WHO/FOOD ADD./70.38



    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, 8 - 15 December 1969.



    Rome, 1970



    Chemical name




    Structural formula


    Other relevant chemical properties

    Colourless white powder or needles MP 229°C - BP. 326°C. V.P. 1.089 ×
    10-5 mm Hg at 20°C - sublimable. Insoluble in water and alcohol.
    Soluble in hot benzene. The technical grade used in agriculture
    contains 98 percent hexachlorobenzene, 1.8 percent pentachlorobenzene
    together with 0.2 percent 1,2,4,5-tetrachlorobenzene.

    Commercial formulations (dusts) contain 10-40 percent HCB alone or
    together with small quantities of lindane (0.5-1.0 percent) added to
    prevent insect attack on stored seed.



    Residues of hexachlorobenzene are stored in the liver and fat of
    birds. The biological half-life of hexachlorobenzene in the quail is
    about three weeks (Vos et al., 1968) (see also "Short-term studies.

    When hexachlorobenzene, 400 mg/kg, was administered orally to rabbits,
    the compound did not appear to be metabolized, because the main
    portion was found in the gut contents five days after dosing, with
    only 6 percent appearing in the faeces. There was no significant
    urinary or pulmonary excretion of metabolites. Hexachlorobenzene did
    not form conjugated glucuronic acids, ethereal sulphates or
    mercapturic acids. Most of a dose (100 mg/kg) given subcutaneously was
    found at the site of injection after five days, with no chlorinated
    benzenes being found in the faeces over this period (Parke and
    Williams, 1960).

    There is evidence that hexachlorobenzene (or a toxic metabolite of it)
    is excreted in milk. Two pregnant female rats were given
    hexachlorobenzene in their food (the dose was not stated, it was
    presumed to be 0.5 percent). One died, but the other was delivered
    normally and reared the young until they died with convulsions after
    seven to eight days. The mother was then given three normal week-old
    rats to foster, which died three to four days later with convulsions.
    The mother also died four weeks later with the usual symptoms of
    hexachlorobenzene intoxication. This study indicated that a toxic
    compound was excreted in the milk (De Matteis et al., 1961).


    Acute toxicity (oral)

    Animal              LD50          References
                   mg/kg body-weight

    Mouse               4000          Savitskii, 1964

    Rat                 3500          Savitskii, 1964

    Guinea pig        > 1000          Melis, 1955

    Rabbit              2600          Savitskii, 1964

    Cat                 1700          Savitskii, 1964

    Short-term studies


    Hexachlorobenzene given at levels of 120-480 ppm in the diet for three
    months, caused no toxic effects in chickens (Melis, 1955).


    A diet containing 0.5 percent of hexachlorobenzene was given to
    guinea-pig and to mice. These two species proved to be remarkably
    susceptible to hexachlorobenzene and developed very marked
    neurological symptoms within eight to 10 days (De Matteis et al.,


    See under "Guinea-pig".


    Five groups of adult Japanese quail (10 females and two males per
    group) were fed a mixed diet containing 0, 20, 100, 500 or 2500 ppm of
    hexachlorobenzene for three months. Eggs from the 100 ppm, 20 ppm and
    control groups were collected during three periods and incubated to
    determine reproduction results. At the 2500 ppm level four birds died
    within a week, the remainder within a month. Pre-mortem effects noted
    were loss of weight, drooping wings, ruffled feathers, trembling,
    ataxia and paralysis. On examination, bones, liver and kidney showed a
    red fluorescence typical of porphyria. Degeneration and necrosis of
    liver cells was seen. At 500 ppm all birds died within a month,
    showing the same symptoms and lesions seen at the higher level. At 100
    ppm, the first bird died on day 20, and 10 were dead within seven
    weeks. An accumulation of porphyrins in liver and kidney was observed
    in all birds in the group. Microscopic examination showed
    degeneration, necrosis and local regeneration of liver cells. At 20
    ppm all birds survived the three months test-period and did not
    develop any visible symptoms. At autopsy one hen showed fluorescence
    of bones and liver in gross ultraviolet examination. Fluorescence of
    liver and kidney cells and swelling and regeneration of liver cells
    was observed in this bird. In the other hens fluorescence of kidney
    cells was seen under ultraviolet microscopy. Significant differences
    were not found in body-weight or in relative weights of liver, spleen
    and brain, nor was there any effect on egg production at 20 ppm. There
    was a significant reduction in the number of chicks hatched, however,
    at this level of hexachlorobenzene. It was tentatively concluded,
    considering the disturbance in porphyrin metabolism and the diminished
    reproduction results, that the no-effect level in this species is
    lower than 20 ppm. (A comparative test in rats indicated the quail to
    be much more sensitive). Residues in the liver of the birds fed
    hexachlorobenzene were as follows:
        TABLE I
                                                         Levels of hexachlorobenzene
       Level of                              Number      in the liver (ppm)
    hexachlorobenzene   History             of birds       Average          Range
      fed (ppm)

