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    INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

    WORLD HEALTH ORGANIZATION





    SAFETY EVALUATION OF CERTAIN FOOD
    ADDITIVES AND CONTAMINANTS



    WHO FOOD ADDITIVES SERIES: 44





    Prepared by the Fifty-third meeting of the Joint FAO/WHO
    Expert Committee on Food Additives (JECFA)





    World Health Organization, Geneva, 2000
    IPCS - International Programme on Chemical Safety

    METHYLMERCURY

    First draft prepared by Dr D.C. Bellinger1, Dr M. Bolger2, Dr C.
    Carrington2, Dr E. Dewailly3, Dr L.P.A. Magos4 and Dr B.
    Petersen5

    1Harvard Medical School, Boston, Massachusetts, USA;, 2US Food and
    Drug Administration, Washington DC, USA; 3Centre de Sante Publique du
    Québec, Québec, Canada, 4TNO BIBRA International Ltd, Carshalton,
    Surrey, United Kingdom; and 5Novigen Sciences Inc., Washington DC,
    USA

    Explanation
    Biological data
         Pharmacokinetics
              Absorption
              Distribution
              Transfer from mother to offspring
                   Placental transfer
                   Lactation
              Clearance
         Biochemical aspects
              Cleavage of carbon-mercury bond
              Complexes with thiol radicals
              Interaction with selenium
         Toxicological studies
              Acute toxicity
              Renal and hepatic toxicity
              Anorexia
              Neurotoxicity
                   Small rodents
                   Non-human primates
                   Domestic animals
              Reproductive and developmental toxicity (other than
              neurotoxicity)
              Developmental neurotoxicity
                   Exposure in utero
                   Exposure in utero and postnatally
                   Exposure after parturition
              Carcinogenicity
              Immunomodulation
              Extrapolation between species
         Observations in humans
              Case series
              Childhood development
                   Neurological status
                   Developmental milestones
                   Early development
                   Development later in childhood
                   Sensory, neurophysiological and other end-points
              Adult neurological, neurophysiological, and sensory function
              Bias: Covariates, confounders and effect modifiers

                   Study in the Faroe Islands
                   Study in the Seychelles
                   Study in the Amazon Basin
                   Study in New Zealand
                   Study in Peru
                   Reanalysis of the study in Iraq
    Estimates of dietary intake
         Environmental mercury
         Biomarkers of exposure
         Intake assessment
              Residues
              National intake estimates
              Estimates based on WHO GEMS/Food diets
              Estimates of intake by fish consumers at the 95th percentile
    Comments
    Evaluation
    References

    1.  EXPLANATION

         The Committee first evaluated methylmercury at its sixteenth
    meeting (Annex 1, reference 30), when it established a provisional
    tolerable weekly intake (PTWI) of 300 µg of total mercury per person,
    of which no more than 200 µg should be present as methylmercury. At
    its twenty-second and thirty-third meetings (Annex 1, references  47
    and  83), the Committee confirmed the PTWI of 200 µg of methylmercury
    (3.3 µg/kg bw) for the general population. At its thirty-third
    meeting, the Committee noted that pregnant women and nursing mothers
    may be at greater risk than the general population from the adverse
    effects of methylmercury. The Committee considered the available data
    insufficient to recommend a specific intake for this population group,
    and it recommended that more detailed studies be undertaken. 

         At its present meeting, the Committee reviewed information that
    had become available since the previous evaluation. The PTWI was not
    reconsidered and was maintained at its present value. Two other WHO
    publications have dealt with the effects of mercury and methylmercury
    on human health (WHO, 1976, 1990). Relevant information from those
    documents and the studies published since the report of the
    thirty-third meeting are summarized and discussed in this monograph,
    and the data were used to estimate the risks associated with exposure
    to methylmercury. It should be noted that the doses given refer to the
    mercury constituent of the organic mercury compound.

    2.  BIOLOGICAL DATA

    2.1  Pharmacokinetics

    2.1.1  Absorption

         The dermal absorption of methylmercury is similar to that of
    inorganic mercury salts (Friberg et al., 1961). Studies of
    occupational exposure and studies on rats (Fang, 1980) and mice
    (Ostlund, 1969) both indicate that pulmonary absorption accounts for
    95% of the dose. Methylmercury in ligated segments of intestines was
    absorbed 17-35 times faster than was an inorganic mercury salt (Sasser
    et al., 1978). In volunteers (Aberg et al., 1969; Kershaw et al.,
    1980), in squirrel monkeys (Berlin et al, 1975a), and in macaque
    monkeys (Rice et al., 1989), the peak concentration in blood was
    reached within 6 h of ingestion; 95% of an ingested dose was absorbed
    by volunteers (Nittinen et al., 1973), squirrel monkeys (Berlin et
    al., 1975a), and rats (Walsh, 1982).

    2.1.2  Distribution

         The distribution of methylmercury has three characteristics: (i)
    a high concentration of mercury in blood and a high ratio of the
    concentration in erythrocytes:plasma; (ii) greater ease of transfer
    across the blood-brain and blood-placenta barriers than any other
    mercury compound with the exception of elemental mercury vapour
    (although the transfer of the latter is limited by its rapid
    oxidation; Magos et al., 1989); and (iii) less renal deposition than
    any other mercury compound.

         Three days after a single intravenous dose of methylmercury to
    rats at 0.5 µg/g, the blood concentration of mercury was 2.1 µg/ml and
    that in the brain was 0.14 µg/g. In rats given the same dose as
    mercuric acetate, the concentration of phenyl and methoxyethylmercury
    was 0.042-0.068 µg/ml in blood and 0.018-036 µg/g in brain. The renal
    concentration was 87% less in rats given methylmercury than in those
    given the other mercury compounds (Swensson & Ulfvarson, 1967).

         A high concentration of mercury in blood is associated with a
    high concentration ratio in erythrocytes:plasma in every species
    tested. The reported ratios are about 20 in humans (Miettinen, 1973;
    Kershaw et al., 1980) and nearly 20 in squirrel monkeys (Berlin et
    al., 1975a), guinea-pigs (Iverson et al., 1973), and sheep (Kostyniak,
    1983). The ratio was 12 in hamsters (Omata et al., 1986), 9 in pigs

    (Gyrd-Hansen, 1981), and 5-9 in mice (Ostlund, 1969; Sundberg et al.,
    1998a) and rabbits (Berlin, 1963). In cats, the ratio was 42 (Hollins
    et al., 1975), and in rats the reported ratios ranged from 128 to 288
    (Ulfvarson, 1962; Norseth & Clarkson, 1970; Vostal & Clarkson, 1973).
    The high ratio in rats has been attributed to the greater number of
    thiol groups in rat haemoglobin (Naganuma & Imura, 1980), which
    results in an eight times greater release of methylmercury from human
    than from rat erythrocytes suspended in albumin (Doi & Tagawa, 1983).
    Rat haemoglobin also has an increased capacity to bind alkyltin, which
    has little affinity for thiol radicals (Rose & Aldridge, 1968). The
    accumulation of methylmercury in rat blood is associated with
    organ:blood concentration ratios of mercury that are lower than in any
    other species.

         Except in rats, the brain:blood ratios were greater than 1 in all
    species tested (Table 1). The ratio was consistently high in monkeys,
    and in all species it was higher after multiple dosing than after the
    administration of a single dose. Differences in strain and sex
    affected the concentration of mercury in blood of mice more than that
    in brain. The concentration in the brain was higher in female than in
    male mice. A similar sex difference in brain mercury concentrations,
    but without a difference in the brain:blood ratio, was seen in rats.
    Six to 12 days after four daily oral doses of methylmercury chloride
    at 8 mg/kg bw, the concentration of mercury in brain was 8.8 µg/g in
    female and 6.7 µg/g in male rats (Magos et al., 1981).

         In heavily exposed squirrel monkeys, the brain stem had
    approximately the same concentration as the cerebellum and most of the
    cerebral regions, with the exception of the occipital lobe, which had
    the highest concentration (Berlin et al., 1975c). The thalamus had
    somewhat higher concentrations than the occipital pole (Vahter et al.
    1994). In pigs, the concentrations in the cerebrum, cerebellum, and
    optic nerve differed only slightly, and all had higher concentrations
    than the spinal cord (Platonow, 1968). In guinea-pigs, the cerebellum
    had the lowest concentration (Iverson et al., 1974). In rats, the
    highest concentration was found in the spinal roots and ganglia,
    closely followed by the cerebral cortex and the cerebellum (Somjen et
    al., 1973a), but the concentrations in the cerebellum, medulla
    oblongata and various areas of the cerebrum differed only slightly
    (Magos et al., 1981).


        Table 1. Organ:blood concentration ratios of mercury after treatment with methylmercury 
                                                                                                         

    Species               Treatmenta      Blood         Organ:blood ratio                  Reference
                                          (µg/g)                                    
                                                        Brain      Liver      Kidney 
                                                                                                         

    Squirrel monkey       Single dose,    0.63          3.1 (cc)   5.9        5.1          Berlin et al.
                          8 days                                                           (1975b)

    Squirrel monkey       < 2 months,     1.4           5.3        -          -            Berlin et al.
                          9-22 days                                                        (1975c)

    Macaque monkey        < 2 months,     2.4           2.7        13         21           Evans et al.
                          1 day                                                            (1977)

    Macaque monkey        < 2 months,     2.0           3.1        -          -            Stinson et al.
                          1 day                                                            (1989)

    Macaque monkey        < 2 months,     1.1           4.4 (o)    -          -            Vahter et al. 
                          1 day                                                            (1994)

    Macaque and           < 2 months,     0.45          3.1        12         47           Kawasaki et 
    rhesus monkeys        1 day                                                            al. (1986)

    Pig                   Single dose,    0.39          3.3        12         17           Gyrd-Hansen
                          28 days                                                          (1981)

    Pig                   4-10 doses,     1.8           1.8 (c)    11         8.5          Platonow 
                          1 day                                                            (1968)

    Rabbit                Single dose,    0.08          5.4        10         17           Petersson et
                          7 days                                                           al. (1989)

    Cat                   < 2 months,     14            2.1 (cc)   5.2        2.6          Charbonneau 
                          1 day                                                            et al. (1974)

    Guinea-pig            < 2 months,     3.4           1.8 (of)   8.2        21           Iverson et al.
                          1 day                                                            (1974)

    Table 1. (cont'd)
                                                                                                         

    Species               Treatmenta      Blood         Organ:blood ratio                  Reference
                                          (µg/g)                                    
                                                        Brain      Liver      Kidney 
                                                                                                         

    Guinea-pig            Single dose,    3.5           1.3        4.0        6.7          Iverson et al. 
                          7 days                                                           (1973)

    Rat, male             Single dose,    3.5           0.08       -          1.2          Farris et al. 
                          7 days                                                           (1977)

    Rat, female           Single dose,    36            0.08       0.4        1.1          Fang (1980)
                          4 days

    Rat, male             Single dose,    3.6           0.08       0.23       1.2          Thomas et al. 
    Rat, female           4-10 days       3.3           0.10       0.25       2.0          (1986)
    Rat, male             4-10 doses,     40            0.07 (c)   0.3        1.7          Friberg (1959)
                          17 days

    Rat, female           < 2 months,     95            0.07       0.03       1.2          Magos & 
                                          1 day                                            Butler (1976)

    Hamster               Single dose,    1.5           1.9        4.2        10           Omata et al.
                          16 days                                                          (1986)

    Hamster               4-10 doses,     9.0           3.8 (cc)   5.5        9.8          Omata et al.
                          9 days                                                           (1986)

    Table 1. (cont'd)
                                                                                                         

    Species               Treatmenta      Blood         Organ:blood ratio                  Reference
                                          (µg/g)                                    
                                                        Brain      Liver      Kidney 
                                                                                                         

    Hamster               Single dose.    1.7           2.5        5.1        12           Petersson et
                          7 days                                                           al. (1989)

    Mouse, CBA            Single dose,    0.17          0.77       3.2        14           Kostyniak
    Mouse, CWV            8 days          0.04          1.4        3.3        16           (1980)
    Mouse, NMRI, male     Single dose,    0.05          0.9        8.1        20           Nielsen at al.
    Mouse, NMRI, female   14 days         0.21          0.6        4.1        3            (1994)
                                                                                                         

    c, cerebrum; cc, cerebral cortex; o, occipital pole; of, occipital and frontal lobes

    a Type of dosing and number of days between last or single dose and sacrifice
    

         Histochemical localization (by silver amplification) of mercury
    showed a different distribution. The first deposits of mercury in rat
    brain became apparent 10 days after exposure to 16 mg/L of
    methylmercury chloride in drinking-water. The deposits were found
    initially in the brain stem, then in the cerebral cortex and
    supraoptic nucleus, and finally in the cerebellum and thalamus. After
    20 days, the deposits in the cerebellar cortex were restricted to
    Purkinje cells and Golgi epithelial cells and those in the spinal cord
    to the anterior motor neurons; the granule cells of the cerebellar
    cortex remained unstained (Moller-Madsen, 1990, 1991). Similar
    staining was seen after daily intraperitoneal administration of
    methylmercury chloride at 0.16-0.8 mg/kg bw (Moller-Madsen, 1990). As
    the cerebellar granule cells are target cells for methylmercury
    (Chang, 1977), the absence of staining indicates that only
    demethylated mercury can be detected with the silver amplification
    method. When the cortex of the calcarine sulcus of macaque monkeys was
    stained by the same method, large deposits were seen in the astrocytes
    and microglia after six months, whereas staining of neurons appeared
    later and remained faint even after 18 months (Charleston et al.,
    1995). In squirrel monkeys given weekly doses of [3H]methylmercury,
    the amount in protein increased, and it was found in damaged but not
    in undamaged neurons (Berlin et al., 1975a).

    2.1.3  Transfer from mothers to offspring

    2.1.3.1  Placental transfer

         Methylmercury passes about 10 times more readily through the
    placenta than other mercury compounds, like mercuric mercury and
    phenylmercury (Suzuki et al., 1967). Consequently, when 2 mg of
    methylmercury chloride were infused intravenously into female rats,
    the whole-body retention 1 h later was higher in pregnant than in
    non-pregnant rats, but the deposition in blood, kidney, liver, and
    brain was lower, as the fetus acted as a sink for methylmercury
    (Aschner & Clarkson, 1987). When pregnant rats were given a single
    (King et al., 1976) or multiple doses of methylmercury (Magos et al.,
    1980a), the fetal content of mercury increased daily, with no increase
    in the fetal concentration.

         The concentration ratio in fetal brain:maternal brain is > 1,
    except in hamsters given a single dose of mercury at 0.32 mg /kg bw on
    day 2 or 9 of gestation. One day before parturition, the maternal
    brain concentration was higher than that of the fetus (Dock et al.,
    1994a). In macaque monkeys (Stinson et al., 1989), rats (Satoh et al.,
    1985a), and mice (Satoh & Suzuki, 1983) given longterm dietary
    exposure to methylmercury, the concentration of mercury in fetal brain
    consistently exceeded that of maternal brain by a factor of 1.5.
    Similarly, when methylmercury was given to dams during gestation,
    1.7-4.8 times higher concentrations were found in fetal than in
    maternal brain in rats (Null et al., 1973; Aaseth, 1976; King et al.,

    1976) and mice (Inouye et al., 1985), although the ratio to whole-body
    concentration. was 1 in rats (Magos et al., 1980a) and < 1 in mice
    (Childs, 1973).

         The fetal:maternal concentration ratio was slightly > 1 in liver
    and 0.3-0.5 in kidney (King et al., 1976; Wannag, 1976; Inouye et al.,
    1986), and the ratio in blood was 1.1 (Burbacher et al., 1984) or 1.2
    in macaque monkeys (Stinson et al., 1989) and 0.6 in rats (Wannag,
    1976). In contrast to animals, the fetal: maternal blood ratio in
    humans is high. Thus, the cord blood:maternal blood ratio in Inuits
    with a high consumption of marine foods (Hansen et al., 1990) and in
    Swedish women who ate large amounts of fish (Skerfving, 1988) was 3.2.

         In the offspring of squirrel monkeys exposed to methylmercury,
    the highest concentration in the brain was found in the pituitary
    gland, followed by caudatus, striatum, thalamus, cerebrum, cerebellum,
    medulla, and cervical spinal cord (Lögdberg et al., 1993). In rats,
    the concentration was higher in the cerebellum than in the cerebrum
    (Yamaguchi & Nunotani, 1974; King et al., 1976).

    2.1.3.2  Lactation

         The passage of methylmercury from blood to milk is low, in
    contrast to passage through the blood-brain and blood-placenta
    barriers. The average concentration ratio for milk:maternal blood was
    0.2 in hamsters (Nordenhäll et al., 1995a), 0.03 in guinea-pigs
    (Yoshida et al., 1994), and 0.04 in mice (Sundberg et al, 1998a) and
    rats (Sundberg et al., 1991). Inorganic mercury passes more readily
    into milk than methylmercury: after injection of equivalent doses of
    inorganic and methylmercury, the concentration of mercury in the milk
    was five times higher with the inorganic form in lactating mice
    (Sundberg et al., 1998b) and 2.5 times higher in guinea-pigs. In
    guinea-pigs, the milk:maternal blood concentration ratio was 0.12 for
    inorganic mercury and 0.023 for methylmercury, but the milk:plasma
    concentration ratios were similar (Yoshida et al., 1994). One
    consequence of the difference in the passage of inorganic and
    methylmercury is that mothers poisoned with the organic form had 95%
    less mercury in their milk than in their blood, and 40% of the mercury
    in milk was inorganic (Bakir et al., 1973). This tendency was
    confirmed in studies on women who ate large quantities of fish
    (Skerfving, 1988; Oskarsson et al., 1996) and in experimental animals
    (Nordenhäll et al., 1995b; Sundberg et al., 1998a,b).

         In the milk of hamsters, the concentration of mercury decreased
    with a half-time of four days, and 5% of the injected dose was
    excreted in milk. The proportion of inorganic mercury in milk was 16%
    on the first day and 22% after five to six days (Nordenhäll et al.,
    1995a). An average of 1.7% of a maternal dose given on the day of
    parturition was transferred to a litter (Nordenhäll et al., 1995b).

         In suckling pups of mouse dams given a single intravenous
    injection of methylmercury chloride at 0.4 mg/kg bw, the
    concentrations of mercury in plasma and brain peaked after six to
    seven days. While that in plasma then immediately decreased, the
    concentration of mercury in brain showed no variation for a further
    four days (Sundberg et al., 1998a). In pups of hamster dams given a
    single dose of 3.2 mg/kg bw methylmercury chloride by gavage on the
    day 1  post partum, the whole-body and tissue concentrations of
    mercury increased for 10-15 days and then decreased. When the pups
    were 21 days of age, 50% of the body burden was in the pelt
    (Nordenhäll et al., 1995a).

         The body burden of mouse pups was increased to a lesser extent by
    exposure during lactation than by exposure in utero. In
    cross-fostering studies, the body burden at 14 days of age was twofold
    higher in pups exposed  in utero than in those exposed by lactation
    (Nielsen & Andersen, 1992). Similar differences were observed in
    hamster pups (NordenhäIl et al., 1998).

         Although loss of methylmercury during lactation is too small to
    affect maternal clearance, the clearance half-time was significantly
    lower in lactating women (Greenwood et al., 1978), rats (Magos et al.,
    1981), and mice (Greenwood et al., 1978) than in non-lactating ones.

    2.1.4  Clearance

         In animals, mercury is cleared by three main routes: in urine,
    faeces, and hair. Faecal excretion predominates over urinary
    excretion. While in humans excretion in hair is important only for
    biological monitoring, in furry animals this route of excretion can
    strongly alter clearance from the whole body and from toxicologically
    important soft tissues (Table 2).

         In volunteers who ingested a single dose, faecal excretion
    reached a peak faster than urinary excretion; faecal excretion peaked
    at 3% and urinary excretion at 0.11% of the dose (Miettinen, 1973). In
    another study, the cumulative faecal excretion was 31% and urinary
    excretion was 4% of the dose (Smith et al., 1994). In pigs, the ratio
    of cumulative faecal:urinary excretion during the first 15 days after
    a single dose was 17 (Gyrd-Hansen, 1981); in rats, the ratio was 4.8
    in the first three days (Swensson & Ulfvarson, 1967). The ratio
    decreased in rats with increasing dose and prolonged exposure (Magos &
    Butler, 1976). In hamsters given a low dose, the ratio was 2.1 (Dock
    et al., 1994a), while urinary excretion was greater than faecal
    excretion after a renally toxic dose (Petersson et al., 1989),
    probably because of loss of mercury with desquamated tubular cells.

         Reports of concentrations in excreta also suggest the importance
    of faecal over urinary excretion, if it is assumed that the difference
    in volume (or mass) is not great. The faecal:urinary concentration
    ratio was 100 for squirrel monkeys (Berlin et al., 1975a) and 50 for
    cats (Hollins et al., 1975). Although faecal and urinary excretion
    could be used to calculate the clearance half-time for the whole body,
    this method (like chemical or radiochemical estimation of whole-body
    burden) results in an underestimate of clearance from the
    toxicologically important soft tissues in furry animals. In rats given
    repeated oral doses of methylmercury, the contribution of blood to the
    body burden declined to 28% and the contribution of the pelt increased
    to 38% (Magos & Butler, 1976); 98 days after a single dose, the body
    burden represented 12% of the dose and nearly 90% of the body burden
    was in the fur (Farris et al., 1993). In hamster pups exposed to
    methylmercury either  in utero or by lactation, 80% of the body
    burden was in the pelt by 28 days of age (Nordenhäll et al., 1998).

         Correction for mercury in fur (Table 2) decreased the clearance
    half-time by 33% in cats and by 44% in rats. As the only toxicological
    significance of methylmercury in fur is as a source of intake during
    grooming (Farris et al., 1993), the clearance half-time in blood is
    more meaningful than that in the whole body; however, even values
    based on total mercury result in underestimates of clearance since
    decomposition is ignored. Clearance half-times show wide interspecies
    variation, depending on the body mass of the species: the larger the
    body mass, the longer the clearance half-time.

         In adult mice exposed to unlabelled methylmercury before and
    after a single dose of [203Hg]methylmercury, the clearance of
    radiolabelled compound was not affected (Nielsen & Andersen, 1996).
    Similarly, in rats, the biliary excretion of labelled mercury was not
    influenced by treatment with unlabelled compound (Cikrt et al., 1984),
    indicating complete distribution of label between the first and second
    injections.

