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    AGP:1970/M/12/1

    WHO/FOOD ADD/71.42

    1970 EVALUATIONS OF SOME PESTICIDE RESIDUES IN FOOD

    THE MONOGRAPHS

    Issued jointly by FAO and WHO

    The content of this document is the result of the deliberations of the
    Joint Meeting of the FAO Working Party of Experts and the WHO Expert
    Group on Pesticide Residues, which met in Rome, 9-16 November, 1970.

    FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS

    WORLD HEALTH ORGANIZATION

    Rome, 1971

    THIABENDAZOLE

    IDENTITY

    Chemical name

    2-(4'-thiazolyl) benzimidazole

    Synonyms

    Thibenzole (R), Tecto (R), Mertect (R), TBZ

    Structural formula

    CHEMICAL STRUCTURE 

    Molecular Weight 201.3

    Other information on identity and properties

    A stable, white crystalline powder. Solubility in water at pH 2.2 =
    3.84 percent. Solubility decreases at higher or lower pH values. Very
    soluble in dimethyl formamide, alcohols and acetone. Soluble in
    chlorinated hydrocarbons, esters and ether.

    No change in ultraviolet absorption occurs in samples stored eight
    days at 100C. Heating at 220C at atmospheric pressure has no
    apparent effect on its antifungal properties.

    Technical grade contains not less than 97 percent thiabendazole. There
    are no related impurities in the technical grade. Melting point is
    296-303C. Pure products melt at 304-305C.

    EVALUATION FOR ACCEPTABLE DAILY INTAKE

    BIOCHEMICAL ASPECTS

    Absorption, distribution, biotransformation and excretion

    Following a dose of 100 mg/kg body-weight, thiabendazole labelled with
    14C in the benzene ring was rapidly absorbed from the
    gastrointestinal tract of rats, with a maximum concentration found

    from two to three hours after initial treatment. Radioactivity
    gradually disappeared from the blood, and approximately 92 percent of
    a dose of 25 mg/kg and 80 percent of a 100 mg/kg dose was excreted in
    the urine and faeces within three days. Most of the drug and its
    metabolites were excreted within the first 24 hours. Of the
    metabolites, 50 percent appeared as the glucuronide of
    5-hydroxythiabendazole (II) and 40 percent as the sulfate ester of the
    same aglycone (III) (see Fig 1). Traces of unchanged thiabendazole and
    5-hydroxythiabendazole were also evident (Robinson, 1965a).

    Dogs given a single oral dose of 50 mg/kg body-weight of 14C-labelled
    thiabendazole were found to have maximum plasma levels within two
    hours. Excretion was essentially complete in eight days with
    approximately 35 percent of the dose appearing in the urine and 47
    percent appearing in the faeces (Robinson 1965a).

    Sixteen male humans were administered thiabendazole at dosages of 1 to
    2 grams per person in the form of tablets, wafers, capsules or
    suspension. The material was rapidly absorbed, with peak plasma
    concentrations observed about one hour after treatment. Plasma drug
    levels declined rapidly thereafter and reached essentially zero values
    between 24 and 48 hours. Thiabendazole and its metabolic products were
    excreted rapidly in the urine and faeces in 48 hours, which accounted
    for 87 to 100 percent of the dose. Approximately half of the material
    in the urine was associated with compounds identified as the
    glucuronide and sulfate esters of 5-hydroxythiabendazole (II and III).
    In plasma, both unchanged thiabendazole and free
    5-hydroxythiabendazole (I) were also found. At the higher dosage
    levels, maximum drug levels in plasma appeared at three hours
    following dosing. These studies were done utilizing 14C-labelled and
    unlabelled thiabendazole (Robinson, 1965a; Tocco et al., 1966).

    Information is also available on the metabolism of thiabendazole in
    sheep goats and cattle. In cows, about 0.1 percent of an oral dose was
    detectable in the milk within 60 hours. Details of this work are given
    in the section entitled "Fate of residues, In animals".

    Based upon all the metabolism studies, the pathway for thiabendazole
    metabolism is represented by Fig. 1.

    TOXICOLOGICAL STUDIES

    Special studies on reproduction

    Mouse

    A multi-generation reproduction study for five generations utilized 25
    male and 25 female mice given diets containing thiabendazole at
    concentrations of 0, 200, 1000 and 5000 ppm. When the mice attained
    the age of eight weeks, they were mated and continued on the same
    diet. The young from these matings, when they were weaned, were
    maintained on the test diet and mated when they reached the age of
    eight weeks. This procedure continued for five complete reproductive

    cycles. No effects were noted at 200 ppm. At 1000 ppm, slight decrease
    in the weights of weanlings was observed in all five generations. At
    5000 ppm, the number of mice born and weaned per litter were reduced,
    and a marked reduction in the average weanling weight of the young was
    observed (Robinson, 1965a).

    Rat

    A three-generation, two-litter reproduction study (10 males and 10
    females per group) at dosages of 0, 20, 40 and 80 mg/kg body-weight
    per day showed no adverse effect on reproduction, lactation or
    histomorphology in the three generations of rats examined. The only
    treatment-related findings were decreased body-weight, but not growth
    rate, and decreased food consumption in the male rats at all dosage
    levels in the F1 and F2 generations. Slight decreases in final
    body-weights and food consumption at the 80 m/kg dose level in the F1
    and F2 generation females was also observed (Vogin,1968).

    Two groups of rats (20 male and 20 female) were placed on a diet
    containing 0 or 500 ppm thiabendazole. There were no abnormalities
    among the young from either mating attributable to thiabendazole at
    500 ppm. It was concluded that no evidence of teratogenesis is present
    in the data from this particular study (Johnson, 1964).

    Sheep

    Pregnant ewes tolerated a single dose of 400 mg/kg body weight at 2
    1/2 to 8 weeks prior to lambing without any effect on the birth rate,
    growth or viability of the lambs. This dose had no effect on the ewes
    throughout the test interval or on viability of the lambs to six weeks
    of age. Miscellaneous tests using 39 ewes produced 29 weanling lambs
    after treatment with thiabendazole at 400 mg/kg, whereas, from a
    control group of 22 ewes, 13 lambs were weaned. Ewes tolerated a
    single dose of 18 grams per animal (equivalent to 235 to 335 mg/kg)
    three to ten days after lambing without any effect on the weight or
    food intake of the ewes or survival growth of the lambs up to weaning
    time of six weeks (Robinson, 1965a).

    Special studies on cardiovascular and respiratory effects

    Oral doses of thiabendazole (4000 mg/kg) produced no striking acute
    pharmacological effects on blood pressure or respiration in either
    cats or dogs. Similarly, there were no alterations in the
    electrocardiogram (Robinson, 1965a, 1965b).

    Special studies on eye irritation

    Other than a very slight erythema observed for approximately one hour
    after application of 1 ml of thiabendazole as a 12 percent suspension
    in sodium carboxymethyl cellulose or 10 mg of the dry powder to the
    conjunctival sac of two rabbits, no evidence of irritation was noted
    (Robinson, 1965a, 1965b).

    FIGURE 

    Special studies on dermal irritation

    There was no evidence of a significant degree of irritation when 0.5
    gm thiabendazole was applied in cold cream at concentrations of 10 and
    50 percent (w/w) for 24 hours to intact rabbit skin (Robinson, 1965a,
    1965b).

    Acute toxicity

    LD50 doses for various animal species are given in Table I.

    Toxic signs observed following administration of large doses of
    thiabendazole by oral or intraperitoneal routes were generally similar
    and consisted of lethargy and stupor. The intravenous administration
    of a large dose of the hydrochloride produced narcosis. Death appeared
    to be due to respiratory failure. Rabbits that survived large doses
    initially lost weight but recovered after several days (Robinson,
    1965a).

    Short-term studies

    Calf

    Five female Holstein calves (seven months old) were fed a diet
    containing 0, 320, 1 000, 3 200 or 10 000 ppm of thiabendazole for a
    14-week test period. Calves tolerated 3 200 ppm thiabendazole in their
    diet without observable effects on growth, food intake or general
    condition. This concentration corresponded to a mean daily intake of
    90 mg/kg body-weight. At the 10 000 ppm thiabendazole level, the
    calves grew normally for the first two weeks, but in the following 12
    weeks the weight gain was about half that observed for the controls.
    Gross examination at the time of autopsy revealed no pathological
    condition, and histological examination of several tissues showed no
    change as resulting from the incorporation of thiabendazole in the
    diet (Robinson, 1965a).