    500                 died                    9            450            180-850

    100                 died                    6            235             85-720

     20                 killed                  6             36             14-94

     20                 killed 33 days          6             13              5-29
                        after termination
                        of hexachlorobenzene-
    Residues were also found in eggs and in the chicks after hatching.
    Several predatory birds, most of them found dead, had liver residues
    of hexachlorobenzene in the same range as those in the 20 ppm group of
    the quail study. It was concluded that hexachlorobenzene used as a
    seed-dressing, may have toxic effects on seed-eating and predatory
    birds (Vos et al., 1968)


    Female rabbits were fed a diet containing 0.5 percent of
    hexachlorobenzene. After about six weeks an increase in urinary
    porphyrins was noted. Unless sacrificed earlier, the animals died in
    eight to 12 weeks after having demonstrated neurological symptoms. (De
    Matteis et al., 1961).


    Groups of 10 male and 10 female rats were fed diets containing
    hexachlorobenzene at levels of 0, 5, 25, 125, and 625 ppm for 13
    weeks. After 13 weeks five males and five females from each group were
    sacrificed for gross and microscopic examination. The rats left in the
    groups given 125 and 625 ppm were transferred to the control diet for
    a further two weeks, then sacrificed and examined. The rats in the 625
    ppm group displayed signs of marked respiratory involvement; slight
    tremors were also observed in several animals and the females
    exhibited small sores in the skin of the head or neck. None of the
    other groups showed these effects. Growth of male rats at the highest
    level fed was reduced. Survival was not affected at any dose-level.
    Total leukocyte counts were elevated during the test in the group fed
    625 ppm. Organ-weight data showed an increase at 625 ppm, in thyroid,
    liver, spleen and adrenals, and in male rats at 125 ppm, in liver.
    Microscopic examination showed consistent effects in the liver
    (lobular distortion, nuclear and cytoplasmic variations, focal loss of
    cell outline, mitotic activity, and focal cellular necrosis) of the
    rats at 125 and 625 ppm. Changes of a less distinct and less
    consistent nature were observed in thyroid, kidneys, adrenals and
    bone-marrow of animals in these two groups. No significant effects
    were observed in organs and tissues of the rats fed at 5 or 25 ppm of
    hexachlorobenzene (Weir, 1962).

    Female rats were fed a standard died containing 0.2 percent
    hexachlorobenzene. After one week, weight-loss and general debility
    was noted in some rats. Within three weeks a few rats showed increased
    urinary excretion of porphyrins, but this manifestation did not become
    general until the seventh or eighth week. Rats with well established
    porphyria were exposed continuously to ultraviolet light. If an area
    of about 25 cm2 was plucked or shaved before exposure, most of the
    animals died within three to 14 days, but a few rats survived three to
    four months of this treatment. Rats on the hexachlorobenzene diet that
    were not shaved or plucked tolerated the ultraviolet light exposure
    well. After receiving hexachlorobenzene in the diet for six to eight

    months, the rats were returned to a normal diet. Urinary porphyrin
    excretion decreased considerably over the first four months but was
    still above normal levels nine months after hexachlorobenzene has been
    removed from the diet (Pearson and Malkinson, 1965).

    A diet containing 0.2 percent of hexachlorobenzene was fed to 26 male
    rats. Groups of two or three animals were killed at weekly intervals
    over a period of one to 12 weeks. Retardation of weight-gain occurred
    in the second and third week of the experiment. Evidence of a
    localized toxic effect was confined to the liver. An increase 
    in liver-weight reached a maximum from the fifth to the ninth
    week and then the weight of the organ declined. Degenerative changes
    in the liver similar to those reported in earlier studies occurred.
    Porphyrinuria and pathological amounts of porphyrin in liver, bones
    and marrow were observed (Campbell, 1963).