         Clearance from offspring is slow during the first weeks of life.
    Thus, the body burden of rats declined by 5% of the dose during the
    first 10 days and by a further 25% during the next 12 days (Thomas et
    al., 1988). Suckling hamsters (Nordenhäll et al., 1998) and mice
    (Rowland et al., 1983; Sundberg et al., 1998a,b) show similar changes
    in distribution and clearance.

        Table 2. Clearance half-times (days) of methylmercury
                                                                                            

    Species                Compartment                         Reference
                                                            
                           Whole body    Brain      Blood
                                                                                            

    Human                  72            -          -          Aberg et al. (1969)
                           -             -          52         Kershaw et al. (1980)
                           74            -          50         WHO (1990)
                           44a           -          44a        Smith et al. (1994)
    Squirrel monkey        134           -          49         Berlin et al. (1975b)
    Macaque monkey         -             47         14         Rice et al. (1989)
                           -             -          24         Stinson et al. (1989)
                           -             -          26         Vahter et al. (1994)
    Pig                    -             -          27         Gyrd-Hansen (1981)
    Sheep                  -             -          14         Kostyniak (1983)
    Cat                    -             -          39         Charbonneau et al. 
                                                               (1974)
                           117 (76b)     -          -          Hollins et al. (1975)
    Guinea-pig             -             -          13         Iverson et al. (1973)
    Rat, female            34            26         14         Magos & Butler (1976)
    Rat, male              24            -          -          Farris et al. (1993)
    Rat, male              13a           -          11         Farris et al. (1993)
    Rat, male              11a,b         -          -          Farris et al. (1993)
    Hamster                7.7           -          -          Dock et al. (1994a)
    Mouse, female          7.3           -          -          Clarkson et al. (1973)
    Mouse, CR1:CD,         -             - 7        -          Sundberg et al. (1998a)
    female
    Mouse, CBA, male       approx. 1     7.4        -          Kostyniak (1980)
    Mouse, CFW, male       3.0           -          -          Kostyniak (1980)
    Mouse, BALB/c, male    -             15         5.0        Doi & Kobayashi (1982)
    Mouse, C57Bl, male     -             16         7.8        Doi & Kobayashi (1982)
    Mouse, NMRI, male      6.3           -          -          Nielsen & Andersen (1991)
    Mouse, NMRI, female    14            -          -          Nielsen & Andersen (1991)
                                                                                            

    a Clearance includes decomposition to inorganic form
    b Half-time for whole body without hair
    
    2.1.5  Biochemical aspects

    2.1.5.1  Cleavage of carbon-mercury bond

         Methylmercury is the most stable organic mercury compound. The
    addition of even one carbon to the alkyl radical can accelerate
    decomposition in brain and other tissues (Magos et al., 1985a). 

    The bond between the alkyl radical and mercury can be broken by 
    hydroxy radicals (Suda & Hirayama, 1992) and other reactive oxygen 
    species (Suda & Takahashi, 1992), which attack ethylmercury more 
    readily than methylmercury.

         In rats given a single dose of methylmercury, the 1:1 ratio of
    organic: inorganic mercury was reached after 50 days in liver and
    after 14 days in kidney, the main site of accumulation of inorganic
    mercury. Inorganic mercury represented less than 4% of the mercury in
    brain during the first 23 days after a single dose (Norseth &
    Clarkson, 1970) and only 3.4% after prolonged daily treatment (Magos &
    Butler, 1976).

         Inorganic mercury in the brain is most likely formed by
    decomposition  in situ, as pretreatment with methylmercury did not
    increase the concentration of mercury in the brains of rabbits treated
    with inorganic mercury (Dock et al., 1994b). The slower clearance from
    the brain may explain the 88% increase in the contribution of
    inorganic mercury to total mercury in the brains of macaque monkeys
    six months after a long exposure (Lind et al., 1988; Vahter et al.,
    1994), and the concentration of inorganic mercury in the hypothalamus
    was 6.6 times higher than that in monkeys exposed to inorganic mercury
    for two to three months (Vahter et al., 1994).

         The main site of decomposition of methylmercury is the intestinal
    tract, where the portion secreted with bile or with cells shed from
    the intestinal wall is decomposed and the remainder is reabsorbed
    (Norseth & Clarkson, 1971). Most of the decomposition is carried out
    by intestinal bacterial flora; disruption of this bacterial activity
    by antibiotics prolonged the clearance half-time and decreased faecal
    excretion in both rats and mice (Rowland et al., 1980, 1983). Both
    demethylation and methylation occur. Caecal bacteria from rats
    methylated 2.3% of inorganic mercury at a dose of < 0.1 µg/g and less
    of doses > 0.1 µg/g (Rowland et al., 1977). Extrapolation of this
    rate of synthesis to human populations with intakes of 4.6 µg of
    inorganic mercury and 2.4 µg of organic mercury would add only 0.1 µg
    to the daily intake of methylmercury.

         The biliary excretion of methylmercury varies by species:
    hamsters, rats, and mice excrete it readily, while guinea-pigs and
    rabbits excrete 5 and 25 times less. The 'slow excretors' also
    excreted significantly less reduced glutathione (GSH) than the 'fast
    excretors' (Stein et al., 1988). Biliary excretion also depends on
    age: in rats, the ability to excrete GSH and methylmercury develops
    between 2 and 4 weeks of age (Ballatori & Clarkson, 1982).

    2.1.5.2  Complexes with thiol radicals

         Mercury compounds favour links with thiol ligands. Thiols of low
    relative molecular mass offer transport, and those of high relative
    molecular mass within cells offer anchorage for methylmercury,
    targeting sulfydryl enzymes (Rothstein, 1970). The change from one
    ligand to another ensures that diffusable thiol compounds can carry
    methylmercury from the extracellular space to intracellular proteins
    and  vice versa. The high concentration of GSH within erythrocytes
    suggests that exchange occurs between protein and GSH in these cells
    (Rabenstein & Evans, 1978); however, other diffusable thiols, such as
    cysteine, and unphysiological ones such as pencillamine,
     N-acetylpenicillamine, and  N-acetylcysteine, removed methylmercury
    from erythrocytes when their concentration approached the
    concentration of GSH (Kostyniak et al., 1975).

         Complexing with L-cysteine or glutathione has particular
    toxicological significance. In rats, injection of L-cysteine with
    methylmercury changed the short-term distribution of the latter by
    decreasing the plasma concentration and increasing the concentrations
    in brain, liver, and kidney. D-Cysteine had similar effects but did
    not increase the concentration of mercury in brain (Thomas & Smith,
    1982). GSH had less effect on the brain uptake of methylmercury than
    L-cysteine (Kerper et al., 1992). Increased uptake by brain was also
    achieved by administration of a cysteine-supplemented diet (Farris et
    al., 1977). The stimulatory effect of L-cysteine may be due to the
    structural similarity between the methylmercury-cysteine complex and
    L-methionine, which permits use of the same L-amino acid transport
    system (Kerper et al., 1992). L-Cysteine can also increase the biliary
    excretion of methylmercury, after a delay for conversion to GSH (Magos
    et al., 1978).

         GSH plays a key role in biliary secretion of methylmercury.
    Consequently, compounds that deplete GSH decrease and compounds that
    stimulate glutathione  S-transferase increase the biliary excretion of
    both GSH and methylmercury (Gregus & Varga, 1985). This effect is
    modulated by the transport molecule ligandin (a glutathione
    S-transferase): the biliary excretion of methylmercury was inhibited
    by indocyanine green, a non-substrate ligand for ligandin (Magos et
    al., 1979a).  trans-Stilbene oxide, which induces glutathione
    S-transferase activity, had no effect on the biliary excretion of GSH
    or methylmercury (Gregus & Varga, 1985) but potentiated the effect of
    GSH in rats (Magos et al., 1985b). The excreted methylmercury is
    attached to GSH (Refsvik & Norseth, 1975) or to its metabolic product
    (Ohsawa & Magos, 1974; Urano et al., 1988).

         GSH also affects the renal handling of methylmercury. Depletion
    of hepatic GSH by 1,2-dichlorobenzene resulted in a reduced blood GSH
    concentration and a reduced renal content of methylmercury. Inhibition
    of renal gamma-glutamyl-transpeptidase, a membrane enzyme that

    catalyses the breakdown of GSH, decreased the renal deposition and
    increased the urinary excretion of methylmercury. The sex difference
    in the renal handling of methylmercury in some mouse strains may also
    depend on the concentration and metabolism of renal GSH (Tanaka
    et al., 1991)

         Physiological agents that complex thiols do not affect urinary
    excretion or whole-body clearance (Magos & Clarkson, 1976), while
    unphysiological thiols of low relative molecular mass, such as
    penicillamine,  N-acetylpenicillamine, dimercaptosuccinic acid, and
    dimercaptopropanesulfonate, accelerate clearance in both humans
    (Clarkson et al., 1981) and experimental animals (Aaseth., 1976;
    Magos, 1976; Planas-Bohne, 1981). Although clearance was accelerated
    by dimercaptopropanol, this chelator consistently increased the
    deposition of methylmercury in the brains of mice (Berlin et al.,
    1965; Ogawa et al., 1976). Thiol-complexing agents mobilize
    methylmercury more efficiently than inorganic mercury, as shown with
    dimercaptosuccinic acid and penicillamine in mice (Friedheim & Corvi,
    1975) and pencillamine in volunteers (Suzuki et al., 1976).

    2.1.5.3  Interaction with selenium

         Methylmercury has a greater affinity for selenium than for
    sulfur, and the reaction of selenite with thiol groups such as GSH and
    the sulfur radicals of proteins extends the possibility of
    associations between selenium and methylmercury. The effects of
    selenite cannot be extrapolated to the effects of selenium present in
    food, however, because of differences in their decomposition to
    selenide in food. Thus, selenite is twice as potent as
    L-selenomethionine in forming volatile dimethylselenide (McConnel &
    Roth, 1977) and four times as potent as selenium-enriched liver in
    forming mercury-selenium complexes (Magos et al., 1983). The rate of
    selenide formation from selenite is important in the formation of an
    unstable adduct, bis(methylmercury)selenide, which carries
    methylmercury through the blood-brain barrier (Magos et al., 1979b;
    Naganuma & Imura, 1980). The availability of selenide for this
    reaction is limited by methylation to dimethyl-selenide, which is
    stimulated by methylmercury (Yonemoto et al., 1985).

         Selenite can change the distribution of methylmercury in rats by
    decreasing deposition in kidney and increasing deposition in brain,
    independently of whether selenite is given 30 min before (Chen et al.,
    1975), simultaneously with, or even days after methylmercury (Magos &
    Webb, 1977). Both low (0.6 mg/L) and high (3 mg/L) concentrations of
    selenium in drinking-water increased the uptake of mercury in the
    brains of mice, the high dose more persistently than the low dose
    (Wicklund Glynn & Lind, 1995). Within the brain, selenium did not
    alter the subcellular distribution of methylmercury, while the mercury
    compound shifted selenium from the cytosol to the mitochondrial
    fraction (Prohaska & Ganther, 1977).

         Selenite increased the fetal brain content of mercury without
    increasing the fetal concentration, depending on the dose of
    methylmercury given to mouse dams (Satoh & Suzuki, 1979). The
    concentration of mercury in rat fetal brain was also unchanged by 1.3
    mg/kg of selenite when the dams were given 1.6 or 4.8 mg/kg bw as
    methylmercury on days 6-9 of gestation, although the fetal blood
    concentration of mercury was increased by 1.4-and 2.8-fold,
    respectively (Frederiksson et al., 1993). Seleno-L-methione had no
    effect on the deposition of methylmercury in pups exposed prenatally,
    but it increased the deposition of mercury in nearly all organs after
    exposure during lactation (Nielsen & Andersen, 1992, 1995).

         The role of selenite in the degradation of methylmercury is
    amibiguous. Addition of selenite to liver homogenates for 90 min
    increased the decomposition of phenylmercury but not of methylmercury
    (Fang, 1974). Selenite in the presence of a reducing agent like GSH
    (Iwata et al., 1982), cysteine (Baatrup et al., 1989), or hydroquinone
     in vitro (Moller-Madsen & Danscher, 1991) induced some
    decomposition, but the conditions were not physiological. Six months
    after exposure of macaque monkeys to methylmercury for 12 months, the
    concentration of inorganic mercury had declined, while the
    concentration of selenium remained unchanged (Björkman et al., 1994),
    which argues against a role for selenium in cleavage of methylmercury
    in the brain. The final molar ratio of inorganic mercury to selenium
    in these monkeys was 0.5. In contrast, persistent deposits with a 1:1
    molar ratio were found in organs of rats treated with inorganic
    mercury and hydrogen selenide (Groth et al., 1976), in the livers of
    marine mammals (Koeman et al., 1973; Martoja & Viale, 1977), and in
    the brains of retired mercury miners (Kosta et al., 1975).

    2.2  Toxicological studies

    2.2.1  Acute toxicity

         Although methylmercury is almost completely absorbed from the
    gastrointestinal tract, the enteric and parenteral LD50 values differ
    widely. Thus, the LD50 values 24 h after intraperitoneal injection
    were 9.5 mg/kg bw in rats, 20 mg/kg bw in hamsters, and > 14 mg/kg bw
    in squirrel monkeys; after 30 days, the values were 8.1, 12, and

    3.8-5.1 mg/kg bw, respectively. In these three species, most of the
    deaths occurred within the first 24 h (Hoskins & Hupp, 1978). The
    LD50 values after oral administration were 25 mg/kg bw in old rats
    (450 g bw) and 40 mg/kg bw in young rats (200 g bw) (Lin et al.,
    1975).

         The difference in the LD50 values after administration by these
    two routes may reflect the corrosive effect of methylmercury at the
    site of contact. The risk of corrosion depends on the route of
    administration, and it decreases in the order intraperitoneal =
    subcutaneous > intubation > dose given in a small volume of water or
    juice > supplemented food. The importance of corrosion is supported
    by the dramatic reaction seen in rats 15 min after intraperitoneal
    injection of doses > 3 mg/kg bw: the animals became lethargic, with
    drooping heads and dulled eyes; some died after developing dyspnoea,
    spasticity, and loss of the ability to walk. Animals given < 3 mg/kg
    bw became drowsy but survived (Hoskins & Hupp, 1978). The rapidity of
    this reaction suggests a corrosive effect and shock. The corrosive
    effect is also reflected by the ulcerative oesophagitis seen in cats
    dosed orally with methylmercury at 1.3 mg/kg bw per day (Davies &
    Nielsen, 1977) and by the necrosis, oedema, and congestion of the
    stomach in dogs dosed with 0.43 or 0.64 mg/kg bw per day (Davies et
    al., 1977) and in pigs given 0.64 or 0.86 mg/kg bw per day (Davies et
    al., 1976). All the affected animals also had renal and hepatic
    damage.

         These local effects should be taken into consideration in
    interpreting more subtle outcomes. In rats dosed orally for two days
    with 12 mg/kg bw per day as mercury or 8 mg/kg bw per day as
    methylmercury chloride, decreased wakefulness and increased slow-wave
    sleep peaked three days after the second dose, while the brain mercury
    concentration peaked after nine days (Arito & Takahashi, 1991). A
    confounding role of gastrointestinal inflammation could not be ruled
    out. A reduced ability of mice to stand on their hind legs and to
    move, seen 1 h but not 72 h after an intraperitoneal injection of 10
    mg/kg bw as methylmercury chloride (Salvaterra et al., 1973),
    indicates peritoneal irritation and possibly peritonitis rather than
    systemic toxicity.

    2.2.2  Renal and hepatic toxicity

         Mice

         Experiments in mice suggest that males are more sensitive than
    females. In long-term experiments, the incidence of chronic
    nephropathy was higher in male than in female mice given diets
    containing 10 mg/kg (equivalent to 1.5 mg/kg bw per day), and only
    male mice had nephropathy when given 2 mg/kg in the diet (Hirano et
    al., 1986; Mitsumori et al., 1990).

         Rats

         Clinical signs of neurotoxicity induced by methylmercury are
    always accompanied by renal damage. In female rats dosed orally five
    times a week with 0.84 mg/kg bw as methylmercury dicyandiamide for
    8-12 weeks, the renal cortex was extensively damaged, with desquamated
    cells in the tubules and inflammatory reactions and fibrosis in the
    surrounding area. At the end of treatment, mild ataxia was observed in
    some but not all animals (Magos & Butler, 1972). Male rats receiving
    0.8 mg/kg bw per day as methylmercury chloride developed severe
    diarrhoea and loss of appetite after two to four days, and necropsy
    after 10 days of treatment showed ultrastructural changes in the pars
    recta of the proximal tubules (Ware et al., 1975). Differences in the
    time of onset of renal damage rather than in its severity were seen in
    male rats given 8.5 or 1.7 mg/kg bw per day as methylmercury
    hydroxide: renal lesions, mainly in the proximal tubules, were seen
    one day after the last high dose and six days after the low dose
    (Klein et al., 1973).

         In comparisons of renal toxicity, male rats were usually more
    sensitive than females, as indicated by increased serum creatinine
    concentration and decreased bromosulphthalein excretion after single
    oral doses of methylmercury chloride ranging from 4 to 40 mg/kg bw
    (Yasutake et al., 1991); proteinurea in rats fed diets containing
    methylmercury chloride at 0.5, 2.5, or 25 mg/kg for 12 weeks
    (Verschuuren et al., 1976a); and deaths and renal lesions in rats
    given 0.05 or 0.25 mg/kg bw per day as methylmercury chloride in food
    (Munro et al., 1980). No difference in renal morphology was found
    between the sexes after exposure to 2.5 mg/kg of diet for two years
    (Verschuuren et al., 1976b), although the results suggested that
    females were more sensitive than males to diets containing 2 mg/kg of
    methylmercury chloride (equivalent to 0.2 mg/kg bw per day) for 84 or
    142 days (Fowler, 1972).

         The effect of methylmercury on the liver can be rapid and
    lasting. Ultrastuctural changes were detected in the liver 1 h after a
    single subcutaneous dose of 8 mg/kg bw as methylmercury chloride to
    male rats, which developed into cytoplasmic degeneration during the
    first day (Desnoyers & Chang, 1975). Similar changes were seen in the
    livers of cats fed tuna fish containing 0.3-0.5 mg/kg of mercury daily
    for 7-11 months (Chang & Yamaguchi, 1974).

    2.2.3  Anorexia

         A frequent response to methylmercury in experimental animals is
    anorexia resulting in decreased weight gain or even loss of weight.
    Anorexia precedes the neurological signs of methylmercury poisoning in

    rats (Hunter et al., 1940; Herman et al., 1973), rabbits (Jacobs et
    al., 1977), guinea-pigs (Falk et al., 1974), and mice (McDonald &
    Harbison, 1977). In cats (Davies & Nielsen, 1977) and non-human
    primates (Shaw et al., 1975; Evans et al., 1977), anorexia occurred
    only after the onset of disorders of coordination and vision.

    2.2.4  Neurotoxicity

    2.2.4.1  Small rodents

         The species studied most extensively for neurotoxicity is the
    rat, and few experiments have been conducted on other species.
    Guinea-pigs were used to study the effect of methymercury on the
    cochlea. Five weekly doses of 1.7 mg/kg bw as methylmercury hydroxide
    for two to six weeks decreased body weight and locomotor activity and
    damaged the outer rows of hair cells of the cochlea at two-and-a-half
    turns from the cochlear base. The auditory tract was not damaged (Falk
    et al., 1974). In a follow-up study, the damage was localized to the
    sensory nerve end (Konishi & Hamrick, 1979).

         The role of the granular layer of the cerebellum and the
    posterior root fibres as a target of methylmercury was identified in
    rats 60 years ago. The report of this study also described the
    clinical course of severe poisoning as weight loss, ataxia, paralysis,
    and death (Hunter et al., 1940). Axoplasmic and myelin degeneration of
    posterior root fibres was produced by daily doses of 0.8 mg/kg bw as
    methylmercury chloride (Chang & Hartmann, 1972), while the ventricular
    root fibres and the dorsal root nerves remained intact after
    administration of 1.6 mg/kg bw per day (Yup & Chang, 1981). The
    vulnerability of dorsal root ganglia was explained by the extent and
    duration of inhibition of amino acid incorporation (Cavanagh & Chen,
    1971).

         Female rats dosed orally five times a week with 0.84, 1.68, or
    3.32 mg/kg bw as methylmercury dicyandiamide showed signs of ataxia
    after 39, 25, and 10-12 doses, respectively. Owing to progressive
    weakness, animals at the intermediate dose had to be sacrificed after
    34 doses and those at the highest dose after 16 doses, when their
    brain mercury concentrations were 10 and 16 µg/g, respectively.
    Histological examination of the brain showed necrosis in the granular
    layer of the cerebellum and, at the two higher doses, oedema in the
    white matter; at the low dose, only mild damage involving few cells
    was seen even after 59 doses, when the brain mercury concentration was
    about 5 µg/g (Magos & Butler, 1972). The lack of damage at this dose
    was corroborated by the lack of gross or histological effects in male

    rats dosed with 0.8 mg/kg bw per day as methylmercury chloride for 28
    weeks. A dose of 4 mg/kg bw per day caused weight loss, early toxic
    signs, and decreased protein synthesis in the granule neurones. These
    pathological changes were precipitated when the brain mercury
    concentration was 3-6 µg/g. At higher concentrations, Purkinje cells
    were also affected (Syversen, 1982).

         After subcutaneous administration of 2 mg/kg bw as methylmercury
    hydroxide five times a week to male rats, the first signs of
    coordination disorder were detected after the fifteenth dose. The
    earliest morphological change in the central nervous system was
    shrinkage of cells and nuclear disintegration in the cerebellar
    internal granular layer, the vermis, and the depth of the folia of the
    hemispheres. These early changes progressed to more widespread
    necrosis involving the putamen, corpus striatum, and the visual cortex
    in the occipital lobe. The most severely affected parts of the
    peripheral nervous system were the dorsal roots and the sciatic and
    sural nerves (Herman et al., 1973). The concentration of mercury on
    the fifteenth day was about 30% lower in the cerebral cortex than in
    the cerebellum or spinal ganglia, but by day 29 the concentrations in
    each of these tissues was nearly 14 µg/g (Somjen et al., 1973b).
    Damage to the nervous system resulted in the formation of
    autoantibodies that reacted with neurotypic and gliotypic proteins,
    such as glial fibrillary acid protein, after seven days of exposure to
    13 or 26 mg/kg of mercury as methylmercury chloride in the diet
    (El-Fawal et al., 1996).

