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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




    CARBOPHENOTHION

    Explanation

    Carbophenothion was evaluated at the 1972 (FAO/WHO, 1973), 1976
    (FAO/WHO,1977), 1977 (FAO/WHO, 1978) and 1979 (FAO/WHO, 1980) Joint
    Meetings.  In 1979 a full-ADI of 0.0005 mg/kg b.w. was allocated.
    Studies on the identity and the relative toxicity of metabolites were
    considered desirable.

    No additional studies concerning the relative toxicity of these
    metabolites have been received.  However, new studies have become
    available on acute delayed neurotoxicity in adult hens, subacute oral
    toxicity in beagle dogs and metabolism of carbophenothion and are
    included in this monograph addendum.

    The 1977 Joint Meeting listed as desirable additional elucidation of
    the nature of terminal residues on crops under field conditions,
    particularly as to the possible presence of photolysis products which
    had been reported in laboratory experiments.  In response to this
    request for information a greenhouse study on the metabolism of
    carbophenothion on orange trees has been submitted and is evaluated in
    this addendum.  Additional information on national use patterns and
    tolerances were also provided.


    DATA CONSIDERED FOR DERIVATION OF ACCEPTABLE DAILY
    INTAKE

    BIOCHEMICAL ASPECTS

    Excretion via faeces, urine and expired air was studied in rats after
    oral administration of carbophenothion [14C]-phenyl (3 mg/kg bw),
    carbophenothion [14C]-thiomethyl (3 mg/kg bw), carbophenothion
    sulphoxide [14C]-phenyl (3 mg/kg bw), and [14C]-4-chlorothiophenol
    (8.3 mg/kg bw).

    The radiocarbon recovered after 66 - 144 hours as % of the
    administered dose in the carbophenothion [14C]-phenyl,
    carbophenothion sulphoxide [14C]-phenyl and on the [14C]-4-
    chlorothiophenol-treated animals were much the same: in faeces
    (17-24%), urine (71-80%), and expired air (0%).  The major route of
    excretion of [14C] in rats receiving carbophenothion [14C]-thiomethyl
    group was via expired air as CO2: 59%, faeces: 4%, and urine 31%.

    The metabolic products following administration of carbophenothion or
    carbophenothion sulphoxide were quantitatively and qualitatively very
    similar.  Therefore it was concluded that there is an equilibrium
    between carbophenothion and the sulphoxide in vivo.  However, no
    sulphoxidation products were detected (Menn et al, 1975).

    TOXICOLOGICAL STUDIES

    Special study on neurotoxicity

    Groups of 10-14 adult hens were administered orally corn oil (2 ml/kg
    bw), carbophenothion 92.4% (330 mg/kg bw) or TOCP (500 mg/kg bw).  The
    treatment was repeated for surviving hens after 21 days.  The animals
    were observed for 42 days.  The toxic signs of the carbophenothion
    treated hens were most severe up to 7 days after either administration
    and they included signs of cholinergic influence, e.g. ataxia,
    salivation and diarrhoea, loss of body weight and hampered egg
    production.  The severity of these signs diminished with time. 
    Minimal leg weakness was noted only one occasion for one hen given c
    carbophenothion.  Histopathological changes in nerve tissue (brain
    stem, cervical, thoracic and lumbal spinal cord distal and proximal
    sciatic nerve) of hens given carbophenothion were similar to those in
    the group given corn oil.  However, one of 14 chickens receiving
    carbophenothion had nerve fibre degeneration, but no specific lesions
    in spinal cord and medulla.  The TOCP treated birds showed progressive
    loss of body weight and paralysis.  Axonal degeneration was observed
    by light microscopy in nerve tissue, including sciatic nerves
    (bilateral), of this group (Miller and Sprague, 1978).

    Short-term study

    Four male and 4 female beagle dogs per dose group were orally
    administered carbophenothion (purity 92.8%) at dose levels of 0, 0.02,
    0.2 or 2 mg/kg bw per day for 22-25 days (Bier, 1979).  The parameters
    evaluated were mortality, general appearance and behaviour, body
    weight, food consumption, growth, gross pathology and plasma,
    erythrocyte and brain cholinesterase activity (white and grey matter).
    The animals in the intermediate and high dose group showed
    dose-dependent emesis and diarrhoea.