    Rats were given hexachlorobenzene at a level of 2 percent in the diet.
    After appearing normal for the first 10-12 year, clinical and
    biochemical signs of porphyria then appeared rapidly. There was a
    considerable loss in body-weight, the mean for 13 rats being 25
    percent of the initial weight. Cutaneous eruptions on the head, back
    and feet occurred. Generalized tremor was observed. Porphyrinuria was
    seen after two weeks of feeding hexachlorobenzene. Many rats died
    after three to four weeks. Adenosine-5-monophosphoric acid, 20 mg
    daily, was given by intramuscular injection to some of the rats,
    starting on the 13th day of the experiment. By the 28th day, there was
    a striking difference between most of the adenosine-5-monophosphoric
    acid-treated and the other rats. Loss of weight was diminished, no new
    cutaneous eruptions appeared and those that had existed healed
    completely. There was a reduction in the urinary excretion of
    porphyrins. Despite these improvements, however, some of the rats
    treated with adenosine-5-monophosphoric acid died towards the end of
    the fourth week of feeding hexachlorobenzene (Gajdos and Gajdos-Torök,
    1961a, 1961b, 1961c).

    A group of 33 male rats were fed a diet containing 0.2 percent of
    hexachlorobenzene. Within the first month 13 rats died, exhibiting
    terminal tremor, ataxia, weakness and paralysis. In the remaining
    rats, a significant increase in urinary excretion of porphyrins and
    porphyrin precursors was first noted after two to eight weeks of
    hexachlorobenzene administration. In rats in which hexachlorobenzene
    was discontinued shortly after peak excretory values were reached,
    there was a return to normal excretion values within a week. However,
    maintenance of high levels of porphyrin excretion by continued feeding
    of hexachlorobenzene for two to three weeks resulted in an apparently
    irreversible porphyric state, in which marked porphyrinuria continued
    in spite of removal of hexachlorobenzene from the diet. Hepatomegaly
    was common in the porphyric rats. Histological studies showed
    liver-cell degeneration (Ockner and Schmid, 1961).

    Long-term studies

    No information available.


    Several published reports have described an outbreak of a cutaneous
    type of porphyria that began in southeastern Turkey in 1955. The total
    number of cases over a five-year period has been estimated at
    3000-5000. Most of these were in children. The clinical manifestations
    consisted of blistering and epidermolysis of the skin in areas exposed
    to sunlight, particularly the face and hands. The lesions healed
    poorly. Hyperpigmentation was invariable, usually accompanied by
    marked hypertrichosis which was not limited to the exposed skin areas.
    The urine contained large quantities of porphyrins. Weight loss and
    hepatomegaly were frequently present. Neurological symptoms did not
    appear to be evident but abdominal pain was reported in some cases and
    liver enlargement was present in 35 percent of the cases surveyed. In
    many cases bone and joint changes occurred, in some patients there was
    osteoporosis of the bones of the extremities and interphalangeal
    arthritis. The outbreak was traced to the consumption of wheat,
    intended for planting, that had been treated with hexachlorobenzene.
    It was estimated that the amount of hexachlorobenzene ingested by the
    persons affected was from 50 to 200 mg/day for a relatively long
    period before the disease became apparent. After stopping the
    consumption of bread made from hexachlorobenzene-treated wheat, the
    acute skin manifestations disappeared in about 20 to 30 days. Urinary
    findings reverted to normal in most patients. Relapses during the
    summer months were often seen. However one to two years after the
    termination of other symptoms of porphyria the joint lesions were
    found to be still present. It was concluded that the disease was the
    result of a metabolic disorder caused by interference with porphyrin
    metabolism in the liver by hexachlorobenzene. Upon recognition of the
    situation, the use of hexachlorobenzene as a fungicide was
    discontinued in 1959. Subsequently the disease gradually disappeared
    (Cam and Nigogosyan, 1963; Cetingil and Ozen, 1960; Dogramaci, 1961,
    1962, 1964; Dogramaci et al., 1962a, 1962b, Schmid, 1960, Wray et al.,


    Evidence is presented that hexachlorobenzene is a highly toxic
    compound. There is insufficient information on metabolism especially
    in relation to the toxic compound excreted in milk. Use effect on the
    bone-marrow gives rise to serious concern. In addition, no long-term
    studies or studies on reproduction are available and there is little
    information on the effect in mammalian species other than the rat. For
    those reasons no acceptable daily intake can be established. However,
    unintentional residues have been found in a variety of food
    commodities in many countries. The Meeting therefore agreed to
    consider the short-term study in rats as a basis for establishing a
    tentative negligible daily intake using an extremely high safety

    factor. It was stressed, however, that the use of this compound is
    highly undesirable and a search for a more suitable substitute is
    strongly recommended. In addition, extreme precautions should be taken
    to prevent treated seeds and seed-grains from being consumed by humans
    or farm animals.


    Level causing no toxicological effect

    Rat:  25 ppm in the diet, equivalent to 1.25 mg/kg body-weight/day


    0 - 0.0006 mg/kg body-weight.