         The dose-effect relationship for neurotoxicity differs in male
    and female rats, as suggested by the response to four or five doses of
    8 mg/kg bw as methylmercury chloride given by intubation. The flailing
    reflex (rotation of the lower body when the animal is held loosely
    under the forelimbs), hind-leg crossing, and damage in the granular
    layer indicated that female rats were more affected than males. The
    difference may be due to the 24-40% higher concentration of mercury in
    the brains of female than of male rats (Magos et al., 1981). A
    difference was seen also with age: in young rats, an intraperitoneal
    injection of 8 mg/kg bw which resulted in approximately 2 µg/g of
    brain, the morphological changes in the cerebellar hemisphere were
    subtle, including nuclear swelling and increased heterochromatin, were
    reversible, and were not accompanied by the formation of dark cells in
    the granular layer. In older rats, 'dark cells' were a conspicious
    feature (Syversen et al., 1981)

         The relative merit of electrophysiology in the grading of
    methylmercury poisoning was the subject of several investigations
    (Somjen et al., 1973a; Fehling et al., 1975; Miyama et al., 1983). The
    results were not consistent, and its advantage over simple clinical
    observations remains questionable.

         In male mice, dietary intake of methylmercury caused no loss of
    body weight when the concentration was 10 mg/kg of diet as
    methylmercury chloride, but 32 mg/kg of diet caused loss after 30 days
    and 100 mg/kg of diet resulted nearly immediately in weight loss. Mice
    at the intermediate dose showed signs of coordination disorders which
    coincided with the onset of weight loss. A slight effect on head
    positioning was observed earlier. No neurological signs were seen in
    mice at the lowest dose (Suzuki & Miyama, 1971). In wild mice exposed
    to methyl-mercury in their natural diet, the concentration of mercury
    in hair correlated with deviant behaviour and decreased ability to
    swim (Burton et al., 1977).

    2.2.4.2  Non-human primates

         In the first experiment performed on the toxicity of
    methylmercury, one female macaque monkey was exposed by inhalation to
    a concentration in air that was so high that it caused respiratory
    irritation. The clinical events were ataxia, prostration, inability to
    eat, and death after 21 days. The main damage to the nervous system
    was in the sciatic nerve, posterior root ganglia, brain stem, and
    cerebrum, where the frontal and occipital cortices were equally
    affected while the cerebellar cortex was spared (Hunter et al., 1940).

         Rhesus macaque and mulatta monkeys were given methylmercury in
    pellets at doses of 0.01, 0.03, 0.1, or 0.3 mg/kg bw per day for 52
    months or until signs of severe poisoning appeared. Exposure to the
    lowest dose had no effect on body weight, and 0.03 mg/kg bw per day
    depressed body-weight gain only marginally; however, daily doses of
    0.1 or 0.3 mg/kg bw per day caused severe poisoning, with ataxia,
    visual disturbances, blindness, tremor, spasms, paralysis, and death
    or moribund condition at six and three months, respectively. At the
    two lower doses, the average monthly concentrations of mercury in
    blood between 12 and 52 months were 0.4 and 1.1 µg/ml. The mean
    concentrations of mercury in the occipital lobe after 52 months were
    0.7 µg/g and 2.3 µg/g at these doses; the cerebellum contained 10-26%
    less than the occipital lobe. Histological examination of the brain
    showed no abnormalities. In monkeys at the two highest doses, the
    concentrations in the occipital lobe were 13 and 24 µg/g; the most
    severe lesions were found in the occipital cortex and, within it, in
    the primary visual cortex. No changes were seen in the cerebellar
    cortex (Ikeda et al., 1973; Kawasaki et al., 1986). In this study, the
    threshold dose of methylmercury that induced toxic effects after
    long-term exposure was 0.03-0.1 mg/kg bw per day, resulting in
    concentrations of mercury of 2-13 µg/g in brain and 0.7-21 µg/ml in
    blood. The NOEL was 0.01 mg/kg bw per day.

         In corroboration of this finding, daily doses of 0.068-0.085
    mg/kg bw as methylmercury hydroxide given orally through a hypodermic
    syringe for 68 weeks caused clinical signs or cortical lesions in only
    3 of 15 macaque monkeys; the blood concentration of mercury was about
    2 µg/ml. A daily dose of 1.1 mg/kg bw, resulting in a maximum blood

    concentration of about 3 µg/g, led to death or a moribund condition.
    The vision of these three monkeys was also affected (Finocchio et al.,
    1980). Another study with macaque monkeys showed loss of peripheral
    vision at blood concentrations > 2.2 µg/ml (Luschei et al., 1977).
    These concentrations of mercury in blood correspond to 6-10 µg/g in
    brain.

         In squirrel monkeys, the visual cortex was invariably damaged,
    and extension to adjacent areas increased with duration of exposure
    and increasing brain mercury concentration. In one monkey who was
    blinded, the maximum concentration in the occipital lobe 40 days after
    termination of exposure was 6.6 µg/g (Berlin et al., 1975b), which,
    assuming a 47-day half-time in brain, corresponds to 12 µg/g at the
    end of treatment.

         In macaque monkeys exposed for a long time but with no clinical
    signs of poisoning, sensitivity to visual stimuli of low luminiscene
    was reduced when the concentration of mercury was 2.6 µg/ml in blood
    and 8.7 µg/g in the primary visual cortex. The concentrations were
    somewhat higher in the calcarine side of the primary visual cortex and
    in the lateral geniculate of the optic thalamus, where the optic
    neurons from the retina are projected onto the primary visual cortex,
    than in the lateral side of the occipital cortex. The borderline
    between effect and no effect was not sharp: unaffected monkeys had
    slightly higher concentrations of mercury in blood and in the
    occipital cortex than monkeys that were moderately affected, although
    they also were exposed for a shorter time. Constricted vision field,
    somaesthetic impairment, and ataxia usually occurred together (Evans
    et al., 1977),

         The lesions seen in the brains of rhesus monkeys given
    methylmercury hydroxide in fruit juice differed when it was given for
    6-17 days or for 6.3-12 months. Two of four monkeys exposed acutely
    and two of six exposed chronically had no clinical or histological
    signs of poisoning. The maximum concentrations of mercury in blood
    were < 1.1 µg/ml in unaffected monkeys and > 2.0 µg/ml in affected
    monkeys. The two acutely poisoned monkeys had a blood mercury
    concentration of 11 µg/ml. After acute poisoning, the most evident
    histological lesions were seen in the lateral geniculate nucleus and
    in large neurons in several areas. The cerebral and cerebellar
    cortices, including the calcarine and insular cortices, were not
    involved. Animals exposed chronically had damage to the cerebral
    cortex which was maximal around the calcarine and lateral cerebral
    sulci (Shaw et al., 1975).

         The entry of methylmercury into brain, even at concentrations
    below those that cause damage, precipitates an increase in the
    reactive glial population. A similar reaction was produced after
    infusion of inorganic mercury (Charleston et al., 1994).

    2.2.4.3  Domestic animals

         Few experiments have been reported on the toxicity of
    methylmercury in domestic animals, and even fewer that would allow an
    approximation of the threshold toxic dose. The oral dose that had no
    effect was 0.1-0.2 mg/kg bw per day for calves exposed for 91 days
    (Herigstad et al., 1972), 0.19-0.35 mg/kg bw per day for pigs exposed
    for 60 days (Triphonas & Nielsen, 1973), 0.06-0.12 mg/kg per day for
    dogs exposed for 55 days (Davies et al., 1977), and < 0.25 mg/kg bw
    per day for cats exposed for 90 days, whether present naturally in
    fish or added in pure form to the diet (Charbonneau et al., 1974). In
    the central nervous system, the damage was more extensive in the
    cerebellar granular layer in calves and cats and in the cerebral
    cortex in pigs and dogs.

         In rabbits given one to four doses of mercury at 5.8 mg/kg bw as
    methyl-mercury acetate, the most sensitive areas of the nervous system
    were the dorsal root and trigeminal ganglia, which showed degeneration
    after two doses. In the cerebral and cerebellar cortices, damage was
    seen two days after four daily doses. The more severely affected areas
    were the II, III, and IV layers of the cerebral cortex and the
    molecular and granular layers of the cerebellar cortex, where mainly
    the cells of the small neurones, including granule and basket cells,
    were damaged and the Purkinje cells spared (Jacobs et al., 1977), as
    in a human case of methylmercury poisoning (Hunter & Russel, 1954).

    2.2.5  Reproductive and developmental toxicity (other than
           neurotoxicity)

         Mice

         Treatment of male mice on seven consecutive days with
    methylmercury chloride at doses of 1, 2.5, or 5 mg/kg bw per day
    before mating with virgin females had no effect on fertility or
    postimplantation losses but marginally reduced the number of viable
    embryos (Khera, 1973). Intraperitoneal injection of male mice with 8.5
    mg/kg bw per day as methylmercury hydroxide and serial matings with
    young virgin females increased the number of dead implants during the
    first 7.4 days in one strain but not in another. The same treatment of
    females of the unresponsive strain slightly reduced the total number
    of live implants (Suter, 1975).

         Exposure to methylmercury chloride prolonged the length of the
    menstrual cycle by 11% in mice fed 3.2 mg/kg of diet and by 27% at 6.4
    mg/kg of diet. Exposure from 30 days before mating to day 18 of
    gestation decreased maternal weight gain at the high dose. The loss
    due to resorptions and deaths increased from 7.1% in the control group
    to 12% in mice at 3.2 mg/kg of diet and to 44% at 6.4 mg/kg of diet.

    The weight of fetuses on day 18 of gestation was also lowered. Both
    doses increased the frequency of malformations to 17% of fetuses at
    the low dose and 56% at the high dose (Nobunaga et al., 1979). A
    follow-up experiment confirmed that exposure to 3.2 mg/kg of mercury
    in the diet can cause postimplantation loss in some pregnant mice
    (Satoh & Suzuki, 1983). If their food consumption is assumed to be 150
    g/kg bw per day, the daily doses of methylmercury were 0.48 and 0.96
    mg/kg bw. When selenite was added to the drinking-water in these two
    studies, no effect was seen on postimplantation loss but the number of
    malformations was increased, at least at the high dose.

         Treatment of mouse dams on days 6-17 of gestation with 5 mg/kg bw
    per day as methylmercury chloride by intubation reduced the number of
    live pups, and pups born live died within two days. The number of live
    pups and survival were not affected by 1 mg/kg bw per day, but there
    was transitory inhibition of cerebellar cellular migration from the
    external granular layer (Khera & Tabacova, 1973). When methylmercury
    chloride was given orally on days 6-13 of gestation, the lowest dose
    of 2 mg/kg bw per day caused only a few malformations, 4 mg/kg bw per
    day decreased fetal weights and caused a large increase in the
    frequency of malformations, and 4.8 mg/kg bw per day also increased
    postimplantation loss (Fuyuta et al., 1978).

         Postimplantation loss was not observed when dams were given 3, 5,
    or 7 mg/kg bw per day as methylmercury chloride subcutaneously on days
    13-15 of gestation, but the postnatal survival rates were 30%, 22%,
    and 0, respectively (Nishikido et al., 1987). The outcome was similar
    when mouse dams were given 16 mg/kg bw per day as methylmercury
    chloride orally on one of days 13-17 of gestation. Postimplantation
    loss was slight or nil, but only 11% of the liveborn pups survived for
    eight weeks, apparently as a consequence of their inability to suck.
    The spontaneous locomotor activity of live pups was depressed between
    3 and 8 weeks, they had defects in righting and tail position, and, at
    the end of 8 weeks, they had smaller brains than controls (Inouye et
    al., 1985). Starvation, undernourishment, and the consequent general
    weakness were probable contributory factors.

         Rats

         Long-term intake of 2.5 mg/kg of diet as methylmercury chloride
    increased testicular but not ovarian weights in rats (Verschuuren et
    al., 1976c). Exposure of males and females had no effect on fertility,
    but the viability of their offspring was impaired (Verschuuren et al.,
    1976b). If their daily food consumption is assumed to be 100 g/kg bw,
    the daily dose was 0.25 mg/kg bw. Exposure of female rats to
    methylmercury chloride at 8 mg/kg of diet from weaning until delivery
    did not affect litter size, the frequency of stillbirths, birth
    weight, survival, or weight gain up to weaning (Nixon, 1977).

         The mating success of male rats declined by seven days after oral
    treatment with methylmercury chloride at 2.5 or 5 mg/kg bw per day but
    not at 1 mg/kg bw per day. The number of viable embryos per litter
    decreased transiently when 2.5 or 5 mg/kg bw per day was given for
    seven days, 1 mg/kg bw per day for 35 days, or 0.5 mg/kg bw per day
    for 90 days (Khera, 1973).

         Oral treatment of female rats with 0.25 mg/kg bw per day as
    methylmercury chloride from weaning had no apparent adverse effect on
    fetuses, and the only abnormality seen postnatally was eyelid lesions
    associated with hardening of the lachrymal glands. A dose of 0.05
    mg/kg bw per day had no effect (Khera & Tabacova, 1973).

         Higher doses were usually used when administration was restricted
    to the gestation period. In rats given 2, 4 or 6 mg/kg bw per day as
    methylmercury chloride orally on days 7-14 or 18-20 of gestation,
    resorptions, deaths, and malformations were observed at 6 mg/kg bw per
    day. Malformations consisting mostly of cleft palate and vertebral
    defects were seen in the offspring of dams at the two higher doses
    (Fuyuta et al., 1978). When a single oral dose of 8, 16, or 24 mg/kg
    bw as methylmercury chloride was given orally in saline on day 7 of
    gestation, maternal body weight declined at all doses. The decrease in
    the number of live fetuses on day 20 of gestation was 60% at 8 mg/kg
    bw and > 90% at 16 mg/kg bw. A dose-dependent decrease in
    ossification centres was seen. The concentrations of mercury in
    maternal brain were 2.6, 9, and 21 µg/g, and those in fetal brain were
    3.5, 11, and 15 µg/g (Lee & Han, 1995).

         Hamsters

         In hamsters, a single subcutaneous dose of 6.4 mg/kg bw as
    methylmercury chloride on day 3, 5, or 9 of gestation caused some
    maternal deaths, a high incidence of resorptions, decreased fetal
    weights, and moderate to severe malformations, consisting mainly of
    clubfoot and hydrocephalus. A dose of 1.6 mg/kg bw had no visible
    effect on dams or offspring, but when given on days 1-14 of gestation
    it increased the numbers of maternal deaths, resorptions, and
    malformations although it did not decrease fetal weights (Harris et
    al., 1972).

         Non-human primates

         In macaque monkeys, a daily oral dose of 50 or 70 µg/kg bw as
    methylmercury hydroxide in fruit juice for 20 weeks decreased sperm
    motility and increased the frequency of abnormal sperm tail forms,
    with no significant change in serum testosterone concentration or
    testicular morphology (Mohamed et al., 1987). In females given doses
    including 90 µg/kg bw per day, exposure did not affect the menstrual
    cycle, conception rate, or size of offspring at birth, but a maternal
    blood concentration > 1.5 µg/ml decreased the number of viable
    deliveries (Burbacher et al., 1988), and a concentration > 2 µg/ml
    was toxic to the dams (Burbacher et al., 1984, 1988).

    2.2.5  Developmental neurotoxicity

    2.2.5.1  Exposure in utero

         Mice

         Mouse dams were given methylmercury hydroxide subcutaneously as a
    single dose of 5.1, 6.8, or 10 mg/kg bw on day 10 of gestation. As the
    rate of mortality of the neonates of dams treated with 10 mg/kg was
    high, an additional group of dams were given 3.4 mg/kg bw on days
    10-12. Central latency in the open-field behaviour test was increased
    in the pups of dams given 10 mg/kg bw as a single or three divided
    doses. Locomotion was decreased by exposure to 6.8 and 10 mg/kg bw at
    postnatal day 24 but not at day 44 (Su & Okita, 1976). The righting
    reflex and walking ability lagged behind those of controls
    consistently in the pups of dams given 6.8 mg/kg bw subcutaneously on
    day 9, but the difference was not significant (Satoh et al., 1985b).

         Rats

         Methylmercury given at a dose of 3.2 mg/kg bw to pregnant rats on
    day 8 of gestation caused no significant change in the appearance of
    pups, but samples of calcarine and cerebellar cortices, especially in
    the granule cells of the cerebellum, showed focal weakening of the
    nuclear envelope, myelin figure formation, focal cytoplasmic
    degradation, and phagocytosis of cellular debris by macrophages (Chang
    et al., 1977). In pups of hamster dams given methylmercury chloride
    either as a single dose of 8 mg/kg bw on day 10 of gestation or 1.6
    mg/kg bw daily on days 10-15, the early cerebellar changes were seen
    in the external granular layer and in more extensively differentiated
    neural elements in the molecular and internal granular layer (Reuhl et
    al., 1981a). The sequelae of the early injuries, such as astrogliosis,
    may have had clinical or physiological significance when the pups
    reached the age of 275-300 days (Reuhl et al., 1981b).

         When methylmercury chloride was given by gavage to rat dams at
    doses of 0.02, 0.04, 0.4, or 4 mg/kg bw on days 6-9 of gestation, the
    highest dose impaired swimming behaviour at 4-35 days of age, and the
    doses of 0.04 and 4 mg/kg bw increased passiveness and decreased
    habituation to an auditory startle 60-210 days postnatally. The
    histological changes seen at the highest dose were mainly in the
    dendritic spines of the pyramidal neurones (Stoltenberg-Didinger &
    Markwort, 1990). Methylmercury chloride given in apple juice on the
    same days of gestation at a dose of 1.6 mg/kg bw per day caused no
    change in the clinical markers of adverse effects up to weaning. No
    deficits in behavioural function, such as spatial learning in a
    circular bath and maze learning for food reward, were seen at four to
    five months of age (Frederiksson et al., 1996).

         The effect of methylmercury on locomotion is ambiguous. When
    given orally to rat dams on day 8 or 15 of gestation at a dose of 4 or
    6.4 mg/kg bw, no consistent changes in spontaneous locomotor activity
    were seen 4, 8, or 15 days postnatally. Activity was increased on
    postnatal day 4 when the low dose was given on day 8 of gestation, on
    postnatal day 8 when the low dose was given on day 15 or the high dose
    on day 8 of gestation, and on postnatal day 15 when the low dose was
    given on day 15 of gestation (Eccles & Annau, 1982a). The higher dose
    given on day 15 did not affect locomotor activity on postnatal day 14,
    21, or 60 (Cagiano et al., 1990), but 8 mg/kg bw given on day 4 of
    gestation depressed locomotor activity at 110-140 days of age.
    Avoidance learning was also depressed (Schalock et al., 1980).

         The effect of 4 or 6.4 mg/kg bw as methylmercury chloride given
    to rats on day 8 or 15 of gestation on two-way avoidance was tested at
    nine weeks of age. Exposure increased the number of trials required to
    reach the preset criterion; however, owing to variations within
    groups, only the higher dose given on day 8 induced a significant
    difference in reacquisition and both doses given on day 15 for
    acquisition (Eccles & Annau, 1982b). The higher dose given on day 15
    decreased the number of cortical muscarinic receptors by 53% in
    14-day-old pups and by 21% in 21-day-old pups. Recovery from this
    defect was complete at the age of 60 days, but the results of a
    passive avoidance test even a few days before that age indicated
    learning and memory deficits (Zanoli et al., 1994).

         Non-human primates

         Female macaque monkeys were given methyl-mercury in apple juice
    at 0.04 or 0.06 mg/kg bw per day for 198-747 days before mating.
    Infants were separated from their mothers at birth and were tested 210
    and 220 days after conception (50-60 days after birth). The test
    indicated randomness in visual attention to novel stimuli. The mean
    maternal blood concentrations at birth were 0.84 and 1.04 µg/ml, and
    the blood concentrations of the offspring were 0.88 and 1.7 µg/ml
    (Gunderson et al., 1988). The offspring of squirrel monkeys exposed to
    methylmercury during the second half or the last third of pregnancy,
    with maternal blood concentrations of 0.7-0.9 mg/ml, showed reduced
    sensitivity to changes in the source of reinforcement, indicating
    learning impairment at five to six years of age (Newland et al.,
    1994).

    2.2.5.2  Exposure in utero and postnatally

         Mice

         Mouse dams were exposed to 3.2 mg/L as methylmercury in
    drinking-water from mating to parturition, and their pups were further
    exposed up to postnatal day 30 during lactation. The litter weights of

    exposed and control pups were similar. Males but not females showed
    some decrease in the width of the external granular layer in a region
    of the inferior lobe of the cerebellum on postnatal day 7, but not
    later. The density of migrating cells in the molecular layer was also
    decreased (Markowski et al., 1998).

         Rats

         The concentration of mercury was 10 µg/ml in blood and 1.4 µg/g
    in the brains of rat pups exposed throughout gestation and lactation
    via their dams and directly to the same concentration of mercury as
    methyl-mercury chloride at 3.9 mg/kg of diet. No adverse effect was
    seen clinically or histologically in the brain, even on morphological
    maturation of neurons and astrocytes. The only deviation from control
    values was an increase in noradrenaline activity in the cerebellum
    (Lindström et al., 1991).

         In a crossfostering study, rat dams received an approximate daily
    dose of 2.5 mg/kg bw of methylmercury chloride in drinking-water, and
    an additional group of pups was exposed only through drinking-water on
    postnatal days 21-30. Only pups exposed prenatally or directly after
    weaning had deficits in learning ability at day 30, and the effect
    lasted at least until postnatal day 50 (Zenick, 1974). At day 30, the
    action potential in the visual cortex was decreased in each group
    (Zenick, 1976). Offspring of rat dams exposed to 1.2 or 4 mg/L as
    methylmercury in drinking-water from two weeks before mating to
    weaning showed a dose-dependent deficit in correct response when the
    feedback was tactile kinetic perception. The methylmercury intake of
    the dams was 0.12 or 0.25-51 mg/kg bw per day before and during
    gestation and 0.22-0.38 or 0.53-0.95 mg/kg bw per day during lactation
    (Elsner, 1991).