    Plasma butyrylthiocholinesterase activity was depressed throughout the
    study in both males and females; from day 1 at the 0.2 mg/kg bw per
    day level, with a plateau of about 60%, and at the 2 mg/kg bw per day
    level a plateau of about 85% decrease was reached on day 3.  The
    acetylcholinesterase activity of erythrocytes was extremely variable.
    In the high dose groups however the depression was consistent
    throughout the study.  The acetylcholinesterase activity of the grey
    matter of the brain measured at necropsy was decreased at the 0.2 and
    the 2 mg/kg bw group in both males and females, whereas the activity
    was unaffected in white matter.

    RESIDUES IN FOOD

    USE PATTERN

    Additional information was provided on carbophenothion use patterns in
    the Netherlands:

                                                                                                  
                             Extent of Use
               Pest          and Economic      Application
    Crop       Controlled    Importance            rate         Formulation       Treatment
                                                                                              

    Grapes1    Red spider       small            0.6 g/m3       aerosol, 14.8%    Apply only
               mite             scale                           and 25.5%         before
                                                                                  blossom
                                                                                  and after
    Peaches1                                                                      harvest

    Plums
                                                                                              

    1 Glasshouse
    

    FATE OF RESIDUES

    Plants

    Available data on the plant metabolism of carbophenothion were
    reviewed by the Joint Meeting in 1972, 1976 (FAO/WHO, 1973, 1977). 
    The principal route of metabolism was judged to be thioether oxidation
    of carbophenothion to form the sulphoxide and sulphone, followed by
    phosphorothionate oxidation to form the oxygen analogue sulphone. Each
    of these products as well as the oxygen analogue of carbophenothion
    and the oxygen analogue sulphoxide were identified in plants.

    Laboratory photolysis experiments have also shown that ethion and four
    "other related organophosphorus products" can be formed although there
    was no evidence to indicate whether those would occur under field
    conditions.

    In the study evaluated in this Monograph Addendum (McBain et al,
    1977) water-emulsified [phenyl-14C] carbophenothion was brush-applied
    to the surfaces of selected fruits and foliage of a greenhouse-grown
    dwarf variety orange tree at a field concentration equivalent of 2.4 g
    active ingredient (labelled + unlabelled) per liter of spray solution.
    0.5 ml aliquots ( ca 0.94 mg after adjusting for losses) were
    applied to each fruit or foliage sample.  Samples were taken at 0, 3,
    5, 10, 26 and 42 days following treatment.

    Residues were separated, characterised and quantified using sequential
    solvent extractions, column chromatography, two-dimensional TLC, LSC
    and incubation with enzymes and acid.  Residues were categorised as
    external (surface) or internal.  Internal residues were further
    characterized as methanol-soluble (water-soluble + methylene
    chloride-soluble) and bound.

    The distribution of 14C residues in internal and external fractions
    as a percentage of those applied are summarised in Table 1.  At
    harvest, 42 days after application, the surface radioactivity had
    fallen to 11.7% and 5.3% of that applied to the fruit and leaves
    respectively. These surface residues were 32% and 23% of the total
    residues at harvest.  The total internal radioactivity at harvest was
    17.4% of that applied to the leaves (77% of the whole leaf harvest
    residue). For fruit at 26 days, 28% of the applied radioactivity was
    within the fruit (59% of the total whole fruit radioactivity) and at
    42 days 25% of the applied radioactivity remained within the fruit
    (68% of the total whole fruit radioactivity).

    14C recovered from fruit and foliage decreased from 98.4 and 102% at
    0 day to 36.2 and 22.6% respectively at 42 days.  Bound radioactivity
    was none to negligible at 0 day and 3.4 and 4.5% of applied (9.3 and
    19.8% of terminal) at 42 days for fruit and foliage respectively.

    There was little translocation of 14C from the surface of fruit to
    pulp and juice (<1% of applied).  It was shown that only a little
    14C translocation occurs from treated foliage to adjacent untreated
    foliage and fruit but it was not possible to determine how much of the
    applied 14C could have translocated to unanalysed fruit and foliage.