    Pre-harvest treatments

    Seed wheat is treated with HCB to destroy seed-borne and soil-borne
    spores of Bunt fungi (Tilletia spp.) and to ensure freedom from Bunt
    in the subsequent crop. HCB has proved outstanding for this purpose
    and has been used widely since being first introduced in 1945. (Costa
    1952, Holton and Purdy 1957, Meagher 1953, Purdy 1961). Wheat infected
    with "Bunt" or "Stinking Smut" has a bad appearance, an unpleasant
    smell and is unsuitable for milling. "Bunt" can be regarded as a most
    important and common disease of wheat.

    Wheat crops become infected after sowing of seed, which has live Bunt
    spores on its surface. These spores germinate and enter the young
    plant, growing inside it throughout the season. At harvest a black
    mass of spores replaces the starch of the grain whilst the skin of the
    grain remains unaffected. During harvest these "Bunt Balls" are
    broken, thus releasing myriads of spores over the clean grain, which
    if sown without treatment, will lead to heavily infected crops.

    HCB is usually applied in the form of dust containing 10-40 percent
    a.i. but in Canada liquid preparations are also used (Houghton 1969).
    Formulations are coloured to assist in application and to distinguish
    treated seed from other grain. Usually, a blue pigment is used but
    sometimes carbon black or a red dyestuff is added to reduce the risk
    of treated seed being used for food. In Turkey where treated seed was
    used for food with disastrous consequences it was proposed that a
    denaturant with a strong taste or smell be added as well as colour.

    In Australia and various other countries, wheat is selected for seed
    at harvest time for the next season and after grading to remove small
    or broken grains, fungicide is added automatically by machinery at the
    rate of 1-2 ozs 30 percent HCB dust per bushel (330 ppm). The treated

    seed is stored in labelled jute bags until required for planting some
    months later. There is no evidence of significant contamination of
    commercial grain or animal feeds during the process of applying the
    HCB dust. The use of untreated seed wheat invariably leads to heavy
    losses from fungus diseases (Bunt or Stinking Smut) and it is
    therefore standard practice to treat sufficient seed for the
    anticipated needs of the next season. It is exceptional for farmers to
    buy seed: they normally produce and save for their own requirements.

    Farmers are directed not to use treated seed wheat for animal feed or
    to allow it to become mixed with commercial grain. Although such
    directions are normally observed, the presence of 1 bushel of seed
    wheat in 10,000 bushels of commercial grain is sufficient to lead to
    significant residues in eggs from hens receiving such grain in their
    ration. Contaminated grain sacks, machinery, storage premises,
    transport and dust from commercial grain all contribute to the
    residues found in animal products.

    In Canada, seed wheat is usually treated just before planting.
    Although some dusts are used, aqueous suspensions suitable for
    application as a coarse spray are favoured. As an alternative,
    "drill-box" treatments have been developed whereby the fungicide dust
    is added to the seed wheat in the feed box of the seed drill where it
    is distributed by simple agitation. Such drill-box treatments are less
    effective but appear capable of reducing cross contamination of other
    grain, (Wallace 1966).

    Statistics on the production or use of HCB are not available for
    countries other than Australia. In Australia 12 million bushels of
    seed wheat are treated annually with HCB dust, requiring 200 tons of
    technical HCB. A smaller proportion of the total crop is probably
    treated in U.S.A., Canada, U.K. and some European countries but there
    is apparently an extensive use in Turkey, Italy, Spain, Netherlands,
    Germany, France and some Eastern European countries.


    At the time of the original introduction of HCB as a seed dressing and
    for many years afterwards, the analytical methods were not capable of
    detecting the level of residues found in commercial grain or animal
    products. The only trial results available are those from Australia.

    In animals

    Craig (1959) fed HCB treated wheat to poultry at the rate of 1´ oz per
    bird per day for six months. At the end of this period the fat of the
    birds contained more than 300 ppm HCB. Craig and Dwyer (1961) fed seed
    wheat containing 660 ppm HCB to aged sheep at the rate of 4 oz and 12
    oz per head per day. After four months those receiving 4 oz per day
    (80 mg HCB/day) had 330 ppm in the fat but none in liver or kidney.
    Those on 12 oz per day (240 mg HCB/day) had 880 ppm in fat and 200 ppm
    in liver and kidney.

    Gardiner and Armstrong (1960) fed two pigs on HCB treated wheat
    containing 660 ppm HCB for 71 days during which each consumed 329 lbs
    of wheat. Animals gained 50-98 lbs in weight and at slaughter fat
    contained 1000 ppm of HCB.