         Non-human primates

         Macaque monkey dams were given methylmercury chloride orally
    three times a week at doses of 10, 25, or 50 µg/kg bw and were mated
    when their blood concentration of mercury approached 90% of the
    steady-state level. Treatment was continued during gestation. Infants
    were separated from their mothers immediately after birth and given
    the same dose of methylmercury until they were four years of age. The
    blood mercury concentration of the infants at birth was 0.45-2.7
    µg/ml, and their steady-state concentration reached 0.22-0.78 µg/ml.
    Two of the five animals were severely intoxicated and could not be
    tested for spatial or temporal visual function. Some inconsistent
    defects in spatial vision at high and low luminescence and in
    temporal vision at high luminiscence were seen. Temporal vision at low
    luminescence was better in exposed than in control monkeys (Rice &
    Gilbert, 1990). The auditory response was tested in the same monkeys
    when they were 11 and 19 years of age, 7-15 years after the end of
    exposure. The deterioration in hearing between 11 and 19 years was
    more pronounced in exposed than in control monkeys. In 19-year-old
    monkeys, the thresholds for all frequencies were higher in exposed
    than in control monkeys (Rice, 1998).

    2.2.5.3  Exposure after parturition

         Mice

         A single oral dose of 8 mg/kg bw as methylmercury chloride to
    two-day-old mice resulted in a brain mercury concentration of 2.7
    µg/g, reductions in the number of cells and the percentage of late
    mitotic figures, and an increase in cells with reduced nuclear
    diameter (Sager et al., 1982). A smaller reduction in cell numbers in
    the granular layer of the cerebellum was seen after 4 mg/kg bw, but
    the number and proportion of late mitotic figures (anaphase) remained
    significantly lower than in controls at day 19  post partum (Sager et
    al., 1982).

         Rats

         Two-day-old rats were given methylmercury chloride at a dose of 5
    mg/kg bw per day subcutaneously until loss of body weight and signs of
    neurological impairment, including impaired vision (weak visual
    placing response), became evident. At this point, the animals were
    necropsied, and their visual cortices were studied. Degenerating
    neurons were concentrated mainly in layer IV and were scattered in
    layers II, III, V, and VI (O'Kusky, 1985). The same treatment led to
    the spastic dyskinetic syndrome and to decreases in the specific
    activity of glutamic acid decarboxylase (which synthesizes
    gamma-aminobutyric acid) in the occipital cortex (O'Kusky et al.,
    1988a) and in the tissue concentrations of serotonin and dopamine and
    their metabolites (O'Kusky et al., 1988b). As the dose used in these
    studies was high, pain and local irritation could have been
    confounding factors at least on the concentrations of catecholamines
    and serotonin  In addition, renal function was certainly affected, as
    even 0.85 mg/kg bw given as methylmercury hydroxide subcutane-ously
    daily from the day of birth impaired the kidneys (Slotkin et al.,
    1985).

         Non-human primates

         Four infant monkeys were given methylmercury chloride at 0.5
    mg/kg bw per day orally for 28-29 days from day 1 after birth. They
    progressively became ataxic, blind, and comatose and were necropsied
    at 35-43 days. Histopathological changes were marked in the cerebrum
    and less severe in the cerebellum, where the Purkinje and granular
    cells appeared normal (Willes et al., 1978). In an experiment in which
    much lower doses were used, the blood mercury concentration of five
    newborn macaque monkeys given 0.05 mg/kg bw per day orally in fruit
    juice peaked at 1.2-1.4 µg/ml and declined after weaning to 0.6-0.9
    µg/ml. At three to four years of age, they had impaired spatial vision
    at both high and low luminescence (Rice & Gilbert, 1982). Continuation
    of exposure to the same daily doses until seven years of age resulted
    in an increased auditory threshold in four of five monkeys, at high

    frequency (25 000 Hz) in one monkey and at middle frequencies in three
    monkeys (Rice & Gilbert, 1992). When these monkeys reached the age of
    13 years, some appeared to be clumsy and hesitant in large exercise
    cages. Exposed monkeys required more time to collect 10 objects from a
    recessed square and had a higher vibration threshold, but their motor
    response time was normal (Rice, 1996).

    2.2.7  Carcinogenicity

         In mice exposed for two years to methylmercury at 0.4, 2, or 10
    mg/kg of diet, renal tumours developed only in males at 10 mg/kg of
    diet. The incidence of renal adenoma was 8% and that of carcinoma was
    22% in B6C3F1 mice, the corresponding figures being 5% and 17% in ICR
    mice. Nearly all male and female B6C3F1 mice at 10 mg/kg of diet had
    chronic nephropathy, while only 78% male and 43% female ICR mice had
    this pathological change (Mitsumori et al., 1990). The chronic insult
    to the kidney may have contributed to the induction of cancer. In
    groups of male and female rats fed diets containing methylmercury at
    0.1, 0.5, or 2.5 mg/kg of diet for two years, the incidence of
    pathological lesions and tumours was not increased (Verschuuren et
    al., 1975c).

    2.2.8  Immunomodulation

         Exposure of mice to methylmercury in the diet at 3.2 mg/kg did
    not affect body weight or kidney, liver, or spleen weight, but the
    weight of the thymus and the number of thymocytes decreased by 22 and
    50%, respectively. The lymphoproliferative response to T-and B-cell
    mitogens was increased in both thymus and spleen, and natural killer
    cell activity was decreased by 44% in spleen and by 75% in blood
    (Ilbäck, 1991). In a cross-fostering study, when rat dams were exposed
    to mercury as methylmercury at 3.2 mg/kg of diet 11 weeks before
    mating, no consistent alterations were seen in the body weight,
    lymphoid organ weights, or cell number and lymphoproliferative
    response to Bcell mitogens of pups at the age of 15 days. The response
    of thymocytes to T-cell mitogens increased by 30-48% and that of
    splenocytes decreased by 12% only in pups exposed throughout
    lactation; natural killer cell activity decreased by 42% after both
    doses (Ilbäck et al., 1991). Mouse dams and pups exposed to
    methylmercury at a concentration of 0.5 or 5 mg/kg of diet from 10
    weeks before mating until weaning showed inconsistent effects on the
    immune system (Thuvander et al., 1996).

    2.2.9  Extrapolation between species

         The doses used in experiments in rats and mice frequently
    exceeded 1 mg/kg bw per day, which is within the range of the intake
    of the Iraqi patients with severe methylmercury poisoning (Bakir et
    al., 1973) and 140-330 times higher than the daily intake that would

    cause paraesthesia in 5% of a population (WHO, 1976). Thus,
    extrapolation of doses on the basis of body weight clearly results in
    nonsense values. Table 3 shows that the clearance half-times of
    methylmercury increase with mass, in the order mouse < rat < macaque
    monkey < human, and that the concentrations in the whole body after a
    unit dose follow the same order. The data in the Table are based on
    the assumption that clearance from blood approximates clearance from
    the whole body, and the clearance half-times are given as the daily
    clearance in percent of the whole-body burden. The units for dose and
    concentration are identical. As the half-time increases with body
    mass, the concentration in the body after identical doses of
    methylmercury also increases with body mass. Thus, allometric
    extrapolation based on surface area can be used. The method of
    correction, for example from the dose for mouse to the human dose, is:

                   human dose = mouse dose (0.3:70)0.3

    With this allometric extrapolation, the dose in mice multiplied by
    0.098 gives the equivalent human dose, while the multiplication factor
    for the dose in rats is 0.20 and that for macaques is 0.42. The effect
    of this extrapolation on steady-state concentrations is shown in Table
    4.

    2.3  Observations in humans

         Investigations of the possible neurodevelopmental effects of
    prenatal exposure to methylmercury followed a sequence similar to
    those of other neurotoxic exposures: case series of children who
    manifested clinical signs of poisoning and then prospective cohort
    studies of asymptomatic children considered to have 'low' exposure or
    at least exposure lower than that at which clinical signs and symptoms
    appear. The populations chosen were mostly those known to consume
    large amounts of fish, which contain variable amounts of methylmercury
    (see section 3). The goal of the latter studies is largely to
    determine whether a dose-response relationship can be identified for
    adverse neurodevelopmental effects associated with exposure to
    methylmercury, in order to assess the significance to public health of
    exposure in various populations.


        Table 3. Clearance half-times and whole-body concentrations of methylmercury
                                                                                              

    Species     Body      Half-time   Clearance     Ratio of concentration:daily dosea
                weight    (days)      (% body                                                 
                (kg)                  burden)       10 days after   After 10      At steady
                                                    single dose     doses         state
                                                                                  (long-term
                                                                                  exposure)
                                                                                              

    Human       70        52          0.014         0.87            9.4           75
    Macaque     4         25          0.028         0.76            8.7           36
    Rat         0.35      12          0.058         0.56            7.6           17
    Mouse       0.03       7          0.099         0.37            6.3           10
                                                                                              

    a Same units as daily dose
    

    2.3.1  Case series

         The mass poisoning of persons living near Minamata Bay in Japan
    in the 1950s first raised awareness of the severe neurological
    sequelae associated with methylmercury poisoning, particularly when
    experienced prenatally. The primary route of exposure in this episode
    was the consumption of fish contaminated by methylmercury, which
    bioaccumulated up the aquatic food chain. According to Harada (1995),
    all children identified as suffering from the most severe form of
    congenital Minamata disease showed mental retardation, primitive
    reflexes, cerebellar ataxia, disturbances in physical growth,
    dysarthria, and limb deformities, and most showed hyperkinesis (95%),
    hypersalivation (95%), seizures (82%), strabismus (77%), and pyramidal
    signs (75%). The incidence of cerebral palsy among children with the
    disease was also increased, involving 9% of 188 births in three
    villages. Some of the signs and symptoms, such as paroxysmal events,
    hypersalivation, primitive reflexes, and ataxia, abated somewhat in
    subsequent years, although others such as reduced intelligence and
    dysarthria did not. Most patients with the severe form of the disease
    were unable to function successfully in society. The mothers of many
    affected children experienced only transient paresthesia, indicating
    that fetal vulnerability exceeds that of mature individuals. Although
    measurements of the body burden of mercury were not available until
    several years after the episode, analyses of the mercury
    concentrations in archived umbilical cord tissue from patients with
    congenital Minamata disease suggest that the mean concentration in
    maternal hair may have been approximately 41 µg/g (25-75th percentile:
    20-59) (Akagi et al., 1998). The uncertainty associated with this
    estimate is likely to be substantial, however, as case ascertainment
    was undoubtedly incomplete, particularly among individuals who
    suffered milder forms of the disease. For example, even if cases of
    known disease are excluded, the prevalence of mental retardation among
    children born between 1955 and 1958 in the contaminated area was 29%,
    which is far higher than would have been expected and suggests that
    congenital Minamata disease was not diagnosed in many children with
    less severe forms. Thus, these data cannot provide precise estimates
    of the minimum concentration of methylmercury required to produce this
    disease.

         A second episode of mass methylmercury poisoning occurred in Iraq
    in the early 1970s, when seed grain treated with a fungicide
    containing this compound was ground into flour and consumed and
    resulted in 600 deaths and 6000 cases of methylmercury poisoning.
    Thus, the exposure was probably more acute and involved higher doses
    than those experienced by the persons living around Minamata Bay. The
    results of early studies of the most severely affected children who
    were exposed during fetal development were concordant with those in
    Minamata: the children manifested severe sensory impairment
    (blindness, deafness), general paralysis, hyperactive reflexes,
    cerebral palsy, and impaired mental development (Amin-Zaki et al.,
    1974). Several follow-up studies of the exposed population were
    conducted. Marsh et al. (1987) identified 81 children who were  in
     utero at the time of the episode and collected information on their
    neurodevelopmental outcomes from two sources: neurological examination

    of each child and an interview with the mother about the age at which
    the child achieved standard developmental milestones such as walking
    and talking. The maximum concentrations of mercury in maternal hair
    during the pregnancy, which were used as the index of fetal exposure,
    ranged from 1 to 674 µg/g. Developmental retardation was defined as a
    child's failure to walk a few steps unaided by 18 months of age or to
    say two or three meaningful words by 24 months of age. A point system
    was devised for the neurological examination, a score > 3 indicating
    a definite abnormality. The prevalences of these indicators of poor
    outcome were related to the concentrations of mercury in maternal
    hair. The most frequent neurological findings were increased limb tone
    and deep-tendon reflexes with persistent extensor plantar responses;
    ataxia, hypotonia, and athetoid movements were also reported. Boys
    appeared to be more severely affected than girls. Seven of the 28
    children with the highest exposure and none of the 53 children with
    lower exposure had had seizures.

         Additional analyses of this data set were performed to identify
    more precisely the shape of the dose-response relationship and, in
    particular, the threshold for adverse neurodevelopmental effects, if
    indeed one exists. Cox et al. (1989) obtained more accurate estimates
    of peak exposure during pregnancy by applying an X-ray fluorescent
    method to single strands of maternal hair. Using logit, hockey-stick,
    and non-parametric kernel smoothing methods, they estimated a
    population threshold of around 10 µg/g for the outcomes investigated.
    The uncertainty associated with this estimate is heavily dependent,
    however, on the estimated background prevalence of the poor outcomes.
    For example, the upper bound of the 95% confidence interval for motor
    retardation increases from 14 to 190 µg/g if the estimate of
    background prevalence is changed from 0 to 4%. For neurological
    abnormality, the upper bound of the 95% confidence interval for the
    threshold estimate was 287 µg/g when a 9% background prevalence was
    assumede. In later re-analyses of these data, Crump et al. (1995) and
    Cox et al. (1995) demonstrated that the estimate of threshold depends
    on the model used and is sensitive to the definition of abnormality.
    In the case of delayed walking, the estimate was influenced by the
    only four cases of delayed walking among the children of women whose
    hair concentration of mercury was < 150 µg/g. The statistical
    variability of the estimates of threshold appears likely to be
    considerably greater than that of Cox et al. (1989). Crump et al.
    (1995) concluded that the data from the Iraqi episode do not provide
    convincing evidence of any adverse neurodevelopmental effect of
    methylmercury at concentrations in maternal hair < 80 µg/g.

         In evaluating these data, it is important to note that the
    interviews were conducted when the mean age of the children was 30
    months, but some of the children must have been considerably older at
    this time, as the age at which children in the sample were reported to
    have walked or talked was as much as 72 months. In addition, the birth
    dates were generally not accorded significance, and maternal
    recollection of the ages at which their children achieved milestones
    were based on external events such as religious holidays. The extent
    of the imprecision of these data is suggested by the strong digit


        Table 4. Steady-state concentrations of methylmercury in humans after allometric 
    extrapolation of unit doses from three experimental species and comparison of
    human and animal steady-state concentration ratios
                                                                                   

    Animal     Concentration   Human concentration    Ratio at steady state
                               after equal doses      (based on mass)
                               (based on surface)
                                                                                   

    Macaque    28              0.09                   2.1
    Rat        12              0.9                    4.4
    Mouse       6              1.0                    7.5
                                                                                   

    Steady-state concentrations in animals are shown in the last column of Table 3.
    
    preferences in the mothers' responses. For instance, an even number of
    months was given for the estimated age at walking for 70 of the 78
    children and for the estimated age at talking for 70 of 73 children;
    75% of the estimates were multiples of six months. Finally, the extent
    of selection bias in this cohort cannot be characterized because the
    size of the base population from which it was drawn and the referral
    mechanism that brought mothers and children to medical attention are
    both unknown. For instance, women who knew that they had consumed
    large amounts of contaminated grain and were concerned about their
    children's welfare may have come forward, while women who consumed
    equally large amounts of contaminated grain but whose children were
    developing well may not. This issue is critical, because calculation
    of a threshold requires a denominator (the size of the exposed
    population) and the background prevalence of the adverse outcome in
    order to estimate the 'added risk' associated with the exposure of
    interest. In this regard, the background prevalence of developmental
    abnormality appears to have been extremely high among the Iraqi
    children who participated in the follow-up studies. The prevalence of
    delayed walking among children whose mothers had concentrations of
    mercury in hair < 10 µg/g, who can be viewed essentially as a control
    group for estimating background prevalence, was 36% (11/31). In
    contrast, in the population of children in the United States on whom
    the Bayley scales of infant development were standardized (Bayley,
    1969), the prevalence of delayed walking by this criterion was
    approximately 5%. Similarly, the prevalence of delayed talking among
    the Iraqi children was 22% (6/27), whereas 95% of 24-month-old
    children in the standardization sample of the MacArthur communicative
    development inventory were saying 50 words or more (Fenson et al.,
    1993).

    2.3.2  Childhood development

    2.3.2.1  Neurological status

         McKeown-Eyssen et al. (1983) studied 234 Cree children aged 12-30
    months living in four communities in northern Quebec, Canada, whose
    prenatal exposure to methylmercury was estimated on the basis of
    maternal hair samples. Hair samples were collected from 28% of the
    mothers during pregnancy, but the prenatal exposure of the rest of the
    cohort was estimated from hair segments assumed to date from the
    period of the pregnancy. The exposure index was the maximum
    concentration of mercury in the segment of hair corresponding most
    closely to the period from one month before conception to one month
    after delivery. The mean concentration of mercury in maternal hair was
    approximately 6 µg/g; it exceeded 20 µg/g in 6% of samples. One of
    four paediatric neurologists who were unaware of the child's status of
    exposure measured height, weight, and head circumference, identified
    dysmorphology, and conducted a neurological examination, assessing
    coordination, cranial nerves, and muscle tone and reflexes. The
    neurologist then made a summary clinical judgement about the presence
    of a neurological abnormality. None of the children was judged to have

    an abnormal physical finding, but 3.5% of the boys ( n = 4) and 4.1%
    of the girls ( n = 5) were considered to have a neurological
    abnormality. The most frequent abnormality involved tendon reflexes,
    which was seen in 11% of boys ( n = 13) and 12% of girls ( n = 14).
    The only neurological finding that was significantly associated with
    prenatal exposure to methylmercury, either before or after adjustment
    for confounding, was abnormal muscle tone ( n = 2; increased tone in
    legs only) or reflexes ( n = 13; five with isolated decreased
    reflexes, six with generalized decreases, and two with generalized
    increases) in boys ( p = 0.05). The risk for abnormal tone or
    reflexes increased seven times with each 10-µg/g increase in prenatal
    exposure to methylmercury (95% confidence interval, 1-51). After log
    transformation of prenatal exposure, however, the p value for this
    association increased to 0.14. When exposure was categorized, the
    prevalence of tone or reflex abnormality did not increase in a clear
    dose-related manner across the categories. In girls, the only
    association identified was an unexpected inverse relationship between
    prenatal exposure to methylmercury and incoordination, with a 60%
    decrease in the probability of incoordination for each 10-µg/g
    increment (odds ratio, 0.3; 95% confidence interval, 0.1-0.9;
    p = 0.02). The authors noted several caveats with regard to the one
    significant adverse association identified: the abnormalities of
    muscle tone and reflexes in boys were isolated, mild, and of doubtful
    clinical importance; the finding is not consistent with previous
    results which suggest that increased exposure is expressed as severe
    generalized neurological disease, including increases in tone and
    reflexes; there was no coherent dose-response relationship; and there
    was no consistency between the sexes. The finding may reflect chance,
    lack of normality in the distribution of the exposure index, or
    residual confounding.

         A study was conducted in Mancora, a fishing community on the
    northern coast of Peru, in which hair samples and clinical data were
    obtained for 131 infant-mother pairs. The mean concentration of
    mercury in maternal hair was 7 µg/g (range, 0.9-28), with an average
    peak of 8.3 µg/g. The small difference between the mean and the peak
    was probably due to the stability of the fish consumption of the
    mothers. The major outcomes measured were anthropmorphic end-points
    (birth weight, head circumference, and height), maternal reports of
    infant development (age at which the infant sat, stood, walked, and
    talked), and neurological status (Marsh et al., 1995). The specific
    elements of the neurological assessment conducted and the age at which
    the infants were examined were not described. Tone was decreased in
    two children, limb weakness was seen in one child, reflexes were
    decreased in one child and increased in four, and an abnormal Babinski
    reflex was seen in one child; increased tone, primitive reflexes, and
    ataxia were not observed. None of the signs was significantly
    associated with either the mean or the peak concentration of mercury
    in maternal hair.

         In the pilot phase of a cross-sectional cohort study of child
    development in the Seychelles, 789 infants aged 5-109 weeks were
    evaluated by one paediatric neurologist who was unaware of their
    exposure status (Myers et al., 1995a). The mean concentration of
    mercury in maternal hair was 6.1 µg/g (range, 0.6-36 µg/g). The
    features assessed included mental status, attention, social
    interactions, vocalization, behaviour, coordination, posture and
    movements, cranial nerves II-XII, muscle strength and tone, primitive
    and deep-tendon reflexes, plantar responses, and age-appropriate
    abilities such as rolling, sitting, pulling to stand, walking, and
    running. The statistical analyses focused on three end-points,
    selected because of their apparent sensitivity to prenatal exposure to
    methylmercury in the studies in Iraq and the Cree population: overall
    neurological status, increased muscle tone, and deep-tendon reflexes
    in the extremities. The result of the overall examination was
    considered to be 'abnormal' if any findings judged to be pathological
    were present, including abnormalities of cranial nerves (pupils,
    extraocular muscles, facial or tongue movement, swallowing, or
    hearing), increase or decrease in muscle tone or deep-tendon reflexes,
    incoordination, involuntary movements, or poorly developed speech or
    functional abilities. Findings that were considered to be neither
    normal nor pathological were categorized as 'questionable'. Because
    the frequency of abnormal findings was low (2.8%), the questionable
    (11%) and abnormal categories were combined. No association was found
    between the concentration of mercury in maternal hair and questionable
    or abnormal results, the frequency ranging from 16% for hair
    concentrations of 0-3 µg/g to 12% for hair concentrations > 12 µg/g.
    The frequency of abnormalities of limb tone or deep-tendon reflexes
    was about 8% and did not vary in a dose-dependent manner with the
    concentration of mercury in maternal hair.

         In the main study, which involved a cohort of 735 children, one
    paediatric neurologist who was unaware of the exposure status of the
    children conducted essentially the same neurological examination that
    had been used in the pilot study but when the participants were 6.5
    months old (Myers et al., 1995b). The results of the overall
    examination were considered to be abnormal or questionable if changes
    in muscle tone, deep-tendon reflexes, or other neurological features
    were pathological or the examiner considered that a child's functional
    abilities were not appropriate for his or her age. Abnormal or
    questionable neurological scores were found for 3.4% ( n = 25) of the
    children, a frequency too low to permit statistical analysis. For both
    limb tone and deep-tendon reflexes, the frequency of abnormalities was
    2%; questionable limb tone was found in approximately 20% of the
    children and questionable deep-tendon reflexes in approximately 15%.
    For neither limb tone nor deep-tendon reflexes was the frequency of
    abnormal or questionable findings significantly associated with the
    concentration of mercury in maternal hair.