    The disappearance of radioactivity is attributed primarily to
    volatilisation, which is consistent with an experiment showing 50%
    dissipation of 14C within 80 h when [14C]-carbophenothion was coated
    on to glass slides as a thin layer.  As would be expected, however,
    this was a faster dissipation rate than was observed on the fruit and
    foliage.

        TABLE 1.  Distribution of 14C Recovered Following Topical Application of [14C]
    Carbophenothion to Orange Fruit and Foliage

                                                                                            

                      Distribution of 14C From Plant Harvest1
                        External2                 Internal
                                                                                        
                                       Methanol-Soluble
                                                           
    Samples and                                  methylene
    time of harvest                    water-    chloride-      total     bound   Total
    (days)                             soluble   soluble                          recovered
                                                                                            
    Treated foliage

    0                    102.3           0.0       0.0           0.0      0.01    102.3
    3                     73.1           6.8       1.3           8.1      0.4      81.6
    5                     37.2           7.2       1.5           8.7      0.7      46.6
    10                    39.0           9.0       1.7          10.7      0.4      50.1
    26                    19.0           8.1       4.8          12.7      3.9      35.8
    42                     5.3           5.0       7.9          12.9      4.5      22.6

    Treated fruit

    0                     98.4           0.0       0.0           0.0      0.0      98.4
    3                     70.7           3.0       2.1           5.1      0.9      76.7
    5                     74.7           7.1       2.2           9.3      0.6      84.6
    10                    64.0           8.7       3.3          12.0      1.3      77.3
    26                    19.7           20.3      5.2          25.5      2.7      47.9
    42                    11.7           12.6      8.5          21.1      3.4      36.2

    Pulp and juice

    0                                                            0.00               0.00
    3                                                            0.09     0.01      0.11
    5                                                            0.13     0.02      0.12
    10                                                           0.09     0.03      0.13
    26                                                           0.22     0.04      0.24
    42                                                           0.49     0.11      0.61
                                                                                            

    1 Expressed as % of applied
    2 External (surface) residues from acetone rinses.
    
    Individual components identified in peel and foliage external residues
    and in peel and foliage internal organosoluble residues were
    carbophenothion, its oxygen analogue and their respective sulphoxides
    and sulphones as shown in tables 2 and 3 (McBain et al, 1977).
    Unidentified polar and non-polar materials were also detected.
    Carbophenothion is quantitatively the most important individual
    component identified except in the 42-day external foliage fraction
    where the carbophenothion sulphoxide and sulphone predominate.
    However, in this same fraction unknown polar compounds exceed any
    individually identified residue.

    Unidentified surface residues have been reported previously (FAO/WHO,
    1973).

    In general, therefore, carbophenothion and its sulphoxide and sulphone
    are the major components of the residues identified with smaller
    quantities of the oxon and its sulphoxide and sulphone.  This is
    consistent with earlier findings.

    More specifically, carbophenothion and these 5 oxidative products
    account for over 90% of the residual 14C in external and internal
    peel extractives at 42 days.  They account for only 59 and 87%
    respectively of residual 14C in external and internal foliage
    extractives at 42 days, reflecting the higher percentage of
    unidentified moieties in the foliage surface residues.

    Carbophenothion dissipated more rapidly from the surface of foliage
    than from peel and the rate of loss from both decreased substantially
    with time as more of the remaining material was transported into the
    internal tissues.  The concentration of oxygen analogues in combined
    internal and external extraction plateaued after 10-25 days.

    There was no evidence of residues of 4-chlorophenylmethyl sulphoxide
    (4-ClPhSOMe), 4-chlorophenylmethyl sulphone (4-ClPhSO2Me), 3-hydroxy,
    4-chlorophenylmethylsulphone (3-0H, 4-ClPhSO2Me),
    4-chlorophenylsulphenylmethyl methyl sulphone (4-ClPhSMeS02Me), or
    4-chlorophenylsulphonylmethyl methyl sulphone (4-ClPhSO2Me), which
    are known to occur as animal metabolites (FAO/WHO, 1978).