    Chicken

    Groups of 0.5 week old male White Leghorn chicks (10 to 20 per group)
    were fed thiabendazole in the diet at dosage levels ranging from 1 to
    10 000 ppm for a period of 2 1/2 weeks. This level corresponded to a
    mean daily intake of thiabendazole ranging from 0.1 mg/kg to 1 200
    mg/kg. A gradual decrease in growth appeared to occur with
    concentrations of 100 ppm thiabendazole in the diet. This dose
    corresponded to approximately 13 mg/kg per day. Gross pathological
    examination at autopsy of the chicks on a dietary level of 4 000 ppm
    thiabendazole showed them to be normal except for a smaller size
    (Robinson, 1965a).

    Dog

    Groups comprising two male and two female dogs were orally
    administered thiabendazole daily for periods of over two years at

    doses of 0, 20, 100 and 200 mg/kg body-weight/day. No clinical signs
    of morbidity were observed; food and water consumption were normal;
    blood and urine chemistry were normal; a slight retardation in
    body-weight gain at the 200 mg/kg level was observed, which was
    accompanied by a slight reduction in total erythrocyte count. At this
    level, changes in haemoglobin concentration and haematocrit were also
    observed. No gross pathological observations were noted at the
    conclusion of this test, and organ-weights appeared normal.

        TABLE I

    Thiabendazole toxicity in various animal species
                                                                                             

                                                  LD50
    Animal     Route     Form     Solvent    (mg/kg body-weight)    Reference
                                                                                             

    Mouse      oral      HCl      Water           2 400             (Robinson 1965a, 1965b)
               ip        HCl      Water             430                 "       "      "
               iv        HCl      Water             150                 "       "      "
               oral      Base     CMC 2% 1        3 810                 "       "      "

    Rat        oral      HCl      Water           3 600                 "       "      "
               ip        HCl      Water           1 850                 "       "      "
               iv        HCl      Water             130                 "       "      "
               oral      Base     CMC 2%          3 330                 "       "      "

    Rabbit     oral      Base     CMC 2%          3 850                 "       "

    Sheep      drench    HCl      Water           2 000                 "       "

    Goat       drench    HCl      Water          >2 000                 "       "
                                                                                             

    1 CMC = carboxy methyl cellulose
    
    A significant haemosiderosis was present in dogs at the 100 and 200
    mg/kg level in the spleen, liver, lymph nodes and bone-marrow. This
    was not associated with an increase in serum haemoglobin. No
    significant toxicological effects were noted at 20 mg/kg/day
    (Robinson, 1965a, 1965b).

    Groups of dogs (three male and three female) were orally administered
    thiabendazole for two years at doses of 0, 20, 50 and 125 mg/kg
    body-weight/day. At the 125 mg/kg/day level, two of six dogs died. One
    of these dogs had marked liver cirrhosis, seminal tubular
    degeneration, bone marrow atrophy and degenerative renal changes.
    Slight to moderate reduction in haemoglobin and packed cell volume
    occurred. Elevated blood urea nitrogen, serum alkaline phosphatase and
    serum glutamic oxal oacetic transaminase were evident. Urinary albumin
    was seen more frequently than in controls. Inspissated bile entwined
    in the gall bladder villi was also observed in one dog. One dog of
    this group was sacrificed and was found to have pulmonary arteritis of
    parasitic origin. At 50 mg/kg/day, growth of male dogs was slightly
    depressed. All six dogs given thiabendazole at 50 mg/kg survived the
    study, and their tissues differed from those of the control dogs in
    that some liver glycogen depletion was observed in three dogs and
    inspissated material was found adhering to the gall bladder mucosa in
    one. Five of six dogs survived thiabendazole at 20 mg/kg for the term
    of the study. Three of the five showed slight liver glycogen
    depletion. A general impression in this study was of an overall
    appearance of mild chronic inflammatory degenerative renal changes in
    all treated dogs (Woodard et al., 1964).

    Pig

    Five groups of four barrows each were given a diet containing 0, 320,
    1 000, 3 200 and 10 000 ppm thiabendazole for 14 weeks. A
    concentration of 320 ppm was tolerated without observable effects for
    the 14-week period. This concentration corresponded to an intake of 15
    mg/kg body-weight per day. Concentration of 10 000 ppm thiabendazole
    in the diet caused reduced weight gains and reduced food consumption
    with no mortality observed. Gross pathology examination revealed no
    abnormal conditions (Robinson, 1965a).

    Rat

    Rats (10 males and 10 females per group) were administered
    thiabendazole once daily at dosage levels of 0, 100, 400, 800, 1 200
    and 1 600 mg/k-g body-weight per day. During the 30-day experimental
    period, rats in the 800 mg/kg per day group decreased their food
    intake and gradually lost weight. They were slightly less active, had
    ruffled fur and became slightly flaccid. Three males and three females
    died during the course of the experiment. All of the rats in the 1 200
    and 1 600 mg/kg groups died during the 30-day test period. Rats dosed
    at 100 and 400 mg/kg appeared normal throughout the test period,
    although a slight depression in body-weight gain was noted in males at
    100 mg/kg per day and in females at 400 mg/kg per day. Haematologic
    studies on rats receiving 800 mg/kg per day showed a mild neutrophilia
    with concurrent lymphopenia. There was also a suggestion of a decrease
    in red blood cell elements. No gross anatomical changes were noted in
    rats dosed with 100 mg/kg per day, whereas male and female rats
    treated with 400 mg/kg per day showed thymic involution. A slight

    enlargement of the liver was also noted in females at 400 mg/kg per
    day. Animals surviving 800 mg/kg per day showed gross signs indicating
    starvation where normal body fat depot and subcutaneous fat appeared
    to be depleted. Liver and adrenals were slightly enlarged in males and
    females and the thymus involuted. Microscopic pathology showed bone
    marrow hypoplasia, thymus haemosiderosis and colloid depletion in the
    thyroid at 400 mg/kg (Robinson, 1965a, 1965b).

    Rats (30 males and 30 females per group) were administered
    thiabendazole suspended in 1.0 percent methocel daily for 180 days at
    doses of 0, 12.5, 25, 50, 100, 200 and 400 mg/kg body-weight. All of
    the animals survived throughout the duration of the experiment. At 400
    mg/kg, a depression in body-weight was observed with both sexes. At
    200 mg/kg per day, the male rats showed a slight depression in weight
    gain. No clinical signs of toxicity were observable at levels below
    100 mg/kg. Haematological studies at 400 mg/kg per day indicated a
    slight suggestion of a fall of the red blood cell elements which was
    not evident at levels below this dose. Biochemical blood studies and
    urinalysis were normal at all dosage levels with a slight polyurea in
    both male and female rats at 400 mg/kg, which was apparently the
    result of a slight increase in water consumption at this dosage level.
    Gross pathology showed thymic involution in both males and females at
    400 mg/kg per day and in females at 200 mg/kg per day. There was an
    increase in liver size at doses of 100 mg/kg and above in males and at
    25 mg/kg per day and above in females. Males dosed with 200 and 400
    mg/kg and females at 400 mg/kg showed an apparent increase in kidney
    weight. Microscopic examination of animals at 100 mg/kg showed a small
    incidence of haemosiderosis of the thymus. At 200 and 400 mg/kg, these
    examinations showed considerably more haemosiderosis of the thymus and
    colloid depletion in the thyroid (Robinson, 1965a, 1965b).

    Sheep

    Thirty weanling wethers were employed in a 16-week study at doses of
    0, 10, 50, 100, 200, 400 and 800 mg/kg body-weight per day.
    Thiabendazole was administered in gelatin capsules. Two of the four
    sheep dosed at 100 mg/kg did not survive the test period. However,
    because of infection, thiabendazole may not have been directly related
    to the cause of their death. All the animals treated at 200 mg/kg per
    day and above died before the end of the test interval. No significant
    effects on blood biochemistry or urinalysis were observed. Doses of 50
    mg/kg and above affected body-weight gain, after approximately 120
    days on the test. Doses of 10 mg/kg had no effect. Food consumption
    was unaffected at doses of 10 mg/kg per day, but a minor decrease was
    noted at 50 and 100 mg/kg per day and a significant decrease at 200
    mg/kg per day and above. No gross pathological changes were observed
    at doses below 200 mg/kg per day. Starvation and loss of body-weight

    complicated the interpretation of data concerning organ-weights at 200
    mg/kg and above. At 200 to 800 mg/kg, a moderate hypoplasia of the
    bone marrow with replacement by adipose tissue was observed. Loss of
    colloid in thyroid was evident at 800 mg/kg, as well as lymphoid
    atrophy at the higher dose levels (Robinson, 1965a).