         In a study carried out in the Faroe Islands (Denmark), a
    functional neurological examination was administered to children at
    the age of 7 years as part of a general physical examination. The
    examination focused in particular on motor coordination and

    perceptual-motor performance (Dahl et al., 1996). The tests for
    coordination included rapid pronation or supination, reciprocal
    coordination (alternately closing and opening the fists), and finger
    opposition (touching the pulpa of the thumb with the pulpa of the
    other fingers of the same hand). The perceptual-motor tests included
    catching a 15-cm ball thrown from a distance of 4 m, finger agnosia,
    and double finger agnosia. The results were scored as automatic or as
    questionable or poor. Exposure to mercury was evaluated on the basis
    of the concentration in maternal hair at delivery, in umbilical cord
    blood, and in children's hair obtained at about 12 months of age
    Exposure to mercury was not significantly associated with the number
    of tests on which a child's performance was considered to be
    'automatic', as < 60% of the children achieved such a score on the
    tests for reciprocal motor coordination, simultaneous finger movement,
    and finger opposition; however, children with questionable or poor
    performance for finger opposition had had significantly higher mean
    exposure to mercury than children with automatic performance (24
    versus 22 µg/L;  p = 0.04) (Grandjean et al., 1997).

    2.3.2.2  Developmental milestones

         The association between the achievement of developmental
    milestones and prenatal exposure to methylmercury was evaluated in the
    main cohort of the study in the Seychelles (Myers et al., 1997; Axtell
    et al., 1998). Information on the ages at which a child was able to
    walk without support and to say words other than 'mama' or 'dada' was
    elicited by means of an interview with the person with whom the child
    spent five or more nights per week (the 'caregiver'), conducted at an
    evaluation at 19 months. Such information was available for 738 of the
    779 children. The statistical approaches explored included standard
    multiple regression in which age at achievement of a milestone was
    log-transformed, hockey-stick models to estimate the threshold
    concentration of mercury in maternal hair associated with delay in
    achieving a milestone, and logistic regression analyses of 'delayed
    walking', a binary variable in which an abnormal response was defined
    as > 14 months. Prenatal exposure to methylmercury was estimated
    from the total mercury in the single longest segment of hair dating
    from the index pregnancy; the mean concentration was 5.8 µg/g (range,
    0.5-27), and 22% of the children had been exposed to > 10 µg/g. The
    mean age at which a child was considered to talk was not significantly
    associated with the concentration of mercury in maternal hair in any
    of the models tested. In regression analyses stratified by sex, a
    positive association was found between age at walking and exposure to
    mercury for boys ( p = 0.043) but not for girls. The interaction term
    'mercury × sex' in analyses of the complete cohort was not
    statistically significant. The magnitude of the delay in the age at
    which boys walked--a 10-µg/gincrease in the concentration of mercury
    in maternal hair associated with an approximately two-week delay in
    walking--was viewed by the authors as clinically insignificant, and
    the association was not significant when four statistical outliers
    were excluded from the analysis. Hockey-stick models provided no

    evidence of a threshold, as the fitted curves were essentially flat. A
    child's risk for 'delayed walking' was not associated with the
    concentration of mercury in maternal hair.

         In a re-analysis of these data, Axtell et al. (1998) used
    semiparametric generalized additive models with smoothing techniques
    to identify lack of linearity. These models are less restrictive than
    those used by Myers et al., which require strong assumptions about the
    true functional form of a relationship. The major finding of Axtell et
    al. was that the association between age at walking and the
    concentration of mercury in maternal hair in boys was not linear,
    walking being achieved at a later age as exposure increased from 0 to
    7 µg/g but at a slightly earlier age as the concentration increased
    beyond 7 µg/g. The size of the effect associated with the increase
    from 0 to 7 µg/g was small, corresponding to a delay of less than one
    day in the achievement of walking. Because no clear dose-response
    relationship was seen at concen-trations > 7 µg/g, the authors
    considered that the association found at lower concentrations did not
    reflect a causal effect of mercury on the age at walking.

         Data on developmental milestones were also collected in the study
    in Peru (Marsh et al., 1995). The ages of the children at the time the
    mothers were questioned about these events was not stated, although
    the study was conducted prospectively and data were apparently
    collected throughout the women's visits to postnatal clinics.
    Regression analyses, including analyses stratified by sex, showed no
    significant association between the concentration of mercury in
    maternal hair and the ages at which the children sat, stood, walked,
    or talked. The rates of developmental retardation were substantial,
    especially for speech (13/131), although the criteria used to define
    this outcome were not stated. The children's birthweight, height, and
    head circumference were also unrelated to the concentration of mercury
    in maternal hair.

         The ages at which children achieved motor milestones were
    investigated in a birth cohort of 1022 children born during a 21-month
    period in 1986-87 in the Faroe Islands (Grandjean et al., 1995a). The
    data were obtained from interviews with the mothers and from the
    observations of district health nurses who had visited the homes of
    the children on several occasions during their first year of life.
    Complete data were available for 583 children (57% of the cohort).
    Three motor milestones commonly achieved between 5 and 12 months of
    age were selected for analysis: sitting without support, crawling, and
    standing with support. The age at achievement of the three milestones
    was not significantly associated with the concentration of mercury in
    cord blood or maternal hair, but a significant negative association
    was found between the age at achievement of all three milestones and
    the concentration of mercury in children's hair at 12 months. The
    authors concluded that this association reflected residual confounding
    by duration of breast-feeding, since nursing was associated with both
    higher hair mercury concentrations in children at 12 months of age and

    more rapid achievement of milestones. This finding suggests that the
    beneficial effects of nursing on early motor development are
    sufficient to compensate for any slight adverse impact that prenatal
    exposure to low concentrations of methylmercury may have on these
    end-points.

    2.3.2.3  Early development

         In the study of Cree people reported by McKeown-Eyssen et al.
    (1983), the Denver developmental screening test (Frankenburg et al.,
    1981)was administered to all children aged 12-30 months. The scores
    were reported as the percentage of items passed on each subscale
    (gross motor, fine motor, language, personal and social) and on the
    entire test. Although quantitative estimates of the associations
    between test scores and the concentration of mercury in maternal hair
    (mean, 6 µg/g; 6% > 20 µg/g) were not provided, the authors reported
    that they found no significant association compatible with an adverse
    effect of methylmercury, before or after adjustment for confounding
    variables.

         Kjellström et al. (1986, 1989) studied a cohort of children in
    New Zealand whose prenatal exposure to methylmercury was estimated on
    the basis of maternal hair samples and dietary questionnaires
    collected during the pregnancy. Although nearly 11 000 women
    participated in the initial phase during which information on exposure
    was obtained, Kjellström et al. focused on 935 women who had reported
    eating fish more than three times per week during the pregnancy. The
    74 children of 73 women whose concentration of mercury in hair was
    > 6 µg/g were considered to have had heavy exposure to mercury. Three
    controls were matched to each of these children on the basis of ethnic
    group, sex, maternal age, maternal smoking, area of maternal
    residence, and the duration of maternal residence in New Zealand. The
    concentration of mercury in maternal hair was 3-6 µg/g for one of each
    of the controls, whose mother ate fish more than three times per week,
    and 0-3 µg/g for the other two, whose mothers had a lower consumption
    of fish. There were 57 fully matched sets of four children and four
    incomplete sets, for a cohort of 237 children. Evaluations conducted
    when the children were four years of age indicated that about 50% of
    children with heavy exposure to mercury and 17% of the children in the
    control group had an abnormal or questionable result on the Denver
    developmental screening test.

         In the pilot phase of the Seychelles study, a revised version of
    the Denver test was administered to 789 children aged 1-25 months by
    one examiner who was unaware of their exposure status (Myers et al.,
    1995a). No association was found between the concentration of mercury
    in maternal hair during pregnancy (mean, 6.6 µg/g) and the results on
    the test when normal and questionable results were combined in the
    conventional manner, although the prevalence of abnormal findings was
    so low (three children, < 1%) that statistical analysis was not
    meaningful. When abnormal and questionable (n = 65; 8%) results were
    grouped, as was done in the study in New Zealand (Kjellström et al.,

    1986), a higher concentration of mercury in maternal hair was
    significantly (p = 0.04; one-tailed test) associated with a poor
    outcome. This result was largely attributable to the higher frequency
    of abnormal or questionable results (13%) among children with the
    heaviest exposure to mercury (> 12 µg/g), in contrast to the
    frequency of approximately 7% among children in each of the other four
    groups (0-3, 3-6, 6-9, and 9-12 µg/g).

         In the main study in the Seychelles, the revised Denver
    developmental screening test was administered to a cohort of 740
    children (mean concentration of mercury in maternal hair during
    pregnancy, 5.9 µg/g; interquartile range, 6 µg/g) aged 6.5 months by
    one examiner who was unaware of their exposure status (Myers et al.,
    1995b). The frequency of results considered to be abnormal (three
    children; 0.4%) or questionable was very low (11; 1.5%), precluding
    meaningful statistical analysis. The Fagan test of infant intelligence
    (Fagan, 1987), an assessment of visual recognition memory or novelty
    preference, was also administered to 723 of the children at the same
    age. The mean percent novelty preference in the entire cohort was 60%,
    which is similar to that observed in other cohorts, and varied by <
    1% across categories of concentration of mercury in maternal hair. The
    index of perfor-mance on visual attention (the time required to reach
    visual fixation criterion in familiarization trials) was also
    unrelated to the concentration of mercury.

         The Bayley scales of infant development were administered to
    children in this cohort at the ages of 19 months ( n =738) and 29
    months ( n =736) by examiners who were unaware of their exposure
    status (Davidson et al., 1995a). Six items of the infant behaviour
    record, a rating scale, were also completed by the examiner for
    children aged 29 months, to assess activity, attention span,
    responsiveness to the examiner, response to the caregiver,
    cooperation, and general emotional tone. The Bayley scales yield two
    primary scores: the mental development index and the psychomotor
    development index. At both ages, the scores for mental development
    were similar to the expected mean for children in the United States,
    but the children's psychomotor development index scores were markedly
    higher: at 19 months, approximately 200 of the children achieved the
    highest possible score. Accordingly, the psychomotor development
    scores at both ages were expressed as a binary variable, dividing the
    distribution at the median score. The mental development scores were
    not significantly associated with the concentration of mercury in
    maternal hair during pregnancy. Similar results were obtained in a
    secondary analysis that included only children with the lowest (< 3
    µg/g) or highest (> 12 µg/g) concentration of mercury in maternal
    hair. The scores at 19 months for the items on the mental development
    scale designed to assess perceptual skills, dichotomized because of
    the skewing, were not associated with exposure to mercury. The scores
    for this index at 29 months could not be evaluated because of 'a
    pronounced celling effect'. A psychomotor development index score
    below the median was not significantly associated with the
    concentration of mercury in maternal hair in the full logistic

    regression model, but was associated with this exposure index
    ( p = 0.05) in a reduced model in which adjustment was made for a
    smaller number of covariates selected a priori. A secondary analysis
    of the psychomotor development index scores of children with the
    lowest and highest exposure to mercury was not conducted because
    statistical significance was not achieved in the full logistic
    regression model. In analyses of the six items for infant behaviour,
    the concentration of mercury in maternal hair was significantly
    associated only with the examiner's ratings of children's activity
    during the test session and only in boys. The score decreased by one
    point (on a nine-point scale) for each 10 µg/g.

         In general, the use of screening tests such as the Denver test in
    studies of neurobehavioural toxicology is not recommended because of
    their insensitivity to variations within the range of normal
    performance (Dietrich & Bellinger, 1994). More detailed instruments,
    such as the Bayley scales of infant development, have proven to be
    sensitive to prenatal exposure to a variety of neurotoxicants,
    including lead (Bellinger et al., 1987; Dietrich et al., 1987;
    Wasserman et al., 1992) and polychlorinated biphenyls (PCBs; Rogan &
    Gladen, 1991; Koopman-Esseboom et al., 1996). The only study of
    exposure to mercury in which the Bayley scales were administered was
    the study in the Seychelles, which found no significant association
    between children's scores and prenatal exposure. It is notable that
    the psychomotor development index scores were so high in this cohort
    that the distribution had be split at the median and analysed as a
    categorical variable. The median value was not provided by Davidson et
    al. (1995a) but was derived from a figure in the paper of Davidson et
    al. (1995b); it appeared to be approximately 130, or two standard
    deviations above the expected population mean. The mental development
    index scores were close to the expected population mean. It is
    questionable, however, whether statistical analysis of scores > 130
    versus < 130 allows assessment of a potential adverse effect of
    prenatal exposure to mercury on early motor development.

    2.3.2.4  Development later in childhood

         In the study in New Zealand, the 237 children in the 57 fully
    matched groups participated in a follow-up evaluation of
    neurodevelopmental status at 6 years of age (Kjellström et al., 1989).
    The mean concentration of mercury in maternal hair in the group with
    heavy exposure was was 8.3 µg/g (range, 6-86 µg/g; all but 16, 6-10
    µg/g). Extensive information was collected on possible confounding
    factors such as social class, medical history, and nutrition. A
    battery of 26 psychological and scholastic tests was administered to
    assess general intelligence, language development, fine and gross
    motor coordination, academic attainment, and social adjustment.
    Multiple regression analyses were conducted of five primary
    end-points: language development and spoken language; the revised
    Wechsler intelligence scale for children for both performance and
    fill-scale intelligence quotient (IQ) (Wechsler, 1974); the McCarthy

    scales of children's perceptual performance abilities; and the
    McCarthy scale of motor ability (McCarthy, 1972). In addition, robust
    regression methods were applied, involving the assignment of weights
    to observations, depending on their position within the distribution.
    In these analyses, the concentration of mercury in maternal hair was
    associated with poorer scores (p values ranging from 0.0034 to 0.074)
    for full-scale IQ, language development, visual and spatial skills,
    and gross motor skills. The findings in the unweighted regression
    analyses were similar in direction although generally less
    significant. The poorer mean scores of the children with heavier
    exposure to mercury were largely attributable to those whose mothers
    had concentrations in their hair < 10 µg/g, for whom the mean average
    concentration during pregnancy was 13-15 µg/g and the mean peak
    monthly concentration was about 25 µg/g. The concentration of mercury
    in maternal hair accounted for relatively little variance in the
    outcome measures and generally less than covariates such as social
    class and ethnic group.

         In additional analyses of this data set, Crump et al. (1998)
    found that when the concentration of mercury in maternal hair was
    expressed as a continuous rather than as a binary variable, none of
    the 26 scores was associated with exposure to mercury at  p < 0.10.
    The results were heavily influenced, however, by the results of a
    child whose mother had a concentration of mercury of 86 µg/g--more
    than four times the next highest level--despite the fact that the
    child's scores were not outliers by the usual criteria. When the data
    for this child were excluded, the scores on six end-points (Clay
    reading concepts or reading letters [Clay, 1979], McCarthy general
    cognitive index, McCarthy perceptual performance index, grammar
    completion, and grammar understanding) were inversely associated with
    the concentration of mercury in maternal hair at the 10% level.

         Several features of the study in New Zealand are noteworthy,
    including the efforts made to collect data on potential confounding
    variables and the broad battery of standardized outcome measures,
    administered by trained examiners. In contrast to the acute, heavy
    exposure of the Iraqi population, the cohort in New Zealand received
    chronic, low exposure, which was probably fairly constant over time,
    reflecting well-established food consumption patterns. In addition,
    the concentration of mercury in maternal hair was measured
    prospectively.

         As part of the pilot phase of the study in the Seychelles,
    children who reached the age of 66 months underwent developmental
    assessment (Myers et al., 1995c). Of the 247 eligible children, 217
    (88%) were given a battery of tests consisting of the McCarthy scales
    of children's abilities, a preschool language scale, and two subtests
    of the Woodcock-Johnson tests of achievement (Woodcock & Johnson,
    1989): letter and word identification and applied problems. The median
    concentration of mercury in maternal hair in 73 children whose mothers
    had had a concentration of mercury in their hair > 9 µg/gor < 4
    µg/g was 7.1 µg/g(range, 1-36). The rate of missing values was

    substantial for some end-points: e.g. 34% for the general cognitive
    index of the McCarthy scales. Increased concentrations of mercury in
    maternal hair were associated with significantly lower general
    cognitive scores ( p = 0.024), the scores declining approximately
    five points between the lowest (< 3 µg/g) and the highest (> 12
    µg/g) categories of exposure. A similar association was found on the
    perceptual performance subscale of the McCarthy scales ( p = 0.013).
    The children's scores on the auditory comprehension scale of the
    preschool language test were also inversely associated with the
    concentrations of mercury in maternal hair ( p = 0.0019), the scores
    declining approximately 2.5 points across the range of exposure to
    mercury. In additional analyses, exclusion of several outliers or
    influential data points reduced the estimates of the effect of mercury
    substantially, sometimes to nonsignificance. It is important to note
    that in the pilot phase of the study information was not collected on
    several key variables that frequently confound the association between
    exposure to neurotoxicants and child development, specifically
    socioeconomic status, maternal intelligence, and quality of the home
    environment.

         In the main study in the Seychelles, 711 children from the
    original cohort of 779 were evaluated at 66 months of age (± 6 months)
    by a battery of standardized neurodevelopmental tests (Davidson et
    al., 1998). The major domains assessed were general cognitive ability
    (McCarthy scales), expressive and receptive language (preschool
    language scale), reading achievement (letter and word recognition in
    the Woodcock-Johnson tests), arithmetic (applied problems test in the
    Woodcock-Johnson tests), visual and spatial ability (Bender Gestalt
    test; Koppitz, 1963), and social and adaptive behaviour (child
    behaviour checklist). The total amount of mercury in a segment of
    maternal hair during pregnancy served as the index of prenatal
    exposure to methylmercury (mean, 6.8 µg/g; SD, 4.5, range, 0.5-27),
    whereas the total amount of mercury in a 1-cm segment of hair obtained
    from a child at 66 months served as the index of postnatal exposure to
    methylmercury (mean, 6.5 µg/g; SD, 3.3; range, 0.9-26). For none of
    the six primary end-points did the pattern of scores suggest an
    adverse effect of either prenatal or postnatal exposure to mercury,
    and in fact the associations that were found indicated enhanced
    performance among children with heavier exposure. Greater prenatal and
    postnatal exposures to mercury were both significantly associated with
    better total scores for expressive and receptive language (both  p =
    0.02), and heavier postnatal exposure was associated with a better
    score for arithmetic ( p = 0.05). Among boys, higher postnatal
    exposure to mercury was associated with fewer errors on the test for
    visual and spatial ability ( p = 0.009).

         In the study in the Faroe Islands, 917 (90.3%) of the surviving
    members of the birth cohort of 1022 singleton births were submitted to
    comprehensive evaluations at approximately 7 years of age (Grandjean
    et al., 1997). The neuropsychological battery included three
    computer-administered tests from the neurobehavioural evaluation

    system (finger tapping, hand-eye coordination, continuous performance
    test), the tactual performance test, three subtests of the revised
    Wechsler intelligence scale for children (digit span, similarities,
    block design), the Bender Gestalt test, the California verbal learning
    test for children (Delix et al., 1994), the Boston naming test (Kaplan
    et al., 1983), and the nonverbal analogue profile of mood states.
    Parents were asked to respond to selected items on the child behaviour
    checklist. The primary index of exposure to methylmercury was the
    concentration of mercury in umbilical cord blood (geometric mean, 22.9
    µg/L; interquartile range, 13-41;  n = 894). Estimates were also
    available of the con-centration of mercury in maternal hair at
    parturition (geometric mean, 4.3 µg/g; interquartile range, 2.6-7.7;
     n = 914); in the child's hair at 12 months of age (geometric mean,
    1.1 µg/g; interquartile range, 0.7-1.9;  n = 527); and in the child's
    hair at 7 years (geometric mean, 3 µg/g; interquartile range, 1.7-6.1;
     n = 903).

         Not all of the children were able to complete all of the tests,
    and in some cases (e.g. finger opposition test, mood test) failure was
    associated with significantly higher mercury concentrations. Sensory
    functions including visual acuity, contrast sensitivity, auditory
    thresholds, and visual evoked potentials were not significantly
    related to prenatal exposure to mercury. Peaks I, III, and V of the
    brainstem auditory evoked potential at both 20 and 40 Hz
    ( p = 0.01-0.1) were slightly delayed in children with higher
    concentrations of mercury in cord blood, although at neither frequency
    was the interpeak latency associated with exposure. In multiple
    regression analyses, an increased concentration in cord blood was
    significantly associated with worse scores on finger tapping
    (preferred hand,  p = 0.05), continuous performance (in the first
    year of data collection only; false negatives,  p = 0.02; mean
    reaction time,  p = 0.001), digit span in the revised Wechsler
    intelligence scale for children ( p = 0.05), the Boston naming test
    (no cues,  p = 0.0003; with cues,  p = 0.0001), and the California
    verbal learning test (short-term reproduction,  p = 0.02; long-term
    reproduction,  p = 0.05).

          For two end-points (block design and visial-spatial copy
    errors), associations with mercury in cord blood ( p < 0.05) were
    found when an alternative approach to adjustment for confounders was
    applied. The results were similar when the 15% of the cohort whose
    mothers had had > 10 µg/g of mercury in their hair were excluded from
    the analyses. No significant interactions between mercury and sex were
    identified, indicating that the associations were similar for boys and
    girls. In general, the children's test scores were more strongly
    associated with the concentration of mercury in cord blood than in
    maternal hair or in samples of children's hair collected at 1 and 7
    years of age, but it was not stated whether any of the associations
    was significant.