    Water-soluble internal extracts, represented up to 9 and 20% of
    applied 14C at 10 and 26 days in foliage and fruit respectively,
    followed by a decline.  The non-polar fraction contained variable
    amounts of carbophenothion sulphoxide and sulphone and unknown
    compounds whereas the polar fractions contained unknown compounds and
    variable amounts of residues tentatively identified as the
    4-chlorobenzene sulphinic and sulphonic acids.  Evidence that these
    two moieties are present in plants apparently has not previously been
    reported to the Joint Meeting although they have been identified in
    animals.

    The proposed biotransformation of carbophenothion in the orange is
    given in figure 1 (McBain et al, 1977).

        TABLE 2.  Products Isolated by TLC From the External and Internal Organosoluble
    Fraction of [14C] Carbophenothion-treated Orange Fruit Peel

                                                                                      

    Products1,2                  Distribution of 14C as % applied dose, at indicated
                                               days after treatment
                                 0         3        5         10         26       42
                                                                                      

    Peel, External 14C

    Carbophenothion            97.65     68.25    71.67     59.94      16.95     9.04
    Carbophenothion-SO          0.17      1.26     1.04      1.91       1.24     1.27
    Carbophenothion-SO2         0.10      0.29     0.58      0.67       0.51     0.33
    Oxon                        0.04      0.23     0.25      0.29       0.11     0.10
    Oxon-SO                     0.00      0.01     0.02      0.03       0.05     0.09
    Oxon-SO2                    0.00      0.08     0.04      0.03       0.03     0.04
    Non-polar unknowns3         0.41      0.39     0.77      0.72       0.37     0.44
    Polar unknowns3             0.03      0.19     0.31      0.43       0.44     0.40
                        Total  98.40     70.70    74.68     64.02      19.70    11.71


    Peel, Internal
    Organosoluble

    Carbophenothion             0.00      1.24     1.82      2.53       3.97     4.14
    Carbophenothion-SO          0.00      0.26     0.11      0.31       0.61     2.21
    Carbophenothion-SO2         0.00      0.04     0.03      0.06       0.10     0.24
    Oxon                        0.00      0.10     0.03      0.07       0.10     0.45
    Oxon-SO                     0.00      0.08     0.02      0.08       0.15     0.94
    Oxon-SO2                    0.00      0.00     0.00      0.01       0.01     0.05
    Non-polar unknowns3         0.00      0.29     0.14      0.18       0.20     0.38
    Polar unknowns3             0.00      0.06     0.03      0.04       0.05     0.10
                        Total   0.00      2.07     2.18      3.28       5.19     8.51
                                                                                      

    1 14C-Products were identified by two-dimensional TLC analysis with the
    reference standards given in Table 1.  The following products did not occur as
    metabolites:  4-ClPhSOMe and 4-ClPhSO2Me; 3-OH, 4Cl, PhSO2Me; 4-ClPhSMeSO2Me;
    4-ClPhSO2MeSO2Me.

    2 TLC analyses of the 14C carbophenothion emulsion used to treat the orange tree
    revealed the following distribution of 14C-products: Trithion, 98.77%; combined
    oxygen analogues 0.14% and two non-polar unknowns positioned near Trithion, 1.10%.

    3 14C that failed to migrate significantly from the origin on chromatography was
    classified as polar unknowns.  The non-polar unknowns represented: (1) minor
    impurities present in the 14C carbophenothion treatment medium, (2) several minor
    unknown metabolites of carbophenothion and (3) unresolved 14C that smeared from
    the identified products along their paths of migration.
    

        TABLE 3.  Products Isolated by TLC from the External and Internal Organosoluble
    Fractions of 14C Carbophenothion-treated Orange Tree Foliage

                                                                                        

                                 Distribution 14C as % applied dose, at indicated
    Products1,2                                days after treatment
                                 0         3        5         10         26       42
                                                                                        

    Foliage, External 14C

    Carbophenothion            101.13     69.00    32.77     34.48      10.77     0.76
    Carbophenothion-SO           0.07      1.05     1.73      1.29       2.68     0.96
    Carbophenothion-SO2          0.04      1.04     0.85      1.01       1.61     1.15
    Oxon                         0.01      0.31     0.25      0.23       0.21     0.04
    Oxon-SO                      0.00      0.05     0.10      0.08       0.13     0.12
    Oxon-SO2                     0.00      0.03     0.08      0.05       0.10     0.07
    Non-polar unknowns3          0.99      1.02     0.82      0.88       1.05     0.59
    Polar unknowns3              0.06      0.64     0.61      1.00       2.44     1.62
                        Total  102.30     73.14    37.21     39.02      18.99     5.31