    Five groups of two ewe lambs (10 to 12 weeks old) and eight wether
    lambs (10 to 19 weeks old) were fed diets containing 0, 100, 320, 1
    000 and 3 200 ppm thiabendazole for periods up to 50 weeks. Sheep 10
    weeks of age tolerated 1 000 ppm thiabendazole in the diet for the
    duration of the experiment. (This dose corresponded to 30 to 50 mg/kg
    body-weight per day.) The sheep tolerated 3 200 ppm in their diet for
    the first 14 weeks but thereafter lost weight in the final stages of
    the experiment (Robinson, 1965a).

    Long-term studies

    Rat

    Groups of rats (35 of each sex) were fed dietary levels adjusted to
    provide 0, 10, 40 or 160 mg/kg body-weight per day of thiabendazole
    for up to two years. At the 160 mg/kg level, a reduction of weight
    gain of about 25 percent, with concomitant reduction in food
    consumption and slightly reduced haemoglobin and microhaematocrit
    values, were the only changes seen attributable to compound
    administration. No effects attributable to compound administration
    were noted with respect to survival, time of death in non-survivors,
    incidents or location of neoplasms and histopathological examination
    of the tissues. No effect on food consumption or haemograms were seen
    at either the 10 or 40 mg/kg levels. Very slight depression in growth
    rate, of questionable significance, was observed in the male rats at
    40 mg/kg but not at 10 mg/kg. At the 10 mg/kg level, the mean absolute
    thyroid weights of the male rats (including the individual thyroid
    weights from five out of eight animals tested) were heavier than the
    mean thyroid weight of the controls. This effect was not observed in
    the rats given 40 or 160 mg/kg (Woodard et al., 1964).

    OBSERVATIONS IN MAN

    Human subjects have ingested a single oral dose of 2 g (approximately
    30 mg/kg body-weight) of thiabendazole in studies designed to
    ascertain the metabolic fate (see "Biochemical aspects"). No
    information was given on the toxicological effects, if any (Robinson,
    1965a).

    COMMENTS

    Metabolic studies indicate a rapid absorption of thiabendazole from
    the gastrointestinal tract of rats, dogs, sheep and goats with
    excretion in urine and faeces complete within 3-8 days. In cows,
    approximately 0.1 percent of an oral dose was detectable in milk
    within 60 hours. In excreta of man and all animals studied,
    metabolites include 5-hydroxythiabendazole, free and conjugated as a
    glucuronide and sulfate ester, as well as traces of unchanged
    thiabendazole. Slight differences in man and other animals were noted
    in quantitative recovery of the various compounds. A common effect for
    several animal species at high dose levels of thiabendazole is
    retarded growth.

    Short-term studies in rate, sheep, pigs, calves and chickens have been
    carried out with oral administration or with incorporation of
    thiabendazole in the diet for periods of 4-50 weeks. At higher doses
    in rats, thymus involution, colloid depletion in the thyroid and
    increase in liver and kidney size was observed. Loss of the colloid in
    thyroid of sheep administered 200 mg/kg body-weight was also evident.
    Oral administration of 12.5 mg/kg for 180 days did not cause
    significant toxicological changes in rats.

    In a two-year dog study a no-effect level of 20 mg/kg body-weight was
    demonstrated. A two-year study in rats showed heavier thyroid glands
    at 10 mg/kg body-weight/day in the male animals; this effect, however,
    was not dose dependent.

    A five-generation reproduction study in mice showed no adverse effects
    at 200 ppm level. In a three-generation study in rats, only decreased
    body-weight in relation to decreased food consumption could be
    observed.

    TOXICOLOGICAL EVALUATION

    Level causing no toxicological effect

    Dog:      20 mg/kg body-weight/day

    Mouse:    200 ppm in the diet, equivalent to 30 mg/kg body-weight/day

    Rat:      10 mg/kg body-weight/day

    ESTIMATE OF ACCEPTABLE DAILY INTAKE FOR MAN

    0 - 0.05 mg/kg body-weight

    RESIDUES IN FOOD AND THEIR EVALUATION

    USE PATTERN

    Administration to animals

    Thiabendazole is widely used as a broad spectrum anthelmintic for
    sheep, cattle, horses, pigs and goats. It is also administered as a
    vermifuge directly to man. Following the announcement by Brown (1961)
    of the chemotherapeutic properties of benzimidazoles, many
    investigators report the efficacy of thiabendazole as an anthelmintic.
    Gordon (1964) generally reported on its properties. Since that time
    very large numbers of sheep and cattle have been treated with
    thiabendazole suspensions. In sheep a dose of 44 mg/kg is adequate in
    moot circumstances. Dosing may be repeated as frequently as once per
    month, depending upon the level of reinfestation. In cattle a dose of
    88 mg/kg is usually employed and in pigs 50-100 mg/kg.

    Usually the dose is administered by drenching (oral administration of
    an aqueous suspension) but for convenience it is possible to
    administer via the feed when dealing with individual animals. At the
    present time, economic considerations probably preclude the use of
    thiabendazole as a feed additive.

    Pre-harvest treatment of plants

    Thiabendazole has been evaluated and has proved effective for use as a
    fungicide on a number of crops including apples, pears, peaches,
    apricots and bananas but, as far as is known, pre-harvest treatments
    have not yet been adopted commercially. Preliminary results of residue
    trials indicate that the residues from such pre-harvest uses will vary
    considerably due to the systemic action of thiabendazole and the
    ability of plants to take it up from the soil (Wernke, 1969).

    Post-harvest treatment

    Bananas

    Scott (1967), Burden (1967), Anon (1967, 1969), Cuille (1968) and
    Shillingsford (1970) have shown that thiabendazole is highly effective
    for control of post-harvest fungal rots of bananas, when the picked
    fruit is dipped in a suspension of thiabendazole containing from 100-1
    000 ppm. A dip containing 200 ppm (0.02%) appears to be the most
    widely used. Post-harvest spray treatments have also proved effective
    in experimental use, but it is not known whether these are yet used in
    practice.

    Thiabendazole is most soluble in acid solution and can be rendered
    soluble by treatment with lactic acid. Considerable experimental work
    was carried out with lactic acid solutions as fruit dips, but in spite
    of the convenience, the acidic solution gave rise to corrosion
    problems in packing houses and during transport and resulted in

    decomposition of the thiabendazole. Such solutions have been abandoned
    in favour of micro-fine powder suspensions which require minimal
    agitation to maintain uniform concentrations.

    After the bunches of bananas have been picked, they are usually washed
    in water or a solution of sodium hypochlorite. The bunches are then
    separated into hands or clusters of 10-12 fruit which is allowed to
    "bleed" in water for approximately 15-30 minutes. This facilitates the
    removal of natural latex which, if allowed to run on the fruit, will
    result in a visual disfigurement. The wet fruit is then dipped for 2-4
    minutes in a suspension containing 200 ppm thiabendazole before being
    drained for packing into cartons. No visible residue results from the
    treatment.

    Citrus

    Crivelli (1966) and his associates in Italy first demonstrated that
    Penicillium decay of oranges could be prevented by dipping fruit in
    solutions containing 750-1 000 ppm thiabendazole dissolved in lactic
    acid. Confirmation by other laboratories indicated that concentrations
    of 200 to 500 ppm are adequate to give excellent control of
    Penicillium decay. Brown (1967), Eckert (1968), Harding (1968),
    McCormack (1968), Seberry (1968) show the efficacy of thiabendazole in
    controlling Penicillium, and Seberry (1968) gives data showing
    reduction in stem end rot caused by Diaporthe citri after dipping
    oranges in suspensions of 500-1 000 ppm. Spraying or dipping citrus
    fruits in 0.5 to 1.0 percent thiabendazole suspensions, without
    subsequent rinsing, prevents sporulation of Penicillium upon the
    surface of decaying fruits. Oranges so treated have a natural
    appearance with no odour or visible residue.