    Watts (1968) reports trials where HCB residues were determined in eggs
    and body fat of poultry receiving HCB treated wheat. Three groups of
    chickens were fed wheat containing 340 ppm HCB for seven, fourteen and
    twenty-eight days before being given untreated grain for twenty-eight
    days. The maximum amount of HCB found in egg yolk was 76, 167 and 146
    ppm respectively. The maximum amount in body fat of the same chickens
    was 178, 349 and 528 ppm respectively. Controls fed on grain believed
    to be free of residues showed 0.84 ppm in egg yolk and 3.3 ppm in fat.
    It was found that the untreated controls received 0.02 ppm HCB in
    their rations.

    Later trials reported by Watts (1968a) showed that four groups of
    chickens fed rations containing 0.02 ppm, 0.08 ppm, 0.7 ppm and 7 ppm
    HCB for two months showed maximum residues of HCB in egg yolk of 0.2
    ppm, 0.3 ppm, 2 ppm and 15 ppm respectively. Body fat from the same
    chickens contained up to 0.7, 0.7, 5 and 29 ppm of HCB. Half of the
    birds receiving the above rations were given treated grain after one
    month. At the end of the second month the HCB residues in egg yolk
    ranged from 0.18 to 0.2, 0.2 to 0.25, 0.7 to 1.2 and 6 to 12 ppm
    respectively. The body fat of these birds contained HCB ranging from
    0.6 to 0.7, 0.7 to 0.9, 1.5 to 2.6, 16 to 24 ppm respectively. It was
    concluded that residues decline only slowly when birds which have
    previously ingested HCB receive untreated feed for one month.

    De Vos et al. (1968) as a result of identifying HCB in tissues of wild
    birds in the Netherlands carried out semichronic toxicity tests on
    Japanese quail and measured the HCB residues in liver, blood and fat.
    Whilst exceptionally high levels were found in birds receiving toxic
    concentrations in their diet, birds fed on diets containing 20 ppm of
    HCB showed HCB residues in the fat of 350 to 520 ppm after three
    months. The author concluded that the half-life of HCB in the quail is
    about three weeks.

    Wit (1969) reports no HCB in an extensive survey of animal fats
    examined in the Netherlands but he notes that HCB would be obscured by
    and reported as alpha BHC.

    In plants

    Johns (1969) reports results from six samples of wheat grown from HCB
    treated seed in different soils. Residues range from 0.003 ppm to
    0.062 ppm. Two samples of wheat grown from seed that had not been
    treated with HCB showed 0.001 ppm and 0.003 ppm but it is not known
    whether HCB treated wheat was grown in the name soil in previous

    Smith (1969) reported trials where wheat treated with 416 ppm HCB was
    planted and the resulting grain analysed. Residues of 0.0033 ppm were
    found. Untreated seed yielded grains containing 0.0022 ppm HCB. Soils
    from twelve wheat farms were analysed and all showed traces of HCB
    ranging from 0.001 to 0.02 ppm though nine were below 0.003 ppm.

    Old (1969) reports investigations with wheat containing 0.05 ppm HCB,
    to determine the effect of milling on the distribution of HCB residues
    in the various milled fractions, and the HCB residues in bread made
    from the flour fraction. Using wheat containing 0.05 ppm HCB it was
    found that residues were distributed in all main milling fractions.
    The total residues recovered were equivalent to 0.049 ppm. Of this 42
    percent was recovered in the bran, 36 percent in the pollard and 22
    percent in the flour. The flour which was found to contain 0.015 ppm
    HCB produced bread containing 0.0024 ppm HCB. Residues in bread were
    thus only 16 percent of the residues in the flour used for the making
    of bread. Further confirmation of the loss during baking was obtained
    from flour containing 0.006 ppm HCB which produced broad containing
    0.0014 ppm HCB or 23 percent of the amount in the flour used.

    There is extensive evidence from the literature that HCB is volatile
    in water vapour even at low temperatures. It is assumed that the loss
    during baking in due to steam volatilisation.


    As far as can be gauged the HCB applied to seeds in not broken down by
    physical, chemical or biological agencies but is simply dissipated and
    diluted in the soil and atmosphere.

    Because of the extreme stability of the molecule, solubility in fatty
    tissue and the ease with which animals extract and concentrate
    residues of HCB from their feed into their fatty tissues residues are
    readily detectable in animal products.

    The inadvertent contamination of commercial grain and animal feeds
    with traces of HCB, though undesirable, appears hard to eliminate.