         In an additional set of analyses (Grandjean et al., 1998), the
    investigators compared the neuropsychological scores of two groups of
    children: 112 whose mothers' hair had contained 10-20 µg/g (median, 12
    µg/g) of mercury at the time of parturition and 272 children whose
    mothers' hair had contained < 3 µg/g (median, 1.8). The two groups
    were matched by age, sex, year of examination, and maternal IQ. The
    median concentrations of mercury in cord blood also differed
    substantially: 59 µg/L versus 12 µg/L, respectively. The group with
    heavier exposure scored significantly lower than the other children on
    6 of the 18 end-points (one-tailed  p value, 0.05): finger tapping
    (both hands), hand-eye coordination (average of all trials), block
    design in the revised Wechsler intelligence scale for children, the
    Boston naming test (no cues, cues), and the California verbal learning
    test (long-term reproduction). The results of these analyses differ in
    certain respects from those of the main analyses. First, the set of
    end-points on which the two groups differed is similar to but not
    completely identical with the set found in the main analyses to be
    significantly associated with the concentration of mercury in cord
    blood. Moreover, in contrast to the main analyses, interaction terms
    between mercury concentration and sex were significant for several
    scores, including errors in the test for visual-spatial ability,
    short-term reproduction in the California verbal learning test, all
    three finger-tapping conditions, reaction time in the continuous
    performance test, and average hand-eye coordination. In all these
    tests, associations were found for boys but not for girls.

         In a cross-sectional study, Grandjean et al. (1999) evaluated 351
    children aged 7-12 who were living in villages in the Amazon Basin. In
    three of the villages, in which the population frequently consumed
    fish contaminated by gold-mining activities downstream, the
    concentration of mercury in the hair of 80% of children was > 10
    µg/g. In a fourth village, where the fish was not contaminated, only
    1% of children had concentrations of mercury in their hair > 10 µg/g.
    The aspects of neurobehavioural function evaluated included manual
    dexterity, short-term auditory memory, nonverbal memory, and
    visual-spatial skills. The concentration of mercury was associated
    with worse performance on the Santa Ana formboard test for manual
    dexterity (Lezak, 1995) and a copying test for visual-spatial skills.

    2.3.2.5  Sensory, neurophysiological, and other end-points

         In the study in the Faroe Islands, the evaluation at seven years
    also included assessments of visual acuity and near-contrast
    sensitivity, otoscopy and tympanometry, neurophysiological tests
    (pattern reversal visual evoked potentials at 30' and 15', brainstem
    auditory evoked potentials at 20 and 40 clicks/s, postural sway), and
    cardiovascular function (Grandjean et al., 1997; Sorensen et al.,

    1999). Peaks I, III, and V of the brainstem auditory evoked potential
    at 20 and 40 Hz were slightly delayed in children who had had higher
    concentrations of mercury in their cord blood ( p < 0.01-0.1),
    although the interpeak latency was not associated with the mercury
    concentration at either frequency. In additional analyses (Murata et
    al., 1999a), data from the second year of data collection (1994) were
    excluded because of concern about the accuracy of electromyography.
    Higher concentrations of mercury in maternal hair and cord blood were
    associated with lower peak III latencies and longer peaks I-III
    latencies. Of the four conditions under which postural sway was
    assessed, only that with the eyes closed and not standing on foam
    approached significance ( p = 0.09). Visual acuity, contrast
    sensitivity, and variation in heart rate were not related to exposure.
    Additional preliminary analyses suggested that both systolic and
    diastolic blood pressure increased with concentrations of mercury in
    cord blood < 10 µg/L (14 and 15 mm Hg for an increase from 1 to 10
    µg/L), and that, in boys, the variation in heart rate decreased with
    increasing concentration in cord blood (47% for an increase in cord
    blood mercury from 1 to 10 µg/L) (Sorensen et al., 1999).

         In a cross-sectional study of 149 children in the Madeira Islands
    (Portugal), Murata et al. (1999b) examined the association between the
    concentrations of mercury in maternal and children's hair and visual
    and brainstem auditory evoked potentials. As the dietary habits were
    stable, the current concentration of mercury in maternal hair was
    assumed to be a reliable estimate of the concentration during
    pregnancy. The children's hair concentrations were not significantly
    associated with any peak latencies and with only one interpeak
    interval. The concentrations of mercury in maternal hair were
    significantly associated with the I-III and I-V interpeak intervals at
    both 20 and 40 Hz and with the latencies of peaks III and V at both
    frequencies. Only the latency of pattern reversal visual enoked
    potential at 15 min was significantly associated with the
    concentration of mercury in maternal hair.

         The relationship between blood mercury concentration and auditory
    function was investigated by Counter et al. (1998) in 36 children and
    39 adults living in a gold-mining region in Ecuador and 15 children
    and 19 adults living in a control area. Mercury is liberated as a
    vapour in the process by which gold is extracted from alluvial
    sediments, making occupational exposure among gold miners a
    significant problem. Some of the mercury is methylated by aquatic
    organisms, enters the food chain, is biomagnified, and is consumed in
    fish. The concentration of mercury in blood was significantly higher
    in the individuals in the gold-mining area (18 µg/L) than in the
    control area (3 µg/L). Neurological and otological examinations were
    carried out on all persons, and audiological evaluations consisting of
    determinations of the conduction thresholds of pure tones in air in
    each ear at 0.25, 0.5, 1, 2, 3, 4, 6, and 8 kHz were carried out on 40
    individuals in the study area; brainstem auditory evoked potentials

    were measured on 19 subjects. The absolute latencies of waves I, II,
    III, IV, and V and the interpeak latencies of I-III, III-V, and I-V
    were measured for the left and right sides. The concentration of
    mercury in blood was significantly associated with the hearing
    threshold at 3 kHz in the right ear for children only. A borderline
    association was found between blood mercury and I-III interpeak
    transmission time on the left side.

    2.3.3  Adult neurological, neurophysiological, and sensory function

         The effects of chronic exposure to methylmercury on adult
    neurological function are being assessed by Lebel and colleagues in
    fish-eating populations living along the Tapajos River in the Amazon
    Basin who were exposed to mercury released during the extraction of
    gold from soil or river sediments. Lebel et al. (1996) studied 29
    young adults aged 15-35 years (14 female and 15 male), randomly
    selected from among participants in a previous survey. The geometric
    mean concentration of mercury in hair was 14 µg/g (range, 5.6-38). The
    subjects underwent a battery of quantitative behavioural sensory and
    motor tests, including tests of visual function (near and far acuity,
    chromatic discrimination, near-contrast sensitivity, peripheral visual
    fields) and motor function (maximum grip strength, manual dexterity).
    Individuals with elevated concentrations of mercury in hair had
    reduced chromatic discrimination; the three persons with
    concentrations > 24 µg/g had reduced contrast sensitivity, while
    those with concentrations > 20 µg/g tended to have reduced peripheral
    visual fields. The associations found between hair mercury
    concentration and motor function were sex-specific, as more heavily
    exposed women but not men tended to have lower scores in tests for
    manual dexterity and grip strength.

         In a subsequent study, Lebel et al. (1998) assembled another
    sample of 91 individuals aged 15-81 years, representing approximately
    38% of the adult population of the village being studied. Four indices
    of exposure were derived on the basis of the concentration of mercury
    in a hair sample: mean total concentration averaged over all 1-cm
    segments of the sample, total mercury in the first centimeter, maximum
    total mercury in any segment, and methylmercury in the first
    centimeter. People who had > 50 µg/g in at least 1 cm of hair were
    excluded. The mean concentration of methylmercury in hair was
    approximately 13 µg/g. The assessments included the same tests of
    motor and visual function that were used in the previous study and, in
    addition, a clinical neurological examination which was administered
    to a random sample of 59 members of the cohort. The examination
    included the Branches' alternate movement task, which requires
    imitation of a prescribed sequence of hand movements. Abnormal
    performance on the test was significantly associated with all indices
    of exposure to mercury, while abnormal visual fields were associated

    with mean and peak concentrations of mercury. Patellar and bicepital
    hyperreflexia were not associated with any index. Higher
    concentrations of mercury, most notably peak concentrations, were
    associated with poorer scores in the intermediate and higher
    frequencies of near visual contrast sensitivity (in the absence of
    near visual acuity loss) and in the test for manual dexterity, with
    greater muscular fatigue. In women, but not men, grip strength varied
    with peak mercury concentration. For many end-points, the associations
    with mercury in hair were stronger in younger (< 35 years) than in
    older subjects.

         Beuter and Edwards (1998) studied the association between the
    concentration of mercury in hair and the frequencies of four types of
    tremor: resting, kinetic, postural with visual feedback (static), and
    postural without visual feedback (proprioceptive). The subjects were
    adults aged > 40 whose mercury levels had been monitored annually
    for 25 years (1970-95). The group with heavy exposure consisted of 36
    Cree people from northern Quebec, Canada (26 women and 10 men). The
    maximum individual concentration of mercury in hair ranged from 2.2 to
    61 µg/g, while the mean annual maximum concentration ranged from 2.2
    to 31 µg/g. The group considered to have light exposure to mercury
    consisted of 30 adults from the Montreal area (18 women and 12 men),
    but the mercury levels for this group were not reported. Significant
    differences between the two groups were found in several aspects of
    static tremor (drift, amplitude differences, skewness, asymmetry,
    one-dimensional entropy, asymmetric decay of the autocorrelation
    function) and of kinetic tremor (mean tracking error, power in the
    3-4-Hz range, peaked velocity distribution).

    2.3.4  Bias: Covariates, confounders, and effect modifiers

         Analysis of the potential biases that may have occurred in the
    major epidemiological studies of exposure to methylmercury forms part
    of the assessment. The three main studies are those in the Faroe
    Islands, the Seychelles, and the Amazon Basin, for which information
    bias, selection bias, and confounding factors are reviewed, with the
    methods of control used. Short descriptions of new data or analyses in
    the studies in New Zealand, Peru, and Iraq are also reviewed.

         Such biases could affect the internal and/or the external
    validity of the studies. Selection bias could occur, for example, if a
    portion of the population had a different probability of participating
    in the study than the general population or if individuals with
    particular health conditions did not participate. Information bias can
    occur when the outcome measure is affected by knowledge of exposure
    status. Confounding factors, the major potential sources of bias in
    these studies, are associated with both exposure and outcome but are
    not part of their causal relationship.

    2.3.4.1  Study in the Faroe Islands

          Selection bias: Selection bias could have occurred in the study
    in the Faroe Islands at the time of recruitment or during the
    follow-up. The children who were examined were comparable to those in
    the full birth cohort, which represented 75% of all 1367 births that
    occurred during during the sampling period. The non-participants were
    born mainly in two small hospitals (with respective participation
    rates of 46 and 33%; Grandjean & Weihe, 1993) and had had heavy
    exposure to mercury, especially in one hospital (275 nmol/L vs 114
    nmol/L in the capital city, Torshavn). Because of this problem, the
    exposure of the overall cohort is lower than that of the background
    population. The loss of subjects from the study was low (9.7%), and
    the non-participants (106 persons) having somewhat lower exposure (18
    µg/L) than the participants (23 µg/L), owing to the lower
    participation rate of children born in Torshavn.

         Some tests, such as the paper-and-pencil and tactual performance
    tests, were not performed by 85 of the children who had heavier
    exposure to methylmercury (30 µg/L) than those who completed these
    tests (22 µg/L). The results of these tests were not considered in the
    final analyses, however, because of problems in scoring and unusual
    distributions of data. In general, the very high participation rate
    obtained in the study reduces the likelihood of major selection bias.

          Information bias: This type of bias was not likely to occur in
    the study since the children's exposure status was not known to the
    examiners, the children, or their parents. The report does not,
    however, state whether the parents were told the concentrations of
    mercury in cord blood or in maternal or children's hair. Some of the
    responses on the questionnaire could have been affected by this kind
    of bias.

          Confounding factors: In such studies, numerous confounding
    factors could affect associations between exposure to mercury and
    neurodevelopmental outcomes. Those most relevant to the study in the
    Faroe Islands are as follows:

    *     Mother's age: Since mercury accumulates in the body and older
         people tend to have a more traditional diet, age is potentially
         positively associated with exposure to mercury. No data were
         provided on the relationship between the mother's age and the
         body burden of mercury, and it is not clear whether maternal age
         is associated with child development. Factors such as prematurity
         and maternal cognitive function may differ according to a
         mother's age at the time of the birth of a child. Maternal age
         was not taken into account in the regression analyses.

    *     Birth weight: Low birth weight is known to be associated with
         delayed neurodevelopment, particularly in premature infants (body
         weight < 2500 g). Birth weight could also be associated with
         fish consumption, as in some observational studies and clinical
         trials, omega-3 fatty acids in fish oil or in fish meal were 
         reported to increase the duration of gestation and increase 
         birth weight (Olsen et al., 1993), although Foldspang and Hansen 
         (1990) reported an association between lower birth weight and 
         mercury concentration in Greenland. Low birth weight is generally 
         rare among newborns in the Faroe Islands, whose weights are on 
         average 200 g higher than in Denmark (Olsen et al., 1993). In the 
         Faroe Islands cohort, the number of fish dinners was positively
         associated with birth weight. This potential confounder would
         bias the results towards the null hypothesis: that seafood
         increases both exposure to mercury and birth weight. Birth weight
         was not taken into account in the final analysis.

    *     Breast-feeding: Breast-feeding was not considered as a
         confounding factor in the analysis, as methylmercury is not
         transferred to nursing infants. The concentrations of mercury in
         hair were two to three times higher among infants who were
         breast-fed for > 12 months than those breast-fed for 0-3 months
         (Grandjean et al., 1995b). Breast-feeding is related to parental
         behaviour that could affect brain development, and breast milk
         provides essential nutrients, such as docosahexanoic acid, which
         are important for brain development. Because the statistical
         analyses were based on the concentrations of mercury in
         children's hair at 12 months of age, however, the effects of
         breast-feeding were taken into account indirectly.

    *     omega-3 fatty acids: omega-3 fatty acids are polyunsaturated,
         long-chain (20-22 C) fatty acids with five or six double-bonds
         starting from the third carbon after the methyl terminal group.
         They are essential lipids and are not synthesized by the human
         organism but are derived from the diet, seafood being the major
         source. The main polyunsaturated fatty acids are eicosapentanoic
         acid and docosahexanoic acid, although docosapentanoic acid is
         also found in sea mammals. In sea mammal blubber or lipids,
         polyunsaturated fatty acids represent 25-30% of all lipids, with
         a lower proportion in pilot whales. Eicosapentanoic acid is
         reported to protect against cardiovascular disease, and
         docosahexanoic acid is a major component of brain lipids and
         retina and is essential for visual acuity and optimal brain

         development. It is now added to most infant formulas. The
         concentration of polyunsaturated fatty acids in the phospholipids
         of newborns and mothers in this cohort is not known but would be
         expected to be higher than that in urban populations. Olsen et
         al. (1986) reported that erythrocyte membranes from pregnant
         women in the Faroe Islands contained 20% more polyunsaturated
         fatty acids than those of women on the mainland and that the
         quantity was related to the consumption of fatty fish and sea
         mammals. Since mercury and PCBs are both found in seafood, their
         concentrations would be expected to correlate with that of
         polyunsaturated fatty acids. Since the latter enhance brain
         development and vision, this confounding effect would bias the
         results towards the null hypothesis. Polyunsaturated fatty acids
         were not considered in the final analysis.

    *     Selenium: Selenium is an essential trace element, of which
         seafood is a good source. The concentrations of selenium vary
         considerably in human populations, presumably reflecting selenium
         levels in the environment. Although the efficacy of this element
         as an antidote against mercury in humans remains controversial
         (WHO, 1990), selenium has been shown to counteract the toxicity
         of methylmercury in many experimental systems (reviewed by
         Whanger, 1992), including neuron cultures (Park et al., 1996).
         Both selenium and vitamin E reduced the toxic response of the
         nervous system to exposure to mercury (WHO, 1990). Studies in
         rodents suggest that supplementation of the maternal diet with
         selenite provides partial protection against some adverse effects
         resulting postnatally from exposure to methylmercury  in utero
         (Frederiksson et al., 1993). In the study in the Faroe Islands,
         the median concentration of selenium in whole umbilical cord
         blood was 1.4 µmol/L, and a slight but significant increase was
         seen with the number of fish dinners per week. The authors
         reported that the concentration of selenium in this population
         was high. Although most of the available data on selenium are
         derived from measurements in plasma samples, and are therefore
         underestimates of the whole-blood concentrations, comparative
         geometric means are available for Norway (Saami; 1.5 µmol/L), the
         Russian Federation (Kola Peninsula; 1.1 µmol/L), and Canada
         (Nunavik; 3.7 µmol/L) (Arctic Monitoring and Assessment
         Programme, 1998). In the Faroe Islands cohort, selenium
         concentration was positively associated with mercury
         concentration ( r = 0.35;  p < 0.001), indicating that
         exposure to mercury is associated with that to selenium. Although
         selenium cannot be considered a confounder per se (because it is
         not known to be associated with the outcomes), it could bias the
         study results towards the null hypothesis. Selenium was not
         considered at the final step of the regression analysis.

    *     Polychlorinated biphenyls and persistent organic pollutants:
         Tooth whales are known to accumulate lipophilic compounds such as
         PCBs and other persistent organochlorine environmental

         pollutants. The average concentration of PCBs (expressed as
         Aroclor 1254) in 39 samples of pilot-whale blubber from the Faroe
         Islands was 17 mg/kg (Simmonds et al., 1994), and the average
         intake was 1700 µg/person per week (3.5 µg/kg bw per day). As the
         total average concentration of DDE, a ubiquitous persistent
         organic pollutant, was 12 mg/kg, the intake of both these
         compounds and PCBs might be expected to be high among pregnant
         women and their offspring in the Faroe Islands. Extensive
         epidemiological data are available on the neurodevelopmental
         effects of prenatal exposure to PCBs and persistent organic
         pollutants (Rogan& Gladen, 1991; Koopman-Esseboom et al., 1996;
         Jacobson & Jacobson, 1997), although controversy persists about
         the long-term effects of prenatal exposure to PCBs. Since PCBs
         may be a risk factor for adverse neurobehavioural development,
         however, and since they are found in large quantities in pilot
         whales, they should be considered an important potential
         confounder. The PCB concentration in cord tissue was strongly
         correlated with that of mercury (r = 0.38 when PCBs were
         expressed on the basis of lipid and 0.44 when expressed as wet
         weight). As serum samples were not taken, PCBs were measured in a
         subsample of 436 stored samples of cord tissue from the 443
         children seen in 1993. This adjustment decreased the size of the
         cohort by 50% and consequently decreased the power of the study.
         The samples were divided, and PCBs were measured in two
         laboratories, but data on the duplicate analysis have not been
         reported. The total concentration of PCBs was calculated by
         multiplying the sum of PCB congeners 138, 153, and 180 by two.

              The relative reliability of measurements in cord tissue as
         compared with plasma or serum lipids is not known. No paired
         measurements of lipophilic compounds were made in cord tissue or
         established tissue matrices (e.g. cord blood and tissue). It
         would have been desirable to express all concentrations on the
         basis of lipids because umbilical cord thickness and lipid
         content vary among newborns; the cord length is generally around
         58 cm but can range from 20 to 100 cm (Abgoola, 1978). A short
         cord is associated with fetal akinesis or maldevelopment of the
         central nervous system (Ente & Penzer, 1991). As the water
         content of cord tissue (89%) varies according to the content of
         Wharton's jelly, this tissue is a poor biomarker for lipophilic
         compounds like PCBs and persistent organic pollutants.
         Furthermore, the water content decreases with length of gestation
         (Sloper et al., 1979); thus, if the lipid content of cord tissue
         increases with gestational age, the amount of PCBs measured in
         whole cord tissue will be affected not only by the actual
         exposure but also by newborn developmental factors. In the Faroe
         Islands cohort, the lipid content of cord tissue was 2.2 mg/g
         (0.22%); no SD was given. The mean total concentration of PCBs in
         the subsamples was 1.1 ng/g wet weight, and the lipid
         concentration can be calculated to be 1.1 × 454 = 508 µg/kg or

         0.51 µg/g. The value should, however, have been reported as 1.02
         µg/g, since the authors did not multiply the sum of the three PCB
         congeners by 2, as was done previously (P. Grandjean, personal
         communication, 1999). This calculated concentration is still less
         than the previously reported concentration of PCBs in breast-milk
         samples from women in the Islands, which were analysed in four
         pools: range, 1.9-3.5 µg/g; average of four pools, 2.5 µg/g lipid
         basis (Grandjean et al., 1995a). This concentration is close to
         those found in consumers of Arctic sea mammals (Dewailly et al.,
         1989, 1993). Thus, umbilical cord appears to be unreliable tissue
         for measuring exposure to PCBs; however, the effect of expressing
         PCBs on a whole weight basis on the statistical power of the
         study is mitigated by the wide variation in PCB concentrations
         between individuals. The fact that mercury correlates less with
         PCBs expressed on a lipid basis than when expressed on a whole
         weight basis probably reflects this imprecision, and PCBs should
         have been adjusted for on a lipid basis. The reliability of cord
         tissue for measuring PCB and persistent organic pollutants needs
         to be clearly demonstrated.

              Because PCBs and persistent organic pollutants are
         associated with both exposure to methylmercury and child
         development in this study, and because any confounding effects of
         PCBs will lead to a false-positive association between exposure
         to methylmercury and child development, the confounding role of
         PCBs and persistent organic pollutants should be reassessed in
         order to determine the role of methylmercury in the adverse
         effects reported in this study.

    *     Smoking: Smoking is unlikely to be an important confounding
         factor in this study as it is not known to be a source of
         mercury. It is, however, strongly associated with low birth
         weight, which in turn is a risk factor for poor cognitive
         development. In the cohort study, 40% of the women smoked while
         pregnant, but smoking was not associated with mercury
         concentration or with outcome.

    *     Alcohol consumption: Alcohol consumption during pregnancy is a
         major cause of abnormal fetal brain development. It is also often
         related to dietary habits. It is well known that alcohol
         consumption among pregnant women is difficult to measure. In the
         study in the Faroe Islands, alcohol consumption during pregnancy
         was considered to be low; occasional consumption was reported by
         24% of the women. Alcohol consumption was negatively associated
         with exposure to mercury and was unexpectedly associated with the
         results of some of the tests. The association may be due to the
         inclusion of women in Torhavn, who drink more and eat less pilot
         whale, thus having less exposure to mercury. The means by which
         alcohol consumption was measured is not described, even in the
         reference cited (Grandjean et al., 1992), and this factor was not

         considered in the final analysis. Since exposure to alcohol and
         mercury were negatively associated, any confounding effect would
         lead to the null hypothesis.

    *    Maternal cognitive level: Since maternal cognition is associated
         with child development and could affect dietary habits, it is
         considered to be an important potential confounding factor. The
         maternal score on Raven's progressive matrices was available for
         92.5% of the mothers, and a significant negative association
         (r = -0.13; p < 0.001) was found with exposure to mercury.
         Maternal cognitive level was included in the final regression
         analysis.