    The most efficient methods for commercial application of thiabendazole
    appear to be as picking bin drenches, processing line spray or as an
    additive to water emulsion waxes. Thiabendazole is registered in
    Australia and U.S.A. as a post-harvest spray or dip treatment of
    citrus fruit with suspension containing up to 5 000 ppm thiabendazole.
    A rinse is not required, but rinsing does not appear to significantly
    reduce the efficacy of a dip treatment.

    Sporulation of Penicillium on the surface of decayed fruit causes
    superficial soiling of sound fruits adjacent to decayed fruits. This
    soiling can completely ruin all the fruit in a whole container with
    only one or two decayed fruits. For this reason, a post-harvest
    fungicide treatment must control both primary decay (prevent infection
    of fruit) and reduce Penicillium sporulation on the surface of those
    fruits which decay due to deep primary infections. Diphenyl and
    sodium-o-phenylphenate, which have been used as standard treatments
    for many years, have serious deficiencies. Thiabendazole on citrus wax
    is used as a means of controlling fungus diseases referred to as green
    mould, blue mould and stem end rot. This treatment is effective in
    controlling both primary decay and sporulation.

    Thiabendazole/citrus wax treatments are used commercially in Israel
    and on an experimental basis in U.S.A. Aqueous emulsions of waxes
    designed for application to citrus have been in use for many years.
    Thiabendazole is suspended in sufficient wax emulsion to yield a
    concentration of up to 0.5% w/w thiabendazole (5000 ppm). The prepared
    emulsion is used without dilution in waxing equipment designed to
    apply citrus wax evenly to freshly washed citrus. In this equipment,
    the wax containing the thiabendazole is applied through nozzles to the
    citrus as it revolves on horsehair brushes below. The nozzle is
    attached to a chain to move back and forth across the width of the
    waxer at a right angle to the flow of the citrus passing along the
    conveyer. The amount of wax applied is governed by the size of the
    nozzle and the pressure at which it is applied. These are equilibrated
    with the flow of the citrus so that 1 litre of emulsion will coat ca.
    800-900 kg of citrus fruit.

    Other fruit

    Bondoux (1967) reports good results from soaking apples in dilute
    suspension of thiabendazole. Fripp (1969) showed thiabendazole
    outstanding for control of brown rot (Sclerotinia fructicola) of
    peaches. It is known that many other investigators are working with
    thiabendazole to control post-harvest losses of a wide variety of
    fruits, including cherries and pineapples, but as yet the available
    data are not sufficient to enable proposals to be made for tolerances.

    RESIDUES RESULTING FROM SUPERVISED TRIALS

    Bananas

    Residue trials were carried out in Honduras. Green bananas treated
    according to recommended practice were shipped under commercial
    conditions at a temperature of 14-15C. Analysis was made 8 days
    after treatment in the case of green bananas and 15 days in the case
    of ripe fruit.

    A review of the residue data showed that at the recommended treatment
    rate of 200 ppm, a maximum of 0.18 ppm thiabendazole was found in the
    banana pulp (see Table II), while a maximum of 1.24 ppm thiabendazole
    was present in the peel (see Table III), when calculated on a whole
    fruit basis. Therefore, at the recommended rate of 200 ppm, a maximum
    of 1.4 ppm thiabendazole is possible in the whole unpeeled banana.

    Pulp and peel residues generally increase slightly with an increase in
    treatment rate, (see Table II) while the lower treatment rate (100
    ppm) results in somewhat lower residue levels of thiabendazole in pulp
    and peel (see Tables II and III). At the 100 ppm treatment level, an
    average of 0.018 ppm and 0.049 ppm was found in ripe banana pulp from
    two trials. This increased to an average of 0.068 ppm - 0.110 ppm at
    the 200 ppm treatment rate. Residues appear to level off at the 200
    ppm treatment rate, since ripe banana pulp contained an average of
    0.126 ppm and 0.094 ppm at the 400 ppm treatment rate.

    TABLE II

    Thiabendazole residues (ppm) in pulp of green and ripe bananas 1
                                                                                   
    Treatment                   GREEN                              RIPE
                      Average, ppm    Range, ppm       Average, ppm     Range, ppm
                                                                                   

    Control    1        0.0095        0.005-0.01          0.011         0.01-0.02
               2        0.013         0.01 -0.03          0.024         0.01-0.04

    100 ppm    1        0.030         0.01 -0.05          0.018         0.01-0.03
               2        0.037         0.03 -0.05          0.049         0.03-0.07

    200 ppm    1        0.074         0.04 -0.10          0.068         0.05-0.09
               2        0.067         0.05 -0.08          0.110         0.05-0.18

    400 ppm    1        0.173         0.05 -0.31          0.126         0.10-0.18
               2        0.126         0.07 -0.17          0.094         0.07-0.13
                                                                                   

    1 Results on treated bananas are not corrected for results on control bananas.

    TABLE III

    Thiabendazole residues in peel of green and ripe bananas 1
                                                                                   

    Treatment                  GREEN                            RIPE
                      Average         Range            Average          Range
                       (ppm)          (ppm)             (ppm)           (ppm)
                                                                                   

    Control    1      0.020         0.013-0.042         0.040         0.024-0.059
               2

    100 ppm    1      0.39          0.33 -0.52          0.55          0.39 -0.68
               2

    200 ppm    1      0.12          0.97 -1.21          0.17          1.12 -1.24
               2

    400 ppm    1      2.24          2.01 -2.64          2.73          2.61 -2.80
               2
                                                                                   

    1 Results on treated bananas are not corrected for results on control bananas.
    
    The analytical method used had a sensitivity of 0.05 ppm with blank
    values for untreated banana peel and pulp in the range of 0.01 - 0.04
    ppm. The recovery of thiabendazole added to banana pulp and banana
    peel ranged from 74.3 percent to 90.2 percent with an average of 87.3
    percent.

    In Australia, experiments were conducted by the Department Agriculture
    to investigate the use of thiabendazole as a banana dip under
    conditions of commercial usage; 100 cases (250 kg) of fruit was
    treated at each of four concentrations of thiabendazole suspension.
    The results were as follows:


        TABLE IV

    Whole fruit residues produced by various treatment rates
                                                                                                                

         Treatment                   On Peel                 In Peel                  In Pulp            Total

    Rate         Variety       Average     Range        Average     Range        Average    Range       Residue
    ppm                          ppm        ppm           ppm        ppm           ppm       ppm          ppm
                                                                                                                

    0            Orange         0.076     0.00-0.33      0.008     0.00-0.03      0.00     0.00-0.01     0.09

                 Lemon          0.02      0.00-0.03      0.004     0.00-0.02      0.00     0.00-0.01     0.02

                 Grapefruit     0.01      0.01-0.02      0.01      0.01-0002      0.01     0.00-0.02     0.03

    5 000        Orange         0.72      0.33-1.28      0.072     0.00-0.25      0.01     0.00-0.04     0.73

                 Lemon          0.31      0.16-0.74      0.045     0.00-0.13      0.005    0.00-0.01     0.36

                 Grapefruit     0.47      0.27-0.72      0.20      0.14-0.4?      0.02     0.00-0.04     0.66

    10 000       Orange         1.25      0.77-2.22      0.098     0.01-0.19      0.01     0.00-0.01     1.36

                 Lemon          0.95      0.31-1.96      0.10      0.00-0.19      0.005    0.00-0.01     1.06

                 Grapefruit     0.98      0.66-1.08      0.29      0.23-0.39      0.04     0.01-0.07     1.28
                                                                                                                
    

                                                                      
    Dip concentration             Thiabendazole residues (whole fruit)
                                                                      
    0.1% (1000 ppm)               2 - 5 ppm
    0.05%  (500 ppm)              1.5 - 2.5 ppm
    0.025%  (250 ppm)             0.8 - 2 ppm
    0.0125% (125 ppm)             0.3 - 1.5 ppm
                                                                      

    Trials at another research station yielded the following results:

                                                                      
    Dip concentration             Thiabendazole residues ppm
    %                             Flesh          Whole Fruit
                                                                      
    0.02 (200 ppm)                0.03           0.7
    0.04 (400 ppm)                0.07           1.5
    0.08 (800 ppm)                0.11           4.1
    0.16 (1600 ppm)               0.14           6.2
    0.32 (3200 ppm)               0.20           12.0
                                                                      

    Limit of detection was 0.04 ppm.

