3      3.0         0.6
    Benomyl 750

    Guazatine 500        0.3      4.45        2.7
    Benomyl Nil

    Guazatine 500        ND       6.2         2.4
    Benomyl 250

    Guazatine 500        ND       5.9         2.35
    Benomyl 500

    Guazatine 500        0.4      5.0         1.75
    Benomyl 750

    Guazatine 500        0.4      5.0         1.75
    Benomyl 1000

    Guazatine 1000       ND       5.2         2.4
    Benomyl 250

    Guazatine 1000       0.3      8.8         3.8
    Benomyl 500

    Guazatine 1000       0.45     7.2         3.2
    Benomyl 750
                                                       
    1 results mean of 4 replicates


    Information was received from Sweden on guazatine residue distribution
    and levels in oranges, mandarins, and lemons received from Australia.
    The fruit had been washed, flooded with guazatine solution (500 mg/kg)
    and waxed in a laboratory packing line before being air freighted to
    Sweden.  The received fruit was stored at room temperature for 1 week,
    then deep frozen until analysis.  Results are shown in Table 10.
    Maximum residues in whole fruit did not exceed 0.3, 0.5, and 0.2 mg/kg
    for oranges, mandarins and lemons respectively.  All residues were
    found on the peel in separate analyses (Kenogard, 1980).


        TABLE 10.  Residue Analysis of Guanidated Amines in Citrus Fruits from
    Australia 1979

                                                                            
                                               Pesticide and    Residue found
                     Sample                     dosage rate         mg/kg
                                                                            
    Washington navel oranges:   whole fruit      Panoctine          0.3
                                peel             500 mg/kg          3
                                pulp             500 mg/kg          0.05

    Emperor mandarins:          whole fruit      500 mg/kg          0.5
                                peel             500 mg/kg          2
                                pulp             500 mg/kg          0.05

    Eureka lemons:              whole fruit      500 mg/kg          0.2
                                peel             500 mg/kg          2
                                pulp             500 mg/kg          0.05
                                                                            
    

    METHODS OF RESIDUES ANALYSIS

    A new spectrophotometric procedure has been developed for guazatine
    residues in fruit, which is sensitive to 0.02 mg/kg.  A 100 g fruit
    sample is chopped, then mixed with 10 g NaOH, 30 g NaCl, 50 ml
    n-butanol, and 10 ml hexane in a blender for 3 min. at 4000 rpm.  The
    contents are then transferred to a centrifuge cup and centrifuged for
    5 min. at 4000 rpm.  The supernatant portion is filtered through a
    glass or cotton wool plug into a separatory funnel and the solids
    re-extracted with a further 30 ml butanol, centrifuged, and filtered.
    The lower aqueous layer is discarded and the solvent layer mixed with
    70 ml hexane and  extracted with 2  15 ml of MH2SO4.  The acid
    extract is mixed with 5 g NaOH and 5 g NaCl and extracted twice with 5
    ml butanol.  The butanol extract is mixed with 10 ml hexane and
    allowed to stand at least 2-3 hours.  To remove traces of alkali the
    solvent mixture is then washed with 10 ml of 0.14 MH3PO4.  Five ml
    of reagent (0.2 g Coomassie Brilliant Blue G250 in 100 ml ethanol; add
    190 ml 85% orthophosphoric acid and 400 ml of 1% starch solution; make
    to 2 l with water, allowed to stand overnight, and filter (Whatman No.

    1) before using) is mixed with 1 ml of the acid extract or 1 ml
    guazatine standard and the absorption measured at 585 nm against a
    blank (1 cm cell, 1 nm bandwidth).  Because of its selectivity and
    sensitivity, the method appears very suitable for regulatory purposes
    (Higginson, 1979; Ahmed et al, 1979).


    EVALUATION

    APPRAISAL

    Residue data from post-harvest trials on citrus and rock melons were
    available.  Citrus fruits (orange, tangor, lemon) and rock melons were
    dipped or, with citrus fruits, flooded with guazatine solutions of
    500-2000 mg/l concentration.  Guazatine residues in fruit were
    determined up to 21 days after treatment, the treated fruit being
    stored at either 20C or 7.5C during this period.

    In no case did the guazatine residue exceed 5 mg/kg in whole fruit.
    With both citrus and rock melon most of the residue was in the peel;
    on average, about 10% of the residue in citrus and 4% in rock melon
    were in the fruit pulps.  The level of residues were always higher
    than those of oranges and tangors.  With rock melons the whole-fruit
    guazatine residue were proportional to the concentration of guazatine
    in the dip solution.

    Residue data were also provided from analyses of oranges, mandarins,
    and lemons treated in Australia and air freighted to Sweden.  Maximum
    residues did not exceed 0.5 mg/kg (whole fruit) in this experiment and
    virtually all residue was located in the peel.

    An analytical method for guazatine residues in fruit based on the
    spectroscopic absorption of a stable blue complex formed by the
    reaction of guazatine with Coomassie Brilliant Blue G250 has been
    developed which is selective and sensitive to 0.02 mg/kg.  It appears
    very suitable for regulatory purposes.

    The 1978 requirements for data on the level and distribution of
    guazatine residues in citrus treated in commercial practice and for a
    suitable regulatory method of analysis are satisfied.  No information
    was available to the Meeting on the level and fate of guazatine
    residues in meat and milk following the feeding of citrus pulp
    containing guazatine residues to cattle.


    RECOMMENDATIONS OF RESIDUES LIMITS

    The 1978 recommendation of a temporary maximum residue limit of 5
    mg/kg for citrus fruits is confirmed and changed to a maximum residue
    limit.  The maximum residue level listed below for melons is suitable
    for establishing a maximum residue limit in addition to those
    previously established.  The numerical values refer to the parent
    compound alone.

                                                          
    Commodity              Maximum Residue Limit, mg/kg
                                                          

    Citrus fruits                    5
                                                          
                           Estimated Maximum Residue Level
                                    mg/kg
                                                          
    Melons                           5
                                                          

    FURTHER WORK OR INFORMATION

    Desirable

    Information on the level and fate of guazatine residues in meat and
    milk following feeding of citrus pulp containing guazatine residues to
    cattle.

    REFERENCES

    Ahmad N. and Milham, P.J. A rapid and sensitive method for measuring
    micro quantities of guazatine in dilute solutions. (1979)
    (Unpublished) Biological and Chemical Research Institute, Rydalmere,
    N.S.W., Australia.

    Australia Guazatine residues in citrus fruits and rock melons
    following post-harvest treatments. (1980) Private communication.

    Higginson, F.R. Determination of guazatine residues in fruit.
    Biological and Chemical Research Institute, New South Wales,
    Department of Agriculture.

    Kenogard, A.B. Residue analysis of guanidated amines in citrus fruits
    from Australia, 1979, 80-AC-0184. Report submitted to 1980 JMPR.

    Rippon, E. Private communication. Gosford Horticultural Post-harvest
    Laboratory. Gosford, N.S.W., Australia.

    


    See Also:
       Toxicological Abbreviations
       Guazatine (Pesticide residues in food: 1978 evaluations)
       Guazatine (Pesticide residues in food: 1997 evaluations Part II Toxicological & Environmental)