    *     Socioeconomic status: Exposure to mercury and consumption of
         pilot whale could be associated with socioeconomic status and
         health outcomes. Most of the association between mercury and
         socioeconomic status is due to the fact that the women in
         Torshavn, who may have been better educated, were less exposed to
         mercury because their diet was less traditional. Thus, more
         highly exposed children were likely to belong to families of
         lower socioeconomic status, as was found in analyses in which
         mothers in unskilled occupations had significantly greater
         exposure while children in day care had significantly less
         exposure. Day care, maternal education, paternal education, and
         paternal employment were therefore included in the final
         analysis. The methods used to measure socioeconomic status were
         not described. Such details would be valuable since the
         association between concentration of mercury and neurological
         outcomes decreased after adjustment for socioeconomic variables.

    *     Age of the children: The age of the children at the time of
         testing was also included in the final analysis since age was
         associated with the results of the neuropsychological tests.

         A review of all the potential confounding factors indicates that
    PCBs and socioeconomic factors were the most probable sources of bias
    in the study in the Faroe Islands. Adjustment for the city of
    residence (Torshavn or settlements) and for PCBs expressed on the
    basis of lipids may decrease the impact of these factors.

    2.3.4.2  Study in the Seychelles

         In the study in the Seychelles, the confounding factors that were
    identified and used in the final regression analyses were the same at
    testing at 6.5, 19, 29, and 66 months. The concentration of mercury in
    maternal hair was used as the marker of prenatal exposure. In general,
    most of the confounding factors were selected for their potential
    association with childhood development but not with exposure to
    mercury and were as follows: intelligence of the caregiver (Raven's
    score), birth weight, gestational age, sex, birth order, history of

    breast-feeding, medical history of the child and of the mother, age of
    the mother, maternal tobacco and alcohol use during pregnancy, home
    environment, parental education, family income, and language spoken at
    home. A reduced model with a limited number of variables was also
    used. No association was found between prenatal exposure to mercury
    and developmental outcomes at 6.5, 19, 29, or 66 months of age. The
    following section focuses on whether potential biases can explain the
    lack of association.

          Selection bias: As the study population comprised 50% of all
    births, some selection bias could have occurred. Recruitment was
    restricted to Mahé Island for practical reasons and because 90% of the
    population live on it. No information was provided about the reason
    for the non-participation of 45% of the eligible population. The
    authors speculated that the reasons may have included no contact with
    the family, lack of understanding of the study, conflicting
    responsibilities, refusal to allow their child to be examined, or
    superstition about removal of hair from themselves or their child. The
    second reason stated raises the possibility that the selected
    population was better educated than the non-participants, as suggested
    by the low percentage (3%) of abnormal or questionable scores on the
    revised Denver developmental screening test when compared with other
    studies. Exclusion of 18 children from the study could have resulted
    in a 'healthy child' effect. There is no major potential information
    bias, because both the mothers and the examiners were unaware of the
    exposure status of the children to mercury.

          Confounding factors: The most relevant confounding factors for
    the study in the Seychelles are as follows:

    *     Polychlorinated biphenyls: PCBs (28 congeners) were measured in
         49 randomly selected serum samples from children aged 5.5 years,
         and no PCBs were detected, as was to be expected on an island in
         the Indian Ocean. It would, however, have been helpful to know
         whether other potentially neurotoxic chlorinated compounds were
         detected, as they are often present in the Southern Hemisphere.
         For example, DDT is used extensively for malaria control in
         Mauritius and Madagascar and other islands of the Indian Ocean.

    *    omega-3 fatty acids: omega-3 fatty acids were not measured in the
         study in the Seychelles. Most studies on the fatty acid content
         of fish have been conducted in the Northern Hemisphere, but
         analysis of the fatty acids in lipid extracts from tropical
         seafood in Australia showed that the content of arachidonic acid
         represented 4.8-14% of the total. The seafood contained almost no
         linoleic acid but was a rich source of omega-3 fatty acids
         (14-31% of the total). Thus, seafood from tropical waters, unlike
         seafood from colder waters, is a natural source of
         polyunsaturated fatty acids in both the omega-6 and omega-3
         series (O'Dea & Sinclair, 1982). Since omega-3 fatty acids are
         concentrated in fatty fishes, and these predators often contain

         high concentrations of mercury, the concentration of omega-3
         fatty acids in the blood of Seychellois can be expected to
         correlate strongly with the concentrations of mercury in blood or
         hair. In a report of a workshop on methylmercury (National
         Institute of Environmental Health Sciences, 1998), it was noted
         that omega-3 fatty acids had been measured in randomly selected
         cord blood samples and found to be 'in the normal range';
         however, there is no abnormal range for these fatty acids, and
         the toxicological paradigm does not apply to nutrients. It
         remains to be determined whether the concentration of
         docosahexanoic acid in cord plasma phospholipids approaches 1.5%,
         as observed in western urban areas with low fish intake, or is
         closer to 7-8%, as observed in Inuit newborns. The range, the
         distribution, and the correlation with mercury should also be
         measured. omega-3 fatty acids thus appear to be a major potential
         confounding factor, which could explain the absence of
         associations, although some positive correlations were seen
         between mercury concentration and child development. Since fatty
         acids can be analysed in only 0.5 ml of serum, plasma, or
         erythrocyte membranes, they could be measured in a large number
         of archived cord blood samples. An alternative option will be to
         measure them in the blood of mothers attending the next
         evaluation.

    *     Selenium: As mentioned above, selenium could counteract the
         neurotoxicity of mercury but is not a true confounder since it is
         not known to be related to child development. A high selenium
         intake could therefore explain the lack of an association. No
         data were available on the selenium status of inhabitants of the
         region or of the concentration in fish. The workshop report
         (National Institute of Environmental Health Sciences, 1998) notes
         that selenium was measured in randomly selected cord blood
         samples and found to be 'in the normal range', but it is unclear
         whether that refers to values of 1.2, 1.5, or 4 µmol/L. It
         remains to be determined whether the concentration of selenium
         correlates strongly to fish consump-tion and to exposure to
         mercury.

         The role of fish nutrients (mainly omega-3 fatty acids and, to a
    lesser extent, selenium) could have masked an association between
    exposure to mercury and child development. It is also possible that
    the participants in the study were better educated than the
    non-participants and that the study did not have sufficient power
    because of the low background prevalence of abnormal test results.

    2.3.4.3  Study in the Amazon Basin

         In the preliminary study (Lebel et al., 1996), neurological
    dysfunction (sensory and motor performance) was investigated in 29
    adults selected randomly from two villages. Little effort was made to
    document potential confounding factors, and only age, location, and

    alcohol and tobacco consumption were included in the regression
    analysis. In the second study (Lebel et al., 1998), on 91 adults in
    one village, near visual contrast sensitivity and manual dexterity
    were investigated. The participation rate was 40%. Although
    sociodemographic information, smoking and drinking habits, medical and
    work histories, and level of education were recorded, the only
    potential confounder associated with the results of clinical
    examination and neurofunctional tests was age, although other relevant
    exposures may not have been considered. As malaria is endemic in the
    region, various pesticides (DDT, organophosphates, carbamates, and
    pyrethroids) may have been used, most of which are neurotoxic. Thus,
    some of the participants may have been exposed to these pesticides
    either directly (sprayed) or from consumption of contaminated fish. It
    is not known whether exposure to pesticides was associated with the
    fish diet or the area of residence. The reliability of the measurement
    of alcohol consumption, another potential confounding factor, is
    questionable since only three participants reported taking two drinks
    or more per week.

    2.3.4.4  Study in New Zealand

         The study carried out in New Zealand has basic weaknesses
    (Kjellström et al., 1986, 1989). Although no confounding was found
    from socioeconomic factors, health status, and maternal smoking
    (Kjellström et al., 1986), smoking was not graded, and neither alcohol
    consumption nor previous pregnancy outcomes were reported. Maternal
    consumption of alcohol can cause borderline mental deficiency (see,
    e.g. Berkow, 1988), and the children of smokers have lower scores than
    those of nonsmokers on most tests of intellectual function and
    intelligence at four and seven years of age. Moreover, minor
    neurological disorders are more common in children of women who smoke
    (Murphy, 1984). Additionally, a matching problem was recognized by the
    authors when they reduced the number of pair comparisons from the
    original 31 to 23 for ethnic differences or pairing of a New
    Zealand-born mother with an immigrant or both, and thus the results of
    11 and not 12 of the Denver tests were positive in the group exposed
    to mercury. The correct numbers show no significant difference between
    children exposed to mercury and reference children: three and not two
    positive results and eight and not ten negative results were found
    (chi2 = (8-2)2/(8 + 2) = 3.6;  p > 0.05). Thus, nine Pacific
    Islander pairs were responsible for 10 of the positive responses, and
    the other 14 pairs were responsible for only six, indicating that the
    effect of ethnic group in this study should be analysed thoroughly.
    Irrespective of exposure, the scores on the Denver developmental
    screening test were significantly higher among Pacific Islanders than
    people of other ethnic groups: 16 positive responses in Pacific
    Islanders (57%) and 6 in others (37%) and 12 negative responses in
    Pacific Islanders and 28 in others (chi2 = 8.8;  p < 0.005). As 25
    of the 28 Pacific Islanders were immigrants, a significant difference
    would also be expected between the children of immigrant and New
    Zealand-born mothers, as was the case: positive responses in six

    children of New Zealand-born mothers (18%) and in 15 of immigrant
    mothers (54%) and negative responses in 28 and 13, respectively (chi2
    = 5.9;  p < 0.02). The Pacific Islanders were at greater risk than
    Europeans or Maoris, with four positive responses among Pacific
    Islanders and one among others and one negative response among Pacific
    Islanders and 20 among others (p = 0.034; Fisher exact test).

         In the second stage of the study, on six-year-old children, the
    exposed group had been increased to 57 and their performance was
    compared with that of three groups ( n = 59, 60, and 58) of reference
    children in a battery of tests. The larger number of children ensured
    satisfactory matching for ethnic group and length of residence in New
    Zealand, but the presentation of the data does not allow verification
    of the distribution of positive responses by ethnic group in the
    exposed and reference groups. This is unfortunate in view of the
    results of the first stage of the study and the fact that the ratio of
    Pacific Islanders to non-Pacific Islanders was 2 (66% of all children
    were Pacific Islanders). Furthermore, the greatest differences between
    the exposed and reference groups were found in the tests for spoken
    language (8.6%) and the Wechsler test for full-scale IQ (5.3%), out of
    16 psychological tests, but the differences in the results of these
    two tests were more than twofold greater between Pacific Islanders and
    Europeans, 25 and 12%, respectively. Comparison of the results of 16
    psychological tests for the children of exposed and of reference
    mothers (48 comparisons) gave only two significant differences ( p =
    0.034 and 0.045), which are approximately those expected by chance.

         Regression analysis showed significant differences in the test
    for spoken language, the revised Wechsler intelligence scale for
    children, and the McCarthy scales for perceptual performance, but a
    clear association with exposure to mercury was seen only at
    concentrations in maternal hair >10 µg/g and only when a weighted
    regression analysis was used and 14 confounding factors were accounted
    for. The reliability of the 'accounting' is questionable when each of
    14 confounding variables introduces its own error; when other
    statistical manipulations, such as adjusting for outliers, although
    they are legitimate for eliminating one source of error, may have been
    the source of another type of error; when at least one confounder
    (ethnic group) was associated with larger differences in two of the
    three tests that correlated with maternal exposure to mercury than was
    maternal exposure; and when the highest concentrations of mercury in
    hair (> 10 µg/g) accounted for only about 2% of the overall variance.

    2.3.4.5  Study in Peru

         The study in Peru was conducted between 1981 and 1983 in a
    fishing community (Marsh et al., 1995), and hair samples and clinical
    data were obtained from 131 mother-infant pairs. Major information
    bias is unlikely to have occurred because the neurologists were
    unaware of exposure status, but it is not reported whether the mothers

    were informed about their exposure to mercury. Recall bias about their
    children's development is possible if the mothers knew their exposure
    status.

         Selection bias could have occurred in this study because hair
    samples were obtained from 369 mothers and only 194 of their children,
    and complete data were available for only 131 mother-infant pairs. The
    reason for this 65% reduction is not described but may have resulted
    in selection bias, with greater participation of healthier infants. No
    data are available to compare participants and non-participants.
    Furthermore, the participation rate among all pregnant mothers in
    Mancora during the recruitment period was not reported, and selection
    of healthy mothers might also have occurred. Although information was
    collected on several potential confounders, including alcohol and
    tobacco use, none was considered in the final statistical analysis.
    The authors stated that Mancora women drank little or no alcohol, that
    the group did not contain any smokers, and that there was little
    socioeconomic diversity.

         In this study, the role of nutrients in fish is of major
    importance. The authors noted that the difference in the results of
    their study and that carried out in Iraq could be due to the
    difference in the origin of mercury. They discussed the possible role
    of selenium in seafood and the possibility that the infants were
    protected against the neurotoxic effects of methylmercury by high
    selenium intake from their mothers during the pregnancy, but no data
    on selenium concentrations in fish or in biological samples from the
    infants or their mothers were available. Another potential nutrient,
    which was not discussed in the report, is polyunsaturated fatty acids
    and especially docosahexanoic acid, which is present in large
    quantities in fatty predator fishes, which are known to accumulate
    methylmercury. Polyunsaturated fatty acids may have acted as a
    confounding factor in this study, as they are associated with both
    exposure to methylmercury and infant development.

         This cohort study was therefore possibly affected by selection
    bias, and fish nutrients may have masked (polyunsaturated fatty acids)
    or mitigated (selenium) the neurotoxicity of methylmercury.

    2.3.4.6  Reanalysis of the study in Iraq

         The importance of the study in Iraq is that it is still used as
    the basis for the assessment of risks to human health risk by WHO and
    regulatory agencies. The consequences of exposure were investigated in
    adults and in 81 30-month-old children who had been exposed prenatally
    (Marsh et al., 1987). Although the heavily exposed infants showed
    deficits similar to those reported in Minamata Bay, Japan, efforts
    were made to investigate the group with lighter exposure. Selection
    bias could have occurred, for example, if the participants had
    experienced patent clinical symptoms and volunteered to participate,
    leading to an overrepresentation of severe cases. No data are
    available on how representative of the background population the

    participating children were. Information and recall bias are, however,
    the most important biases in this retrospective study, since precise
    information on ages at walking and talking and even age at testing was
    difficult to obtain, as there was no birth registry, and it had to be
    obtained from the mothers. It has been reported elsewhere that mothers
    underestimate the age at walking by 0.4 months (Piles, 1935, cited by
    Marsh et al., 1987). If such imprecision is equally distributed with
    respect to exposure status, this error will decrease the power of the
    study and bias the results towards the null hypothesis. It is unlikely
    that the mothers were less accurate in their answers to questionnaires
    because of exposure to mercury. The incident resulted in contaminated
    bread, and this basic food is expected to be consumed by the entire
    population regardless of socioeconomic status, level of education, or
    intelligence.

    3.  ESTIMATES OF DIETARY INTAKE

    3.1  Environmental mercury

         In the environment, methylmercury is produced from inorganic
    mercury in natural and anthropogenic sources as a result of microbial
    activity. Microbial methylation of inorganic mercury occurs in the
    upper sedimentary layers of lakes and sea bottoms, and the
    methylmercury formed is rapidly taken up by living organisms in the
    aquatic environment. A number of studies of mercury forms in air have
    shown that, except in industrial areas, near volcanoes, and mercury
    ore deposits, the concentration of total mercury in air is < 10
    ng/m3, of which mono-and dimethylmercury account for approximately
    22%. As the intake of the general population of methylmercury from air
    is estimated to be < 0.04 µg/day, air is considered to be an
    insignificant source (WHO, 1990).

         The mean concentrations of total mercury in rivers, lakes, and
    groundwater range from 10 to 50 ng/L. It can form stable complexes
    with various organic ligands in water, but the resulting methylmercury
    compounds are rapidly taken up by biota, since < 1 ng/L has been
    found in unpolluted waters. If consumption of 1.5-2 L of water daily
    is assumed, the intake of methylmercury from this source would be <
    0.002 µg/day (WHO, 1990).

         Most foods except fish contain very low concentrations of total
    mercury (< 0.01 µg/g), which is almost entirely inorganic mercury.
    Fish and shellfish contain higher concentrations, and over 90% is in
    the form of methylmercury because fish feed on aquatic organisms that
    contain this compound, ultimately originating from microorganisms
    which biomethylate inorganic mercury. The amount of methylmercury in
    fish correlates with a number of factors, including the size and age
    of the fish, the species (e.g. larger, older, predatory species like
    shark and swordfish usually contain higher concentrations), and, for
    freshwater species, the mercury content of water and sediment and the
    pH of the water. The concentration of methylmercury in most fish is

    generally < 0.4 µg/g, although fish species higher up the aquatic
    food chain, such as swordfish, shark, walleye, and pike, may have
    concentrations up to several micrograms per gram. The intake of
    methylmercury from fish depends on fish consumption and the
    concentration of methylmercury in the fish consumed. Many people eat
    < 20-30 g of fish per day, but certain groups eat 400-500 g per
    day. Thus, the daily dietary intake of methylmercury can range from
    < 0.2 to 3-4 µg/kg bw (WHO, 1990). The ranges of concentrations of
    methylmercury in various fish species are shown in Table 5.

    3.2  Biomarkers of exposure

         Two approaches are used currently to assess the body burden of
    methylmercury: one based on dietary modelling and the other on
    biomarkers. Each has limitations which prevent their use in making
    unequivocal estimates of exposure. The outcomes of dietary models
    depend on differences in approach and in assumptions, and their
    reliability remains to be confirmed. Despite the limitations of the
    existing data sets, biological measures of exposure, such as the
    concentrations of methylmercury in hair or blood, provide a useful
    start for discussions of exposure since they allow a biologically
    based validation of dietary models.

         The two most popular media for quantifying methylmercury in the
    body are blood and hair. Quantitative relationships between exposure
    (daily intake) and concentrations in blood and hair were first
    established in the study in Iraq and naturally involved many
    assumptions, for instance that hair grows at a rate of 1 cm per month.
    The relationship between the concentrations of mercury in blood and
    hair was verified in several studies, in which the concentrations in
    hair ranged from 0-13 µg/g (Netherlands) to 20-325 µg/g (Japan). In
    two communities in the United Kingdom, one being a fishing community,
    the concentrations in hair ranged from 0.1 to 21 µg/g. These studies
    showed that every microgram increase in blood concentration resulted
    in a 140-370-µg/g increase in the concentration in hair, although the
    regression lines in six of ten studies gave hair:blood concentration
    ratios of 230-280. Thus, a ratio of 250 gives a relatively acceptable
    extrapolation from one media to the other (WHO, 1990).

         Under stable dietary conditions, the concentrations of
    methylmercury in blood and hair can be used to predict the possible
    effects of methylmercury on health, since the concentrations are
    directly proportional to the concentrations of methylmercury in the
    brain (Phelps et al., 1980; Cernichiari et al., 1995). The
    concentration in hair is about 250 times greater than that in blood at
    the time the hair strand is formed. Once formed, a strand grows at a
    rate of approximately 1 cm per month and thus provides a record of
    previous exposure to methylmercury. Approximately 80% of the mercury
    present in the strand is methylmercury (Phelps et al., 1980;
    Cernichiari et al., 1995). Hair and blood can both be used to document
    exposure, but hair is preferred because it involves a simple,

    uninvasive sampling procedure that allows monitoring of the intake of
    methylmercury. Total mercury concentrations are typically used to
    characterize exposure to methylmercury from fishery products, since
    total mercury and methylmercury concentrations are linearly related
    and it is less costly to determine total mercury. Although
    measurements of total mercury can provide an upper bound of the
    concentration of methylmercury in hair, the dose to the brain, and
    intake from the diet, they may provide misleading evidence of exposure
    and dose if certain hair treatment formulations (cold-wave solutions
    and hair relaxers) which extract methylmercury have been used.
    Reductions of over 60% have been observed (WHO, 1990).

    Table 5. Estimated concentrations
    of mercury in fish 
                                    

    Species         mg/kg of fish
                                    

    Mackerel        0.07-0.25
    Sardine         0.02-0.3
    Tuna            0.03-1.2
    Swordfish       0.06-0.8
    Shark           0.004-1.8
    Other           0.03-0.3
                                    

    From WHO (1990)

         Studies of the concentrations of mercury in hair provide a set of
    data for describing the range of exposures. Two large studies of
    women's hair were conducted during the early 1980s. One involved 2000
    women aged 15-45 who were part of a dietary panel that was intended to
    be geographically and demographically representative of the population
    of the United States (Smith et al., 1997). They maintained monthly
    diaries of seafood consumption, recording species and amount. At
    three-month intervals, hair samples were obtained by cutting strands
    of hair close to the scalp from the occipital region. These were
    provided by 1437 of the women (72%), were cut into 4-cm segments
    corresponding to the three-month period associated with each diary
    (i.e. one month before and one month after the month covered by the
    diary), and analysed for methylmercury by an electron capture gas
    chromatographic method. The concentrations in the hair of women who
    had eaten some seafood during the one-month period covered by the
    diary were compared with those of women who ate no seafood during that
    period. The authors reported that the distribution of methylmercury
    concentrations in the two groups was approximately log-normal and that
    99.72% of all hair samples had concentrations < 3.9 µg/g.

    The concentrations in four samples exceeded 3.9 µg/g (4.4, 5.9, 6.0,
    and 6.3 µg/g), and the arithmetic mean for all samples was 0.48 µg/g.
    The distribution of concentrations reveals that 90% of all values were
    < 1.3 µg/g.

         In another analysis, the results were statistically weighted to
    reflect the population and were adjusted by season to provide annual
    population estimates. The mean, median, and maximum weighted annual
    concentrations of mercury in hair were 287, 204, and 3505 µg/kg,
    respectively; the 90th percentile concentration was 531 µg/kg. The
    mean concentration for men (260 µg/kg) was about 20% lower than that
    for women (315 µg/kg), suggesting that use of hair treatments that can
    alter hair mercury concentrations was not widespread in the study
    population. The mean and median concentrations for children nine years
    and younger were 177 and 133 µg/kg, respectively. For the 59 women of
    child-bearing age, the mean was 347 µg/kg and the maximum was 1585
    µg/kg. A four-day dietary survey included in the study showed that 21%
    of the people surveyed reported eating fish during the survey. Those
    who had eaten fish at least once during the survey had a mean hair
    concentration of 418 µg/kg, while those who had not eaten fish had a
    mean concentration of 326 µg/kg(Smith et al., 1997).