    Citrus fruits

    Residue trials were conducted in conjunction with the Florida Citrus
    Experiment Station on oranges, grapefruit and lemons. The following
    products were assayed before and after processing: whole unwashed
    fruit, single strength juice, finisher pulp, citrus oil, molasses,
    dried citrus pulp. The fruit was processed according to typical
    practice, including a two-minute flood treatment with a suspension
    containing 0, 5 000 and 10 000 ppm thiabendazole. The fruit was then
    waxed according to commercial practice.

    The detection sensitivity of the analytical method used was 0.03 ppm
    for the pulp and 0.40 ppm for the peel with blank values for untreated
    pulp and peel in the range 0.00 - 0.02 ppm and 0.00 - 0.40 ppm
    respectively.

    A review of the whole fruit residue data, which is summarized in Table
    IV, shows that at the application rate of 5 000 ppm, a maximum of 0.04
    ppm thiabendazole is found in the pulp of the citrus, while a maximum
    of 1.28 ppm and 0.41 ppm thiabendazole is present on and in the peel
    respectively. This is the maximum residue found in pulp or peel of all
    oranges, lemons or grapefruit assayed. Therefore, at the treatment
    rate of 5 000 ppm, a maximum of 1.73 ppm is possible on the whole
    unpeeled fruit.

    In all cases, the peel residues increase with increasing treatment
    rates. However, in the case of pulp assays, the level of residue is at
    or below the sensitivity of the assay method with no indication of
    increases with higher dosages. These results are corroborated by

    radioactive studies reported later, which show that the level of
    residues in the pulp is approximately equivalent to the assay
    sensitivity and appears to result from inadvertent contamination
    during peeling or sectioning. The result of assays on dried citrus
    pulp (summarized on Table V) indicated the maximum residues range from
    1.55 ppm in lemons to 2.87 ppm in the case of grapefruit, with dried
    orange pulp being 2.61 ppm.

    From yet another research station, it was reported that dipping green
    bananas in a suspension containing 800 ppm thiabendazole resulted in
    residues of 3.3 ppm in the whole fruit and 0.4 ppm in the pulp.

    FATE OF RESIDUES

    In animals

    Radioactive thiabendazole administered orally to sheep at a dose of 50
    to 100 mg/kg body-weight or to goats at a single dose of 150 mg/kg was
    rapidly absorbed and metabolized to its 5-hydroxy derivative and
    excreted as the aglycone and glucuronide and sulfate ester. Peak
    concentrations in plasma occur approximately four hours after
    administration, with 90 percent of the radioactivity eliminated in the
    excreta in three to four days. Thiabandazole and small concentrations
    of two unknown metabolites were observed (Robinson, 1965a; Tocco et
    al., 1964).

    Residues observed with eight sheep receiving 14C-or 35S-labelled
    thiabendazole at 50 mg/kg are given in Table VI, where concentrations
    reported as zero are equal to or less than a detection limit of 0.06
    ppm.

    TABLE V

    Citrus byproducts - residues following treatment
    (Treatment rate 5 000 ppm)
                                                         

    Byproduct           Variety       Average residue, ppm
                                                         

    Finisher pulp       Orange               0.02
                        Lemon                0.01
                        Grapefruit           0.01

    Single strength     Orange               0.01
    juice               Lemon                0.01
                        Grapefruit           0.02

    Pulp, dry           Orange               2.54
                        Lemon                1.4
                        Grapefruit           2.53

    TABLE V (cont'd)

    Citrus byproducts - residues following treatment
    (Treatment rate 5 000 ppm)
                                                         

    Byproduct           Variety       Average residue, ppm
                                                         
    Oil                 Orange               1.05
                        Lemon                0.16
                        Grapefruit           1.38

    Molasses            Orange               1.77
                        Lemon            Not assessed
                        Grapefruit           1.97
                                                         

    Trials conducted in Australia at the Citrus Wastage Research
    Laboratory showed the following results when oranges were dipped in
    thiabendazole suspensions under commercial conditions:

                                                                      
    Dip concentration      Thiabendazole residues ppm
    %                      Flesh          Whole Fruit
                                                                      

    0.02                   N.D.1          0.49
    0.04                   N.D.           0.21
    0.08                   N.D.           0.52
    0.10                   N.D.           0.39
    0.16                   N.D.           0.73
    0.32                   N.D.           1.68
                                                                      

    1 N.D. = not detected (less than 0.04 ppm)

    Analytical studies in lactating goats showed that although
    thiabendazole was absorbed rapidly, less than 1% of the dose was
    excreted in their milk within 24 hours after administration of 150
    mg/kg body-weight. Peak concentrations were found in the milk within
    24 hours, and no thiabendazole or its metabolites were detectable in
    the milk four days later (Robinson, 1965a; Tocco et al., 1965).
    Studies with goats given single oral dosages of tritium-labelled
    thiabendazole at 150 mg/kg, demonstrated that the compound was
    absorbed rapidly, reaching peak concentrations in the plasma about 4
    hours after administration. The compound was rapidly excreted in the
    urine and faeces. Residues of thiabendazole were not detectable in any
    of the 15 tissues from goats examined 30 days after treatment.

    Thiabendazole is less rapidly excreted by pigs than by several other
    animals. Following a single dose of 50 mg/kg body-weight,
    approximately 76 percent of the dose was found in the excrete within

    about two weeks (Robinson, 1965a; Tocco et al., 1965). Thiabendazole
    (14C-labelled) administered to cattle at doses of 50 mg/kg and 200
    mg/kg body-weight was rapidly excreted. Plasma levels reached a
    maximum in four to seven hours, after which they decreased rapidly,
    with no residual thiabendazole or metabolites detectable in tissues 30
    days after administration of the lower dose. Traces of radioactivity
    were demonstrable in calves two months following doses of 150 and 200
    mg/kg. Three fluorometrically detectable metabolites were observed,
    corresponding to 5-hydroxythiabendazole and its sulfate and
    glucuronide conjugate. Approximately 0.1 percent of an oral dose of 3,
    5 or 10 grams per 100 lb was secreted in the milk as thiabendazole and
    its metabolites within 60 hours after the animals were dosed. The
    highest concentration appeared within 24 hours. Over 99 percent of the
    compound was metabolites, and residues were not detectable 2 1/2 days
    after the cows were administered the compound (Robinson, 1965a; Tocco
    et al., 1965).

        TABLE VI

    Thiabendazole residues in sheep
                                                                                      

      Sample           6 hours    5 hours   8 days1/  16 days   24 days   30 days1/
                                                                                      

    Abomasum             5.1        0.0       0.15      0.0       0.0       0.0
    Blood                7.7        0.0       0.0       0.0       0.0       0.0
    Brain                1.0        0.09      0.14      0.0       0.0       0.0
    Caecum              34.4        0.0       0.08      0.0       0.0       0.0
    Fat                  2.8        0.0       0.04      0.0       0.0       0.0
    Heart                2.7        0.15      0.14      0.08      0.0       0.0
    Kidney              13.9        0.28      0.22      0.0       0.0       0.0
    Large intestine      4.6        0.0       0.10      0.0       0.0       0.0
    Liver                9.6        0.62      0.62      0.15      0.0       0.0
    Lung                 2.4        0.0       0.04      0.08      0.0       0.0
    Muscle               2.0        0.09      0.06      0.13      0.0       0.0
    Pancreas             2.6        0.18      0.13      0.0       0.0       0.0
    Skin                 3.2        0.0       0.19      0.0       0.0       0.0
    Small intestine     33.6        0.0       0.16      0.0       0.0       0.0
    Spleen               3.4        0.0       0.0       0.0       0.0       0.0
                                                                                      

    1/ Two animals slaughtered at each of these intervals. Results given in
       table are means of the two values.
    
    Table VII shows thiabendazole residues found in calves treated at 110
    mg/kg. Results are the total of thiabendazole and its metabolites
    expressed in ppm, determined fluorometrically.