    Evidence of residues in food in commerce or at consumption

    A considerable amount of the date available results from surveys
    conducted to determine the level and source of HCB residues in eggs,
    meat fat and dairy produce from individual farms. Except in isolated
    instances where farmers have not observed instructions not to feed
    treated seed grain to domestic animals, the contamination invariably
    results from the inadvertent feeding of grain or milling fractions
    containing low levels of HCB residues. It is not possible for farmers
    to know that such animal rations are contaminated and in view of the
    slow rate of excretion the retention of residues in fatty tissues of
    such animals presents serious economic and administrative problems.

    The following table given results of an extensive survey of HCB
    residues in animal products calculated on a fat basis.

        TABLE II
    HCB residues in Animal Products

    Commodity         No. of      No.       Less      0.1       0.25      0.5       Over
                      Samples     with      than       to        to        to
                                  HCB       0.1 ppm   0.25      0.5       1.0       1.0

    Beef Fat          2,029        14       12          1        1         0        0

    Mutton Fat          833        20        2          8        9         1        0

    Butter              514        15        8          5        0         2        0

    Cheese              977         4        3          0        1         0        0

    Frozen liquid       393       227       81        106       25        11        4
    whole egg

    Frozen liquid       154        90        8         42       17        19        4
    egg yolk
    A survey of commercial wheat samples carried out in Australia revealed
    that HCB residues can be found in a significant proportion of the
    samples in amounts ranging from traces (less than 0.005 ppm) to a
    maximum of 0.05 ppm.

    In the United Kingdom positive evidence of the presence of HCB has
    been obtained in several shipments of imported wheat. Three samples
    from one shipment contained 0.67, 1.3 and 0.48 ppm HCB. In two other
    shipments the combined HCB and alpha BHC was less than 1 ppm.

    Surveys of cheese imported into Australia reveal HCB residues in a
    significant proportion of samples from many countries. More than 10
    percent of such imports showed residues in excess of 0.1 ppm, with
    many samples ranging from 0.3 to 0.9 ppm.

    Examinations of food products imported into U.S.A. (Duggan, 1969)
    showed a very low incidence of HCB residues for 1967 and 1968. One 
    and cheese in 1968). However, in 1969 the incidence increased, HCB
    being reported in 170 samples of the 1,866 examined. The increase
    probably reflects improvements in analytical technique more than an
    increase in the incidence and level of contamination.

    The majority of these findings were in manufactured dairy produce with
    HCB being reported in 149 of 608 samples mainly of cheese. The range
    of values for HCB in the positive sample was from a trace to 0.81 ppm
    on a fat basis. The average of 608 samples was 0.01 ppm. Processed
    foods, other than dairy produce, showed 12 positive findings in 345
    samples examined. The range of positive findings in these commodities
    was from a trace to 0.23 ppm.

    One sample of butter was found to contain the following residues (on a
    fat basis):-

            BHC      -  0.24 ppm
            lindane  -  0.32 ppm
            HCB      -  0.37 ppm

    It is obvious that rather more than average care must be taken in
    resolving such mixtures of residues which can easily become confused
    by certain GLC analytical procedures.

    HCB residues have been found in small proportion of the samples of
    domestic food production. In 1967, HCB was reported in 12 samples out
    of approximately 20,000 examined. In 1968 it was reported in only one
    sample out of approximately 10,000 examined and in 1969 in 10 samples
    out of approximately the name number tested. The level in positive
    findings varied from a trace (less than 0.01 ppm) to 0.5 ppm.


    On foods

    Most chlorinated pesticides can be successfully isolated from fatty
    substrates by methods based on the acetonitrile-hexane partition of
    Mills (1959). However, HCB 10 exceptionally non-polar, and is
    distributed preferentially into the hydrocarbon phase. Its partition
    ratio by experiment using equal volumes of the two solvents in
    approximately 0.75 to hexane at 20°C. In three counter-current
    separations with equal volumes, recovery would be about 10 percent.
    Increasing the volume of acetonitrile to increase extraction of the
    pesticide would also increase extraction of unwanted fatty matter.

    Methods using direct extraction with adsorption cleanup, but without
    partition, give acceptable recoveries of HCB from eggs, Onley and
    Mills (1962), butterfat, Moats (1963), Langlois et al. (1963) and meat
    fat. A method using elution through florisil with methylene
    chloride-hexane is now in use in the Australian Commonwealth
    Laboratories (Taylor 1969, direct communication).

    In wheat

    The analytical problem here is somewhat different, as the significance
    of the presence of HCB in grain for consumption is related to the
    potential for concentration of residues in animals from low levels in
    the wheat, as well as to that used for direct human consumption. Thus
    the levels of interest are much lower than those in animal products.