    Foliage, Internal
    Organosoluble 14C

    Carbophenothion              0.00      1.11     1.21      1.41       3.27     2.84
    Carbophonothion-SO           0.00      0.09     0.15      0.15       0.80     1.82
    Carbophenothion-SO2          0.00      0.01     0.02      0.03       0.19     0.49
    Oxon                         0.00      0.01     0.01      0.02       0.04     0.16
    Oxon-SO                      0.00      0.02     0.04      0.03       0.14     0.49
    Oxon-SO2                     0.00      0.00     0.00      0.00       0.01     0.08
    Non-Polar unknowns3          0.00      0.02     0.05      0.04       0.24     0.54
    Polar unknowns3              0.00      0.01     0.02      0.01       0.11     0.50
                        Total    0.00      1.27     1.50      1.69       4.80     7.92
                                                                                        

    1 14C-Products were identified by two-dimensional TLC analysis with the reference
    standards given in Table 1.  The following products did not occur as metabolites:
    4-ClPhSOMe and 4-C1PhSO2Me; 3-OH, 4Cl,PhSO2Me; 4-ClPhSMeSO2Me; 4-ClPhSO2MeSO2Me.

    2 TLC analyses of the 14C carbophenothion emulsion used to treat the orange tree
    revealed the following distribution of 14C-products: Trithion, 98.77%; combined
    oxygen analogues 0.14% and two non-polar unknowns positioned near Trithion, 1.10%.

    3 14C that failed to migrate significantly from the origin on chromatography was
    classified as polar unknowns.  The non-polar unknowns represented: (1) minor
    impurities present in the 14C carbophenothion treatment medium, (2) several minor
    unknown metabolites of carbophenothion and (3) unresolved 14C that smeared from
    the identified products along their paths of migration.
    
    FIGURE 1

    NATIONAL MAXIMUM RESIDUES LIMITS

    The Netherlands reported the following national tolerances to the
    Meeting.

                                                                      
                           Commodity                Tolerance (mg/kg)
                                                                      

    In force               fruits and vegetables    0.01

    Under consideration    fruit                    0.0511/

                           other commodities
                           of plant origin          0.05
                                                                      

    1/ At or about the limit of determination.


    EVALUATION

    COMMENTS AND APPRAISAL

    Present carbophenothion metabolism studies confirm and extend the
    experiments previously reported.  Both carbophenothion and its
    sulphoxide are metabolised both quantitatively and qualitatively very
    similarly.  The acute oral toxicity of the majority of the metabolites
    of carbophenothion in the rat have been evaluated previously.  They
    are considerably less toxic than that of the present compound.  These
    results indicate that carbophenothion is metabolised to less toxic
    compounds.

    In an acute delayed neurotoxicity study with technical carbophenothion
    at an extremely high dose one adult hen out of 14 showed minimal leg
    weakness, accompanied by nerve fibre degeneration.  However, no
    specific histopathological lesions were observed in spinal cord and
    medulla.  Furthermore, in a previous study with adult hens no
    neurotoxic effects had been observed.  It seems therefore very
    unlikely that carbophenothion induces delayed neurotoxicity.

    In a subacute study with dogs a no-effect level, based on
    cholinesterase inhibition, of 0.02 mg/kg bw/day was confirmed,
    assuring there was no reason to alter the previously established ADI
    for man.

    In response to requests listed as desirable by the 1977 Joint Meeting,
    a carbophenothion metabolism study on orange trees has been submitted
    and evaluated.  Additional information on national use patterns and
    tolerances was also submitted.

    In the metabolism study using greenhouse-grown orange trees (14C
    phenyl) carbophenothion was applied to selected fruits and foliage,

    which were harvested for analysis at 0, 3, 5, 10, 26 and 42 days.  It
    was found that initial residues were almost entirely surface residues
    with a high initial rate of loss, attributed primarily to
    volatilisation.  After some time, internal residues became the highest
    percentage of the total terminal residue in foliage and peels.  There
    was little translocation of residues from treated foliage to adjacent
    untreated foliage and fruit, although there was no basis on which to
    determine how much of the total terminal residues would be in the
    fruit and foliage not adjacent to that treated.  There is minimal
    translocation of residues on treated fruit into the pulp and juice.