        TABLE VII

    Thiabendazole residues in calves
                                                                                                 

      Days                      No.
      Post                      of            Muscle              Liver              Kidney
    Treatment     Group       Animals          (ppm)              (ppm)               (ppm)
                                                                                                 

                  Control       3         0.13(0.09-0.17)     0.13(0.13-0.14)     0.12(0.10-0.15)

    3             Treated       4         0.12(0.05-0.17)     0.17(0.15-0.19)     0.16(0.13-0.18)

    7             "             4         0.10(0.03-0.19)     0.17(0.15-0.19)     0.12(0.10-0.13)

    14            "             4         0.08(0.06-0.12)     0.15(0.14-0.18)     0.15(0.07-0.35)
                                                                                                 

    It may be noted that in no instance does the average value exceed the control level by
    more than 0.05 ppm.
    

    In plants

    Bananas

    There is nothing to suggest that thiabendazole residues in bananas
    undergo any change during application, shipping, storage or ripening.

    Citrus

    Radioactive thiabendazole 14C was employed to determine the uptake of
    thiabendazole by Valencia oranges after dipping in 0.1% suspension,
    before and after storage under conditions simulating customary
    shipping and use practices (Merck, 1969; Rosenblum, 1970). Peal and
    edible pulp were analysed separately. Peel was divided into inner and
    outer portions. At the end of the maximum storage period, which
    extended for two weeks at 10C followed by an additional two weeks at
    21C,  peel was assayed by the reverse isotope dilution method for
    intact thiabendazole as an estimate of the stability of the latter in
    the fruit.

    The pulp was essentially free of thiabendazole even after four weeks
    of storage. The small apparent residues recorded are probably due to
    uncontrollable contamination from the peel during sectioning of the
    orange for analysis. Peel representing 25% of the whole fruit
    contained approximately 50 ppm of thiabendazole (on weight of peel).
    All the thiabendazole applied as a suspension was still present as
    intact thiabendazole after the four weeks of storage.

    In several instances the stability of the sorbed thiabendazole - 14C
    was investigated by extraction and by thin layer chromatography, with
    and without prior addition of unlabelled carrier compound. Since the
    radioactivity of the pulp did not increase with temperature or time of
    storage above the initial value prior to storage, it may be concluded
    that the thiabendazole content of the pulp due to diffusion of
    absorbed fungicide into the interior during the four weeks of storage
    is less than or equal to a detection limit of 0.01 ppm. Diffusion from
    the surface of the fruit into the peel is appreciable an from 7-20% of
    the measurable activity is observed in the inner peel. This diffusion
    does not, however, extend to the pulp at the interior of the fruit.

    Because of the widespread practice of waxing citrus after washing and
    cleaning, the application of thiabendazole in the aqueous wax emulsion
    is a convenient, practicable and efficient method of conferring
    protection against fungal spoilage. Trials carried out in California
    under controlled conditions in commercial packing houses resulted in
    acceptable protection. The residues found on the treated fruit ranged
    up to 4.3 ppm, and the results are set out in Table VIII. It is not
    anticipated that the penetration into the peel will be any greater
    when the treatment is applied via wax emulsion that it would from a
    flooding, dipping or spraying treatment.

        TABLE VIII

    Residues of thiabendazole on whole fruit following application to citrus
    of thiabendazole in wax emulsion
                                                                                         

                                           Storage             Time
    Variety        Treatment                                 Interval     Residue
                                        Temp.    Humidity     (days)       (ppm)
                                                                                         

    Valencia
    Oranges      0.3% TBZ in wax       21-24C      90%          8         1.25

      "          0.5% TBZ in wax       21-24C      90%          8         2.16

      "          0.3% TBZ in wax       21-24C      90%         10         1.76

      "          0.4% TBZ in wax       21-24C      90%         10         2.20

      "          0.5% TBZ in wax       21-24C      90%         10         2.43

      "          0.35% TBZ in wax      21-24C      90%         10         2.15

      "          0.5% TBZ in wax       21-24C      90%         10         3.91

      "          5267 ppm TBZ in wax    9-20C      90%         31         4.3
                                                                                         
    
    Evidence of Residue in Food In Commerce

    Shipments of commercially processed grapefruit which had been treated
    by dipping in thiabendazole before being exported from U.S.A. were
    sampled in Europe and systematically analysed for thiabendazole
    residues. Ten fruits were homogenized from each shipment and analysis
    was carried out on the homogenate. The results which are set out in
    Table IX are slightly lower than those obtained from controlled
    experiments carried out in Florida. It is notable that the residue on
    grapefruit treated by flooding in a bath containing 3 000 ppm
    thiabendazole are approximately twice as high as those found in fruit
    treated in a similar manner in a bath containing 1 000 ppm
    thiabendazole.

    In order to determine the fruit-to-fruit variation in any one
    consignment, nine separate fruit were taken from a consignment and
    these were analysed individually. There was close agreement between
    the results (range 0.26-0.55 ppm - standard deviation 0.09 ppm) with
    the mean agreeing closely with the result obtained by analysis of a
    homogenized sample of the same consignment.

        TABLE IX

    Residues of thiabendazole on whole citrus exported from U.S.A. and
    analysed in Europe
                                                                             

    Shipment    Variety        Treatment     Residue on whole fruit (ppm) 1/
       No.                       Rate           Average        Range
                                                                             

       1        Grapefruit     1 000 ppm         0.22       0.18 - 0.25
                               3 000 ppm         0.44       0.32 - 0.63

       2            "          1 000 ppm         0.23       0.20 - 0.29
                               3 000 ppm         0.57       0.46 - 0.86

       3            "          1 000 ppm         0.17       0.14 - 0.18
                               1 000 ppm         0.13       0.10 - 0.16
                               3 000 ppm         0.27       0.23 - 0.30

       4            "          1 000 ppm         0.17       0.15 - 0.19
                               1 000 ppm         0.18       0.16 - 0.21
                               3 000 ppm         0.24       0.21 - 0.28

       5            "          1 000 ppm         0.39       0.37 - 0.42
                               1 000 ppm         0.33       0.21 - 0.28
                               3 000 ppm         0.86       0.72 - 0.96
                                                                             

    1/ Variation in thiabendazole residue levels between individual fruits

    Analysis of composite sample                =      0.39 ppm

    Analysis of individual fruits
    0.53     0.37     0.38           Mean       =      0.39 ppm
    0.38     0.55     0.30        Standard Deviation   0.09 ppm
    0.26     0.36     0.34           Range             0.26 - 0.55
    
    Shipments of a variety of citrus exported from Israel to Europe were
    sampled and analysed to determine the fruit-to-fruit variation.
    Samples were taken of citrus prepared and packed by 13 separate
    packing houses. In this case, the thiabendazole was applied as a
    suspension in the wax emulsion used to treat the fruit after washing
    and prior to packing in cartons. The wax contained 0.3% (3 000 ppm)
    thiabendazole. The results given in Table X show a much higher level
    of residues than those found following dipping. The fruit-to-fruit
    variation was considerably greater and there was a significant
    variation between different packing houses. Table XI shows results of
    analysis of individual fruits from 7 packing houses in Israel
    following treatment with 0.3% thiabendazole in wax emulsion.

    TABLE X

    Thiabendazole residues on Israeli citrus following application
    of 0.3% thiabendazole in citrus wax
                                                                   

    Packing    No. of Fruit    Average    Standard      Range
     House       Analysed        ppm     Deviation       ppm
                                                                   

       1            20           1.79       0.31      1.21 - 2.36
       2            20           0.71       0.20      0.43 - 1.13
       3            10           2.65       1.10      1.51 - 4.81
       4            10           4.41       1.10      2.76 - 5.89
       5            10           4.76       1.2       3.39 - 6.40
       6            10           3.89       1.7       0.95 - 7.35
       7            10           4.75       1.0       3.37 - 5.73
       8            10           2.97       0.69      2.12 - 4.19
       9            10           3.34       0.53      2.45 - 4.28
      10            10           1.47       0.45      0.92 - 2.24
      11            10           3.86       0.81      2.96 - 5.56
      12            10           1.12       0.53      0.59 - 1.48
      13            10           4.06       0.83      2.98 - 5.44
                                                                   

    TABLE XI

    Distribution of results of analysis of individual citrus
    fruits treated with 0.3% thiabendazole in wax and analysed
    one week after application
                                                                 

    Range of residue levels     Number of fruits     Percent
                                                                 

      less than 1.0 ppm               24               10
          1.0 - 1.9                   64               26
          2.0 - 2.9                   59               23
          3.0 - 3.9                   41               18
          4.0 - 4.9                   22                9
          5.0 - 5.9                   25               10
         above 6 ppm                  10                4

          Total                      245              100
                                                                 

    Samples of oranges treated in South Africa by dipping in a suspension
    containing 0.5% (5 000 ppm) thiabendazole which were analysed after
    arrival in the United Kingdom contained between 0.28 ppm and 0.35 ppm
    thiabendazole. The lower residue levels were found on fruit which had
    received a water rinse after dipping. The higher residues were on

    unrinsed fruit. Fruit dipped in suspensions at 43C were found to have
    residues of 0.80 ppm thiabendazole, twice the level found in those
    dipped cold.