    Extraction of HCB from wheat was achieved by cracking in a blendor,
    refluxing with "hexane" (Shell X4) and filtration through anhydrous
    sodium sulphate. Beside the chlorinated residue, this extract contains
    fat-soluble plant extractives. Passage through a column of activated
    florisil cleans the solution sufficiently for determination by GLC and
    confirmation by, TLC, if the levels are comparatively gross, (over 0.1
    ppm). However, when the concentration is 0.01 ppm or less, the ratio
    of interfering substances to pesticide becomes large enough to require
    further cleanup if TLC is to be used. Alternatively, other means of
    confirmation are required.

    Investigation has therefore followed two lines: to Improve cleanup and
    to devise other confirmatory tests.


    Hexachlorobenzene is stable to concentrated sulphuric acid. A hexane
    solution of residual substrate and HCB, after florisil treatment, was
    washed with the acid several times until no further darkening
    occurred. The hexane layer was washed with water until neutral, and
    dried with anhydrous sodium sulphate. The infra-red spectrum of the
    residue after removal of solvent appeared to be that of a hydrocarbon.
    Gas chromatography produced meaningful peaks at retention times
    corresponding to HCB, but resolution by TLC was prevented by the
    streaking effect of the oily residue. This cleanup in inadequate for
    low levels.

    Attempts at separation of HCB from hydrocarbon material on alumina and
    on activated carbon columns with a variety of eluting solvents did not
    result in sufficiently clean products.

    Refluxing of wheat extracts containing HCB with alcoholic potash
    resulted in varying degrees of breakdown of the pesticide as well as
    substrate. Although this precluded alkaline cleanup, it suggested
    possibilities for confirmation of identity of HCB (vide infra).

    Steam distillation of standard solutions of HCB was found to give high
    recovery. Wheat extract previously partly cleaned by treatment with
    sulphuric acid was therefore distilled from a large (twenty-fold)
    excess of water, the vapour rising through a Vigreux column and
    descending through a water-cooled condenser before collection under
    hexane. The hexane extract from 20 ml of aqueous distillate produced

    clear spots on TLC plates for both hexachlorobenzene and "benzene
    hexachloride" the former at a level of 0.02 ppm (referred to the
    original wheat). Recovery tests by adding known amounts of HCB
    produced similar results, with recoveries close to 100 percent.

    In spite of the high sensitivity of this method it is recommended that
    the sensitivity be developed still further to provide for the need to
    determine residues in cereal products at levels below 0.01 ppm.


    Ideally, confirmation of identity should rest upon two or more methods
    using properties as widely different as possible. For chlorinated
    pesticides, chromatography by GLC and TIC are usually considered
    suitable. However cleanup difficulty, together with small residues,
    may preclude the use of TLC. It is acceptable, then, to use GLC on
    columns of such different stationary phases that the substance has
    substantially different retention characteristics, use being made of
    different combinations of stationary phase-solute interactions.

    Experimentally, the combination of retention values on a silicone
    (low-polar) and polyester (polar) columns were found to provide useful
    confirmations of the presence of HCB.

    A number of experiments were carried out to make use of the
    degradation of HCB by alkali. Both alcoholic potash and sodium
    methylate were used, the latter being more dependably effective. When
    small traces of the substance, of the order of 0.1 ppm or less, are
    present, the pattern of degradation is consistent, in that two major
    products appear in both TLC and GLC, one corresponding in retention
    values with HCB, the other being lower. However, when greater amounts
    of HCB are treated, the result is often obscured by the appearance of
    several other products. The reaction is not, therefore, a reliable
    confirmatory test for all samples.

    A gas-chromatographic column commonly used in estimation of multiple
    chlorinated pesticide residues contains the mixture of DC200 silicone
    and QFI fluosilicone proposed by Burke and Holswade (1966). This
    column does not separate HCB and the alpha isomer of "BHC", the latter
    being the major constituent used in estimation of "BHC". In addition,
    alpha-BHC does not produce a strong reaction on the silver nitrate
    impregnated Thin layer used. Thus BHC may not be detected if in low
    concentrations (at 0.1 ppm levels).

    To distinguish and confirm these compounds, a stationary phase of a
    more polar nature may be used. Separate peaks are produced on Reoplex
    (poly propylene glycol adipate), XE60 (nitrile silicone) and Zonyl E7
    (fluoroalkyl pyromellitic ester). Therefore an extract showing a peak
    of HCB/BHC on the Burke column may be reanalysed on one of these
    columns to distinguish between the two compounds.