    Of the individual components identified in the residue,
    carbophenothion was at the highest concentration followed by its
    sulphoxide and sulphone.  The only exception was the external foliage
    residue at 42 days from application where the carbophenothion residue
    was exceeded by both that of its sulphoxide and sulphone.  Residues of
    the oxon and its sulphoxide and sulphone occurred in lesser amounts.

    In addition to the identified carbophenothion metabolites, relatively
    large amounts of unidentified compounds remained, especially in the
    external foliage residue where polar unknowns constituted the largest
    single part of the terminal surface residue (1.62% of the applied
    radioactivity).  This is however less than the 3.84% and 1.82%
    respectively of carbophenothion and its sulphoxide found in foliage
    internal organosolubles.  A small portion of the residue was bound
    into insoluble components.  There was no evidence of the S-methylation
    of the 4-chlorothiophenol moiety that has been observed in animals.  A
    significant proportion of the internal residues was water soluble, and
    similarly some of these were identified while others were not.  Among
    those identified were carbophenothion sulphoxide and sulphone and,
    tentatively, the 4-chlorobenzene sulphinic and sulphonic acids.  The
    latter two have not been previously reported at Joint Meetings as
    plant metabolites although they are known to be animal metabolites.

    This study is consistent with earlier findings on the metabolism of
    carbophenothion in plants and adds significantly to the understanding
    of plant metabolism.  No additional information was provided to
    elucidate the terminal residues or photolysis products of
    carbophenothion under field conditions, as previously considered
    desirable.  However, the metabolism study provided data that, along
    with information previously evaluated, enabled the Meeting to conclude
    that unidentified terminal residues or photolysis products under field
    conditions are unlikely to be more than a very small portion of the
    total residues.  The meeting confirms that residues currently measured
    are sufficient.

    Level causing no toxicological effects

    Rat:    3 mg/kg in the diet, equivalent to 0.15 mg/kg bw/day.

    Dog:    0.02 mg/kg bw/day.

    Human:  0.8 mg/human/day corresponding to 0.01 mg/kg bw/day.

    Estimate of acceptable daily intake for man

    0 - 0.0005 mg/kg bw/day.

    RECOMMENDATION

    It is not necessary to revise limits or the expression of limits
    previously recommended.


    REFERENCES

    Bier, C.B. A subacute (21-day) oral toxicity study of Trithion in the
    beagle dog. Bio-Research Laboratories TLD, (1979) Unpublished report
    submitted to WHO by Stauffer Chemical Company, USA. T-10015.

    McBain, J.B., Wren, J.P. and Menn, J.J. Metabolism of 14C phenyl
    Carbophenothion by Orange Trees. (1977) Unpublished study provided by
    Stauffer Chemical Company, Mountain View Research Center, Mountain
    View, Ca. 94042. MRC-B-69, July, 1977.

    Menn, J.J., De Baun, J.R., Hoffman, L.J. and Ross, J.H. Metabolism of
    thioaryl organo-phosphorus insecticides. Environmental quality and
    safety, Supp. Vol III, Pesticides, 382-388, (1975).

    Menn, J.J., De Baun, J.R. and McBain, J.B. Recent Advances in the
    Metabolism of Organo-phosphorus Insecticides. Fed. Proc. 35, 2598.

    Miller, J.L. and Sprague, G.L. Acute delayed neurotoxicity study with
    technical TrithionR in adult hens. Report Woodward Research
    Corporation, 1978 (unpublished) T-6404.

    


    See Also:
       Toxicological Abbreviations
       Carbophenothion (ICSC)
       Carbophenothion (WHO Pesticide Residues Series 2)
       Carbophenothion (Pesticide residues in food: 1976 evaluations)
       Carbophenothion (Pesticide residues in food: 1977 evaluations)
       Carbophenothion (Pesticide residues in food: 1979 evaluations)
       Carbophenothion (Pesticide residues in food: 1983 evaluations)