    A further twenty samples representing various treatment levels, times,
    temperatures and after-treatments showed residues ranging from 0.2 ppm
    to 1.62 ppm. The lower levels were found in fruit treated at 1 000 ppm
    and the higher level in fruit treated at 5 000 ppm. After-treatment
    with citrus wax tended to reduce the residue levels to 0.15 ppm and
    0.5 ppm, respectively.

    METHODS OF RESIDUE ANALYSIS

    Citrus

    The method developed for the determination of residues in and on
    citrus and bananas has been published by the U.S. Food and Drug
    Administration (1969). The whole fruit is rinsed with ethyl acetate to
    remove wax and thiabendazole from the surface. The pulp and peel are
    homogenized with water. The thiabendazole is extracted from the
    homogenates with ethyl acetate. The ethyl acetate solutions from
    rinse, pulp and peel are combined. The solutions are purified by a
    series of extraction procedures. The final solution in 0.1N
    hydrochloric acid is measured spectrophotofluorometrically.

    For routine testing of treated citrus, it is advisable to homogenize
    about 10 fruits in a large chopper and to take a sub-sample of 60g of
    puree for analysis. This is blended with an equal weight of sodium
    acetate/sodium chloride solution, and from the slurry a 20 g sample is
    taken for analysis. A number of minor variations have been introduced
    into the extraction procedure to deal with various citrus fruits, the
    separate components of the fruit and citrus products. When analysing
    whole fruit, the method is sensitive to 0.1 ppm, but with pulp the
    sensitivity is estimated to be 0.03 ppm and with peel 0.4 ppm. Blank
    values for untreated pulp and peel range from 0.00-0.02 ppm and
    0.00-0.40 ppm, respectively.

    After purification, the extract is dissolved in 0.1N hydrochloric acid
    and the fluorescence of the final acid solution is read on an
    Amico-Bowman spectrophotofluorometer having an excitation wavelength
    set at approximately 300mu and the mission wavelength set at 360mu.
    Other instruments may operate more effectively at slightly different
    wavelengths. The method is suitable for regulatory purposes but
    requires a suitable spectrophotofluorometer.

    Where a suitable spectrophotofluorometer is not available, the method
    of Szalkowski (1965) has been modified by Gilbert (1969) to yield a
    reliable colorimetric method with a sensitivity of 0.08 ppm and
    recoveries between 93% and 110%. The blank varies between 0.02 and

    0.04 ppm for both pulp and whole fruit. The thiabendazole is extracted
    from homogenized fruit with acidic methanol and interference removed
    with an alkaline wash followed by alumina column cleanup. The
    thiabendazole is reduced to form hydrogen sulphide which complexes
    with p-phenylene diamine. This complex in oxidized with ferric
    solution to give a blue thiazine dye, which is determined
    colorimetrically.

    Bananas

    The method described for the determination of thiabendazole in citrus
    has been adapted to determine residues in banana pulp and peel by the
    U.S. Food and Drug Administration (1969b).

    The thiabendazole in extracted into ethyl acetate from a Ph 4.5
    buffered suspension of ten grams of the banana pulp or peel. The
    combined ethyl acetate extract is washed twice with 0.05N sodium
    hydroxide. Then the thiabendazole is returned to an aqueous phase by
    extraction with 0.1N hydrochloric acid and determined by fluorescence,
    the method described for citrus. The sensitivity of this method is
    0.05 ppm.

    Animal tissues

    The spectrophotofluorometric method has been used successfully for the
    determination of thiabendazole and its 5-hydroxy derivative in animal
    tissue. For liver and kidney tissue a preliminary digestion with
    enzyme in necessary to convert the metabolite conjugates to 5-hydroxy
    thiabendazole. The sensitivity is about 0.1 ppm.

    Milk samples from animals receiving thiabendazole therapy or being fed
    on citrus residues containing thiabendazole may be analysed for
    thiabendazole by spectrophotofluorometric method using 1-5 ml samples.
    For milk, as little an 0.05 ppm may be recovered.

    NATIONAL TOLERANCES
                                                                        

    Country                  Commodity                     Tolerance,ppm
                                                                        
    U.S.A.                   Bananas                       3
                             Banana pulp                   0.4

                             Citrus                        2
                             Citrus from wax treatment     6 (proposed)
                             Citrus pulp (dried)           8
                             Edible tissues of cattle,
                             sheep, goats and pigs         0.1
                             Milk (negligible residue)     0.05

    (Cont'd)
                                                                        
    Country                  Commodity                     Tolerance,ppm
                                                                        

    Canada                   Bananas                       3
                             Banana pulp                   0.4
                             Citrus                        2

    Australia                Bananas                       3
                             Banana pulp                   0.4
                             Citrus                        2
                             Meat of cattle, sheep,        0.2
                             goats and pigs
                             Milk                          0.05

    France                   Bananas                       3
                             Banana pulp                   0.4
                             Citrus (provisional)          6

    Netherlands              Citrus (whole)                6
                             Citrus with diphenyl          3

    Sweden                   Banana (whole)                3
                             Banana (pulp)                 0.4
                             Citrus (whole)                6

    E.E.C.                   Bananas (whole)               6
                             Citrus (whole)                6

    Germany                  Citrus                        6
                             Bananas                       3

    Belgium                  Bananas                       3
                             Citrus                        6

    New Zealand              Bananas                       3
                             Citrus                        2

    Norway                   Citrus                        6
                             Bananas                       3

    Finland                  Citrus                        6

    Italy                    Citrus (when used alone)      6
                             Citrus (when used with
                             other antifungals)            3
                             Bananas                       3
                                                                        

    APPRAISAL

    Thiabendazole is a particularly effective fungicide for the
    post-harvest treatment of bananas and citrus. It has been used very
    extensively since 1960-62 as an anthelmintic for sheep, cattle, pigs
    and horses and later as a vermifuge in human beings.

    Post-harvest dips containing 200 ppm thiabendazole protect bananas
    against the most important fungal rots. Treatment of green bananas
    during picking and packing operations ensures that the fruit may be
    shipped to distant markets with minimum loss or deterioration.
    Likewise post-harvest sprays, dips and wax coatings containing
    thiabendazole at concentrations of 1 000 to 3 000 ppm have proved
    outstandingly useful.  Diphenyl impregnated wraps and interlinings
    have been used for many years to reduce Penicillium sporulation, and
    sodium ortho-phenylphenate dips have been used against stem and
    rots. Both treatments have many disadvantages and objections, and it
    is anticipated that thiabendazole will provide a more acceptable
    alternative to both treatments.

    Data on the level of residues in whole fruit, peel, pulp and processed
    citrus products, as well as in green and ripe bananas, were available
    from U.S.A., West Indies, Australia, Israel and Europe. Results of
    residue analysis of commercial citrus shipments to Europe were also
    available and were not significantly different from those resulting
    from supervised trials.

    Thiabendazole has high stability on and in fruit and under conditions
    of use no metabolites or degradation products are likely to occur. The
    residue levels do not decline significantly in storage.