    The polar phases mentioned are, however, not suitable for all the
    chlorinated hydrocarbons under investigation. On some, DDT is degraded
    or in indistinguishable from DDD. On others, DDE and Dieldrin are not
    separated. Work is currently on hand to select a column suitable for
    separating all the chlorinated hydrocarbons for which goods are at
    present analysed.


    No national tolerances are known.

    Australia, Canada and the U.S.A. approve the use of HCB seed dressings
    on the basis that the seeds are not used for food or feed for animals.


    HCB has been used extensively as a fungicide to control Bunt
    (Tilletia caries, T. tritici and T. foetida) in wheat
    since 1945. There are many reports of its action on other seed borne
    and soil borne fungi of cereals and as seed treatment for onions but
    these uses do not appear to be of great importance.

    HCB formulations for seed treatment are sold in most wheat growing
    countries. To date the only alternative materials are organomercurial
    compounds. The rate of application is usually within the range of 1-3
    ozs. of 30 percent dust per bushel of wheat seed equivalent to 330-990
    ppm on the seed.

    HCB is a comparatively pure material containing 98 percent
    hexachlorobenzene. The impurities are tetrachlorobenzene and

    The data available to the meeting were obtained from the published
    literature and by correspondence with more than 100 authors,
    administrators and manufacturers throughout the world. In spite of the
    wide search there is only limited information on residues in food
    commodities in international trade or following good agricultural
    practices, other than from Australia. One reason for this is that when
    examining food commodities by several GLC analytical procedures HCB
    has been obscured or confused with BHC.

    All available data indicates that HCB is not metabolized to any
    significant extent by plants or animals and that it persists in soil
    and in animal tissues for long periods. There is an indication that
    HCB might be translocated from treated seed into plant material and
    subsequent grain but the level of contamination from this source is

    Contamination of commercial grain from soil, seed, machinery and bags
    may be minimized by strict attention to agricultural practices but it
    is difficult to avoid all traces of HCB in commercial grain.

    Animals feeding on grain or milling fractions containing traces of
    HCB, concentrate and store the residues in body fat, and excrete
    significant quantities in milk and eggs. The residue level in such
    animal products ranges from ten to one hundred times the concentration
    in feed.

    The literature includes several methods of residue analysis which
    measure HCB in grain and animal products by GLC but the results can be
    confused as and by isomers of BHC. The sensitivity for the
    determination of HCB is 0.005 ppm but special extraction and cleanup
    procedures are necessary to ensure reasonable recovery from plant
    products and especially from animal fats. However, no referee method
    has been evaluated.

    In view of the importance of HCB seed dressings in countries where
    Bunt is endemic, and the lack of suitable alternatives, the Joint
    Meeting recognized that recommendations for administrative action
    levels were required.



    The following temporary practical residue limits are to apply to raw
    agricultural commodities moving in commerce unless otherwise

    Raw cereal (wheat)                 0.05 ppm

    Cereal products from wheat         0.01 ppm

    Fat of cattle, sheep, goats,
    pigs and poultry                   1.0 ppm

    Milk                               0.012 ppm

    Milk products                      0.3 ppm

    Eggs (shellfree basis)             1.0 ppm


    REQUIRED (before an acceptable daily intake or tolerances can
             be established)

    1. Metabolic studies in animals including identification of the toxic
       product or products excreted in milk.

    2. Short-term studies in non-rodent mammalian species and long-term
       studies especially in relation to the effect on bone-marrow.

    3. Reproduction studies in rats.

    REQUIRED before 30 June 1973 (for 1973 review of
             Practical Residue Limits)

    1. Data from countries other than Australia on residues in raw
       agricultural commodities.

    2. Data from surveys of residues in commodities moving in
       international trade.


    1. Further information on analytical procedures capable of
       distinguishing HCB from BHC isomers suitable for use in 
       laboratories engaged in general regulatory work.

    2. Collaborative studies on analytical methods capable of recovering
       and identifying HCB residues in the above food commodities.


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    See Also:
       Toxicological Abbreviations
       Hexachlorobenzene (EHC 195, 1997)
       Hexachlorobenzene (HSG 107, 1998)
       Hexachlorobenzene (ICSC)
       Hexachlorobenzene (PIM 256)
       Hexachlorobenzene (WHO Pesticide Residues Series 4)
       Hexachlorobenzene  (IARC Summary & Evaluation, Supplement7, 1987)
       Hexachlorobenzene  (IARC Summary & Evaluation, Volume 20, 1979)
       Hexachlorobenzene  (IARC Summary & Evaluation, Volume 79, 2001)