    Several methods of residue analysis are available. The most convenient
    and sensitive method employs spectrophotofluorometric measurement of
    the thiabendazole in dilute hydrochloric acid solution. The method may
    be adapted to determine thiabendazole residues in a variety of fruit
    products and in animal tissues and milk. The sensitivity varies with
    the substrate but ranges from 0.01 ppm to 0.04 ppm. This sensitivity
    is adequate for the level of residues likely to occur and the proposed
    tolerances. The method is suitable for regulatory purposes and is
    specific to thiabendazole. de Vos and Bosma examined the possibility
    of using gas chromatography with sulphur specific detector; they
    showed that it was possible to determine thiabendazole residues to a
    limit of 0.1 ppm by using the ethyl acetate extract of raw fruit for
    injection into the chromatograph. They found that the method using
    concentrated crude ethyl acetate extracts was satisfactory, but it led
    to rapid fouling of the chromatographic column.

    The administration of thiabendazole to sheep and cattle as an
    anthelmintic gives rise to low level transient residues in several
    edible tissues especially kidney and liver, but these residues are
    eliminated within three days. Lactating dairy animals receiving a

    therapeutic dose of thiabendazole excrete detectable amounts in the
    milk for 24 hours post treatment. However, animals with severe
    helminthiasis and liable to be dosed with thiabendazole are rarely
    suitable for slaughter or for production of milk for human
    consumption. Furthermore, it would be very unusual to treat more than
    a small proportion of a herd at any one time and thereby to exceed the
    detectable level of about 0.05 ppm in meat or milk. Under these
    circumstances it does not appear necessary to recommend a tolerance
    for thiabendazole residues in meat or milk.

    RECOMMENDATIONS FOR TOLERANCES

    TOLERANCES

    The following tolerances are recommended for thiabendazole:

                                                               
              Crop                     Residues ppm
                                                               

              bananas, whole           3
              banana pulp              0.4
              citrus, whole            6
                                                               

    FURTHER WORK OR INFORMATION

    DESIRABLE

    Studies to determine the effect of thiabendazole on the thyroid gland.

    REFERENCES

    Anon. (1967) Control of squirter disease of bananas by thiabendazole.
    Agricultural Gazette of New South Wales, 78:604

    Anon. (1969) New treatments reduce fruit wastage - Rural Research in
    CSIRO, 67:7-12

    AOAC. (1966) Analysis of thiabendazole in feeds. J.A.O.A.C.
    49:238-239 and 312

    Bondoux, P. (1967) Acac. Agric. France, Compt. Rend. Hebd.  Seances,
    53: 1314-1323

    Brown, G.E., McCormack, A.A. and Smoot, J.J. (1967) Thiabendazole as a
    post-harvest fungicide for Florida citrus fruit. Plant Disease
    Reporter, 52:95-97

    Brown, H.D., et al. (1961) Journal of American Chemical Society, 
    83:1764

    Burden, O.J. (1967) Studies on grown rot on bananas. Queensland
    Agricultural Journal, March, p. 186

    Crivelli, G. (1966) Part V Tests with the fungistat thiabendazole
    against Penicillium of oranges (in Italian). Il. Freddo, 20:25-29

    Cuille, J. and Bur-Ravault, L. (1968) Fruits, (Paris), 23:351-356

    Eckert, J.W., Kolbenz, M.J. and Kraght, A.J. (1968) Proc.
    International Citrus Symposium, Vol 3, Riverside, Cal., 13 March 1968

    Fripp, Ivonne J. and Dettman, E. Belinda. (1969) Thiabendazole as a
    post-harvest treatment against Sclerotinia fructicola in dessert
    peaches. Australian Journal of Experimental Agriculture and Animal
    Husbandry, 9:9-11

    Gilbert, W.S. and Wright, Julia. (1969) Method for determining
    thiabendazole residues on oranges

    Gordon, H. McL. Studies of anthelmintics for sheep - thiabendazole.
    Australian Veterinary Journal, 40(1):9-18

    Harding, P.R. Jr. (1968) Plant Disease Reporter, 52: 623-625

    Johnson, C.D. (1964) Evaluation of teratogenic potential in the rat.
    Unpublished Report (April 1964) from Woodard Research Corporation
    through Merck Institute for Therapeutic Research to FDA

    McCormack, A.A. and Brown, G.E. (1968) Thiabendazole, an experimental
    fungicide for fresh citrus fruit. Proc. Florida State Horticultural
    Soc., 80:232-237

    Merck and Company, Inc. (1969) Rahway, New Jersey, U.S.A. 
    thiabendazole residues in bananas and citrus. Submission to U.S. Food
    and Drug Administration

    Merck and Company, Inc. (1970) Rahway, New Jersey, U.S.A.
    Thiabendazole residues in citrus following application in citrus wax.
    Submission to U.S. Food and Drug Administration

    Robinson, H.J., Stoerk, H.C. and Graessle, A.E. (1965b) Studies on the
    toxicological and pharmacological properties of thiabendazole.
    Toxicol. appl. Pharmacol., 7:53-63

    Rosenblum, C. and Meriwether, H.T. (1970) Determination by the 
    radioactive indicator Method of the retention and stability of
    thiabendazole in treated Valencia orange. Journal of Radioanalytical
    Chemistry, (in press)

    Scott, K.J., and Roberts, E.A. (1967) Control in bananas of black end
    rot caused by Gloeosporium musarium. Australian Journal of
    Experimental Agriculture and Animal Husbandry, 7:283-286

    Seberry, J.A., and Baldwin, R.A. (1968) Thiabendazole and
    2-amino-butane as post-harvest fungicides for citrus. Australian
    Journal of Experimental Agriculture and Animal Husbandry, 8:440-443

    Seberry, J.A. (1968) Proc. Int. Citrus Symposium, Riverside, Calif.,
    13 March 1968

    Shillingsford, C.A. (1970) Banana fruit rot control in Jamaica. 
    P.A.N.S.,16(1)

    Szalkowski and Kanora. (1965) Spectrophotometric determination of TBZ
    in feed. JAOAC,48(2):288-295

    Tocco D.J., Buhs, R.P., Brown, H.D., Matzuk, A.R., Mertel, H.E.,
    Harman, R.E. and Trenner, N.R. (1964) The metabolic fate of
    thiabendazole in sheep. J. med. Chem., 7:399-405

    Tocco, D.J., Egerton, J.R., Bowers, W., Christensen, V.W. and
    Rosenblum, C.J. (1965) Absorption, metabolism and elimination of
    thiabendazole in farm animals and a method for its estimation in
    biological materials. J. Pharmacol. exp. Therap., 149:263-271

    Tocco, D.J., Rosenblum C., Martin. C.M. and Robinson, H.G. (1966)
    Absorption, metabolism and excretion of thiabendazole in man and
    laboratory animals. Toxicol. appl. Pharmacol., 9:31-39

    U.S. Food and Drug Administration. (1969) Thiabendazole residues in
    citrus fruits. Pesticide analytical manual, Vol II, Section 120.242

    U.S. Food and Drug Administration. (1969b) Thiabendazole residues in
    banana peel and pulp. Method A. Pesticide analytical manual, Vol II,
    Section 120.242.

    Vogin, E.E. (1968) Multigeneration reproduction and lactation studies
    with thiabendazole. Unpublished reports (Dec. 1967 and March 1968).
    Food and Drug Research Laboratories, through Merck Institute for
    Therapeutic Research to FDA

    de Vos, R.H. and M.P. Bosma. (1970) Residues of thiabendazole in
    citrus fruit. Report 3199. TNO Central Instituut voor
    Voldingsonderzoek, Ziest, Netherlands

    Weinke, K.E., Lauber, J.J., Greenwald, B.W. and Preiser, F.A. (1969)
    Thiabendazole, a new systemic fungicide. Proc. 5th Brit. Insectic.
    Fungic. Conference

    Woodard, M.W., Cockrell, K.O. and Woodard, G. (1964) Safety evaluation
    by oral administration to dogs and rats for 104 weeks. Unpublished
    Report (April 1964). Woodard Research Corporation through Merck
    Institute for Therapeutic Research to FDA
    


    See Also:
       Toxicological Abbreviations
       Thiabendazole (WHO Food Additives Series 39)
       Thiabendazole (WHO Pesticide Residues Series 1)
       Thiabendazole (WHO Pesticide Residues Series 2)
       Thiabendazole (WHO Pesticide Residues Series 5)
       Thiabendazole (Pesticide residues in food: 1977 evaluations)
       Thiabendazole (Pesticide residues in food: 1979 evaluations)
       Thiabendazole (Pesticide residues in food: 1981 evaluations)