    3.3  Intake assessment

         Estimation of the intake of a contaminant is complicated by the
    skewed distribution of residues, since contaminants do not reach food
    through controlled or predictable agricultural or manufacturing
    processes. It is often possible to control contamination, and those
    controls should achieve the maximum impact on potential intakes.
    Rational decision-making requires estimates of the major contributors
    to intake and the likely impact of proposed controls.

         Methylmercury can be ingested as a result of the presence of
    mercury in food, water, or air. This assessment is limited to food and
    is based on the conservative assumption that all of the results
    reported were for methylmercury. National governments may wish to
    consider other potential sources of intake and add them to estimates
    of intake from foods in order to estimate total intake. Virtually all
    of the available data derived from monitoring are for total mercury.
    The Committee had received data from 25 countries representative of
    all regions of the world, and several countries submitted estimates of
    the intake of mercury by their populations. When data on infant or
    child intake were available, they were included.

         The WHO Global Environment Monitoring System-Food Contamination
    Monitoring and Assessment Programme (GEMS/Food) has collected data on
    food contamination through a network of participating institutes in
    over 70 countries around the world since 1976. It has also developed
    five regional and cultural diets for use in estimating the intakes of
    a wide range of the world's populations. The diets were derived from
    food balance sheets compiled by FAO, and thus provide data that are

    comparable across different countries and regions of the world. They
    are based on the countries' annual food production, imports, and
    exports and do not take into account waste at the household or
    individual level; they are thus expected to be overestimates of
    consumption of actual food intakes, by about 15%. The data do not,
    however, permit the analysis of intakes by subgroups such as children
    and infants.

         These diets include estimates of fish consumption. The Committee
    used the diets in combination with available data on residues of
    mercury to estimate typical mercury intakes. The mercury intake of
    high consumers of fish was determined on the basis of analyses by
    Australia and the United States. The potential impact of establishing
    limits was determined in a Monte Carlo simulation model.

    3.3.1  Residues

         Most of the data on residues were available to the Committee in
    summarized form. The United States Total Diet Study and the Australian
    Market Basket studies indicate the ranges of residues in a variety of
    fish species and products (Table 6; Food & Drug Administration,
    1993-96; Australia New Zealand Food Authority, 1998). The United
    States also provided information on residues in fresh tuna, swordfish,
    and shark from a survey by the National Marine Fisheries Services.
    Although data were reported for various oceans, no clear differences
    were found. These data are similar to those reported by WHO (1990) and
    also show that some species have higher concentrations than others,
    including the commonly consumed tinned tuna, flake, and estuarine
    fish. A true representative average value cannot be selected.

        Table 6. Mercury in fish from Australia and the United States
                                                                           

    Fish                      Concentration (mg/kg)         95th percentile
                              Mean           Range
                                                                           
    Australia

    Calamari rings            0.02           Trace-0.03     NA
    Fish, estuarine           0.12           0.09-0.15      NA
    Flake fillet (fried)      0.33           0.04-0.80      NA
    Tuna, tinned              0.22           0.08-0.56      NA

    United States

    Tuna, tinned              0.18                          0.46
    Tuna, fresh               0.2                           0.45
    Shark                     0.96                          2.4
    Swordfish                 0.7                           1.1
                                                                           
    Data for Australia from the Australia New Zealand Food Authority (1998)
    and those for the United States from Food & Drug Administration (1993-96).

    Total mercury and not methylmercury was measured in these studies.

    NA, not available
    
         An average or mean concentration of mercury residue is
    appropriate for estimating the intake of methylmercury in the WHO
    GEMS/Food regional diets. The Committee concluded that concentrations
    based on estuarine fish, tuna, or flake fillet would be appropriate
    for this purpose, and the average values for tinned tuna and flake
    fillet were used to provide a range of estimates of regional intakes
    of mercury.

    3.3.2  National intake estimates

         Estimates of the intake of mercury are available for the
    populations of 25 countries (Table 7), which provide a good measure of
    differences in intake across populations and subgroups, including
    infants and children. More than one study was available for some
    countries. For example, Australia provided estimates of intake from
    their market basket study (Australia New Zealand Food Authority, 1998)
    and from their 'diamond' model, and the United States provided an
    assessment based on their total diet study and a Monte Carlo
    simulation that included additional data on residues in fish
    (Carrington, 1999). The 'diamond' model permits assessments for
    individuals, including high consumers, because it includes data from
    the 1995 National Nutrition Survey and data on water consumption. The
    intakes ranged from 0.7 to 5.6 µg/kg bw per week, depending on the
    method used, the subgroup evaluated, and the residue data used.
    Australia and New Zealand estimated intake for a variety of age
    groups, including young children.

         Slovakia determined the concentrations of mercury in a study which
    duplicate samples of meals were consumed and statistically representative
    samples were collected four times a year; estimates of the mercury intake
    of infants who were breast-fed or who consumed milk formula were also 
    provided (Ursķnyovį & Hladķkovį, 1997, 1998).

    3.3.3  Estimates of intake based on WHO GEMS/Food diets

         The Committee estimated the typical intakes of mercury by
    consumers by using the average total consumption of all species of
    fish and shellfish from the GEMS/Food regional diets and a range of
    typical concentrations of methylmercury in fish. For these analyses,
    it was assumed that all species would contain one of two
    concentrations of methylmercury: the first analysis (Table 8) assumed
    a concentration of 0.2 mg/kg of fish, and the second analysis (Table
    9) assumed 0.33 mg/kg of fish. These concentrations are in the range
    of the average and median values in several countries. As some
    species, such as shark and swordfish, often contain concentrations of
    mercury residues above these two limits, frequent consumers of these
    fish will have correspondingly higher intakes of mercury.

    3.3.4  Estimates of intake by fish consumers at the 95th percentile

         Australia and the United States also estimated the intake of high
    consumers (Table 10). Australia used the actual consumption and two
    assumptions about the concentrations of mercury in the fish that were
    consumed. In the first analysis, it was assumed that fish contained
    0.2 mg/kg, and in the second analysis it was assumed that predatory
    fish contained 0.64 mg/kg of fish. The United States took into account
    variability in both residue concentrations and food consumption
    patterns, using a Monte Carlo simulation to predict the most likely
    distribution of mercury intake across each population subgroup. The
    analysis was repeated after exclusion of residues at concentrations
    over certain limits to simulate the effect of prohibiting fish
    containing > 1 or > 0.5 mg/kg.

    4.  COMMENTS

         Although methylmercury can occur in other foods, it is found
    primarily in fish. In other foods, mercury is present mainly as
    elemental mercury. The Committee noted the variation in concentrations
    of methylmercury in fish, both within and between species, and also
    noted that fish from polluted waters usually have higher mercury
    concentrations than those from unpolluted bodies of water. When
    intakes of total mercury were provided, the Committee assumed
    conservatively that all of the mercury was methylmercury. A 'typical'
    concentration of methylmercury must be established to permit
    estimation of intake from the WHO GEMS/Food regional diets. A
    'typical' concentration should correspond to the concentrations that
    are consumed 'on average' by consumers and should therefore represent
    the usual concentrations in commonly consumed species of fish. The
    Committee concluded that concentrations based on estuarine fish, tuna,
    or flake fillet would be appropriate for this purpose. For these
    analyses, the average concentrations found in tinned tuna and flake
    fillet were used to derive a range of estimates of regional intakes of
    methylmercury.

         Data on the concentrations of mercury residues in food and/or
    assessments of mercury intake were submitted to the Committee by 25
    countries which represent the major regions of the world. The WHO
    GEMS/Food diets include estimates of fish consumption in each of five
    regional diets. The Committee used information from these sources to
    estimate typical methylmercury intakes of 0.3-1.1 µg/kg bw per week,
    depending on the region of the world. These values are predicated on
    the assumption that all fish and shellfish contain methylmercury at
    200 µg/kg of fish. If all fish and shellfish that are consumed contain
    methylmercury at 330 µg/kg of fish, the intake ranged from 0.5 to 1.8
    µg/kg bw per week.

         The methylmercury intake of consumers in Australia who were
    considered to have a high intake of fish was estimated on the
    assumption that the fish contained methylmercury at either 200 or 640

    µg/kg of fish. The estimated intakes for consumers in the 95th
    percentile were 2.1 and 5.6 µg/kg bw per week, respectively. As these
    values are based on the assumption that all fish contain these
    concentrations, they are highly conservative estimates of extreme
    intake. A probability analysis was conducted in the USA to provide a
    more realistic estimate of the intake of methylmercury by consumers in
    the 95th percentile, by taking into account variation in both fish
    consumption and residue concentrations in the fish that are consumed.
    The analysis covered the entire distribution of consumption and
    methylmercury residues in fish. An estimate was also provided from a
    simulation model of the potential impact of establishing limits on
    intake of methylmercury, by repeating the analysis after excluding
    residue concentrations that exceeded theoretical regulatory limits of
    1000, 500, or 200 µg/kg of fish. The results of the analysis are
    presented in Table 10 for consumers in the 95th percentile in three
    population groups. These results suggest that the intake of the adult
    population will be below the PTWI as long as individuals eat fish with
    'typical' concentrations of methylmercury.

         The Committee also specifically evaluated the potential intake of
    children and infants. The WHO GEMS/Food diets do not include separate
    estimates for children, but several countries provided estimates of
    the intake of mercury by children and infants. Comparison of the
    intake by adults and children in each country shows that children
    consume two to three times more mercury than the adult population on
    the basis of unit body weight. Nevertheless, the concentrations of
    mercury in the hair of children are similar to those in adult hair,
    indicating that children have similar body burdens to those of adults.
    Therefore, the higher intakes of children would not necessarily result
    in an equivalent increase in risk, and, if children are not more
    sensitive than adults to methylmercury, the PTWI would be appropriate
    for both adults and children. In simulations conducted in the USA,
    children were found to have intakes below the PTWI. Although data were
    not available to permit equivalent analyses for other countries, the
    results can be expected to be similar as long as the concentrations in
    fish and the fish consumption are similar to those seen in the USA.

         Studies of the kinetics of methylmercury showed that its
    distribution in tissues after ingestion is more homogeneous than that
    of other mercury compounds, with the exception of elemental mercury.
    The most important features of the distribution pattern of
    methylmercury are high blood concentrations, high ratios of
    erythrocyte:plasma concentration and high concentrations of deposition
    in the brain. Another important characteristic is slow demethylation,

        Table 7. Estimates of intake of methylmercury, assuming all residues
    measured as mercury are actually methylmercury
                                                                                            

    Country             Estimate       Populationa                        Reference
                        (mg/person
                        per week)
                                                                                            

    Australia           0.7-4.3b       9-month-old infants                Australia New
                        0.7-3.4b       2-year-old children                Zealand Food
                        0.4-1.7b       Adult women                        Authority (1998)
                        0.3-1.8b       12-year-old girls
                        0.3-1.7b       Adult men
                        0.3-1.6b       12-year-old boys
    Australia           0.3            Adults                             WHO (1992)

    Belgium             1.63           All                                Jorhem et al. (1998)
    Belgium             1.6            Adults                             WHO (1992)

    China               1.20           All                                Chen & Gao (1993)
    China               0.63           Standard man (58 kg)               Gao (1999)
                        1.61           2-7 year-old children (16.5 kg)
                        1.69           8-12-year-old children (29.4 kg)
                        0.42           20-50-year-old men (63 kg)
                        0.41           20-50-year-old women (53 kg)

    Cuba                1.6            Adults                             WHO (1992)

    Denmark             0.09           All                                Jorhem et al. (1998)
    Denmark             1.8            Adults                             WHO (1992)

    Finland             0.22           All                                WHO (1992)
    Finland             0.3            Adults                             WHO (1992)

    France              1.4            Adults                             WHO (1992)

    Germany             0.07           All                                Jorhem et al. (1998)
    Germany             1.6            Adults                             WHO (1992)
    Germany             0.6-0.7c       Adult                              Becker et al. (1998)

    Table 7. (continued)
                                                                                                 

    Country             Estimate       Populationa                        Reference
                        (mg/person
                        per week)
                                                                                                 

    Guatemala           1.26           All                                WHO (1992)
                        1.5            Adults

    Italy               1.5            Adults                             WHO (1992)

    Japan               0.50           All (55 kg bw)                     Jorhem et al. (1998)

    Netherlands         1.05           All                                Jorhem et al. (1998)
                        0.23           All
                        0.08           All
    Netherlands         1.2            Adults                             WHO (1992)

    New Zealand         0.6            Adults                             WHO (1992)

    Poland              2.0            Adults                             WHO (1992)

    Slovakiad           0.9            Children (vegetarian)              Ursķnyovį &  
                        0.8            Children (non-vegetarian)          Hladikova (1998)

    Sweden              0.7-0.82       All                                Jorhem et al. (1998)
    Sweden              0.3            Adults                             WHO (1992)

    Thailand            0.3            Adults                             WHO (1992)

    United Kingdom      0.3            Adults                             WHO (1992)
    United Kingdom      0.35           All                                Ministry of Agriculture, 
                                                                          Fisheries & Food (1991)

    Table 7. (continued)
                                                                                                 

    Country             Estimate       Populationa                        Reference
                        (mg/person
                        per week)
                                                                                                 

    United States       0.30           60-65-year-old men                 Food & Drug 
                        0.23           60-65-year-old women               Administration 
                        0.23           70-year-old men                    (1993-96)
                        0.20           2-year-old children
                        0.16           40-45-year old men and women
                        0.15           25-30-year-old men
                        0.14           6-year-old children
                        0.12           70-year-old women
                        0.11           25-30-year-old women
                        0.10           14-16-year-old boys
                        0.08           10-year-old children
                        0.06           14-16-year-old girls
                        0.01           Infants
                                                                                                  

    a  Body weights in parentheses are assumptions.
    b  Low end of range based on assumption that samples with no detectable
       mercury have none; high end of range based on assumption that the lowest
       observable concentration of residue is present in samples with no
       detectable mercury
    c  Low end of range, people who do not eat fish; high end, fish consumers
    d  Low end of range, breast-fed infants; high end, infants fed cow's milk;
       formula-fed infants had intermediate values.

    Table 8. Mercury intake if all fish contain 200 µg/kg
    (mean residue in tuna in Australia and the United States)
    and consumption levels are those of GEMS-Food regional diets
                                                                                                   

    Code   Commodity                         Fish intake (g/person per day)

                                             Middle    Far       African   Latin       European
                                             Eastern   Eastern             American
                                                                                                   

    Fish and seafood

    WC     Crustaceans, fresh frozen         0.3       2.3       0.0       1.5         3.0 
    MD     Dried fish                        0.3       2.8       4.4       4.8         0.8 
    WS     Demersal, frozen whole            0.0       0.0       0.9       0.5         3.8 
    WS     Demersal, frozen fillets          0.0       0.0       0.0       1.3         5.0 
    WS     Demersal, cured                   0.0       0.3       0.6       4.5         0.5 
    WS     Demersal                          2.0       3.0       2.4       0.0         9.0 
    WF     Freshwater, tinned                0.0       0.0       0.0       0.0         0.8 
    WF     Freshwater, frozen whole          0.0       0.0       0.0       0.0         0.3 
    WF     Freshwater, cured                 0.3       0.5       1.4       0.0         0.0 
    WD     Freshwater diadrom, fresh         1.3       5.3       4.7       1.3         1.5 
    WS     Marine fish (not otherwise        2.8       5.2       5.1       18.3        2.8
           specified), fresh frozen 
    WS     Marine fish (not otherwise        0.0       1.0       0.0       0.3         0.0
           specified), cured 
    IM     Molluscs except                   0.0       4.0       0.5       0.8         8.3
           cephalopods, fresh
    IM     Molluscs, tinned                  0.0       0.0       0.0       0.0         0.8
    WS     Pelagic, tinned                   1.8       0.8       0.5       4.8         4.8
    WS     Pelagic, frozen whole             0.3       2.0       0.7       0.3         1.3
    WS     Pelagic, cured                    0.0       1.0       2.4       0.0         0.3
    WS     Pelagic marine fish, fresh        4.3       5.8       13        7.0         3.8

    Total fish intake per day (g/person)     13        35        36        45          46

    Table 8. (continued)
                                                                                                   

    Code   Commodity                         Fish intake (g/person per day)

                                             Middle    Far       African   Latin       European
                                             Eastern   Eastern             American
                                                                                                   

    Estimated intake of mercury
      Total (µg/person per day)              2.6       7.0       7.2       9.0         9.2
      Total (µg/person per week)             18        49        50        63          64 
      Total (µg/kg bw per week)              0.3       0.8       0.9       1.1         1.1 
      (for 60-kg adult)

    % of PTWI (3.3 µg/kg bw)                 9%        25%       26%       32%         33%
    for 60-kg adult

    Residue concentration that would         2.2       0.82      0.78      0.63        0.61
    be less than PTWI (mg/kg of fish) 
    assuming a 60-kg adult consumes 
    fish with this concentration on a 
    long-term basis
                                                                                                  

    Table 9.  Mercury intake if all fish contain 330 µg/kg
    (mean residue in flake fish in Australia) and consumption
    levels are those of GEMS-Food regional diets
                                                                                             

    Commodity                    Fish intake (g/person per day)

                                  Middle     Far        African      Latin          European
                                  Eastern    Eastern                 American
                                                                                             

    Fish and seafood

    Crustaceans, fresh frozen     0.3        2.3        0.0          1.5            3.0
    Dried fish                    0.3        2.8        4.4          4.8            0.8
    Demersal, frozen whole        0.0        0.0        0.9          0.5            3.8
    Demersal, frozen fillets      0.0        0.0        0.0          1.3            5.0
    Demersal, cured               0.0        0.3        0.6          4.5            0.5
    Demersal                      2.0        3.0        2.4          0.0            9.0
    Freshwater, tinned            0.0        0.0        0.0          0.0            0.8
    Freshwater, frozen whole      0.0        0.0        0.0          0.0            0.3
    Freshwater, cured             0.3        0.5        1.4          0.0            0.0
    Freshwater diatom, fresh      1.3        5.3        4.7          1.3            1.5
    Marine fish (not otherwise    2.8        5.2        5.1          18.3           2.8
    specified), fresh frozen
    Marine fish (not otherwise    0.0        1.0        0.0          0.3            0.0
    specified), cured
    Molluscs except cephalopods,  0.0        4.0        0.5          0.8            8.3
    fresh
    Molluscs, tinned              0.0        0.0        0.0          0.0            0.8
    Pelagic, tinned               1.8        0.8        0.5          4.8            4.8
    Pelagic, frozen whole         0.3        2.0        0.7          0.3            1.3
    Pelagic, cured                0.0        1.0        2.4          0.0            0.3
    Pelagic marine fish, fresh    4.3        5.8        13           7.0            3.8

    Total fish intake
    per day (g/person)            13         35         36           45             46

    Table 9.  (continued)

                                                                                             

    Commodity                    Fish intake (g/person per day)

                                  Middle     Far        African      Latin          European
                                  Eastern    Eastern                 American
                                                                                             

    Estimated intake of mercury

      Total (µg/person per day)   4.3        12         12           15             15
      Total (µg/person per week)  30         80         84           100            110
      Total (µg/kg bw per week)   0.5        1.3        1.4          1.7            1.8
      (for 60-kg adult)

    % of PTWI (3.3 µg/kg bw)      15%        40%        43%          53%            54%
       for 60-kg adult
                                                                                             

    Table 10. Estimated intake of methylmercury by fish consumers at the 95th percentile
                                                                                           

    Country         Population                         95th percentile consumer
                                                                                           

    1. Using point estimates for consumption and mercury residues in fish

    Australia       Total population                   2.1-5.6 µg/kg bw per week
                    Women of child-bearing age         1.4-4.9 µg/kg bw per week

    2. Using distributions of consumption and mercury residues in fish and a Monte Carlo
    simulation model

    United States

    Scenario 1: No limit, e.g. assuming current distribution of residues in fish as sampled
    in the United States

                    Children 2-5 years                 All seafood   1.5 µg/kg bw per week
                    Women                              All seafood   0.8 µg/kg bw per week
                    Total US population                All seafood   0.9 µg/kg bw per week

    Scenario 2: Limit of 1 mg/kg of fish, assuming all residues above the limit are
    eliminated from the food supply

                    Children 2-5 years                 All seafood   1.4 µg/kg bw per week
                    Women                              All seafood   0.7 µg/kg bw per week
                    Total US population                All seafood   0.9 µg/kg bw per week

    Scenario 3: Limit of 0.5 mg/ kg of fish, assuming all residues above the limit are
    eliminated from the food supply

                    Children 2-5 years                 All seafood   1.4 µg/kg bw per week
                    Women                              All seafood   0.6 µg/kg bw per week
                    Total US population                All seafood   0.8 µg/kg bw per week

    Table 10. (continued)
                                                                                           

    Country         Population                         95th percentile consumer
                                                                                           

    Scenario 4: Limit of 0.2 mg/kg of fish assuming all residues above the limit are
    eliminated from the food supply

                    Children 2-5 years                 All seafood   0.8 µg/kg bw per week
                    Women                              All seafood   0.4 µg/kg bw per week
                    Total US population                All seafood   0.5 µg/kg bw per week
                                                                                           
    

    which is a critical detoxification step. Methylmercury and other
    mercury compounds have a strong affinity for sulfur and selenium.
    Although selenium has been suggested to provide protection against the
    toxic effects of methylmercury, no such effect has been demonstrated.

         A variety of effects have been observed in animals treated with
    toxic doses of methylmercury. Some of these, such as renal damage and
    anorexia, have not been observed in humans exposed to high doses. The
    primary tissues of concern in humans are the nervous system and
    particularly the developing brain, and these have been the focus of
    epidemiological studies.

         Methylmercury induces neurotoxicity in small rodents such as mice
    and rats at doses that usually also affect other organ systems.
    Moreover, the maternal dose that damages the nervous system of
    offspring exposed in utero also results in maternal toxicity. The main
    neurological signs are impairment of coordination and pathological
    changes in selected areas of the brain and spinal cord. Similar
    effects are seen in domestic animals. In cats, no difference in
    toxicity was observed between methylmercury naturally present in fish
    and methylmercury added in pure form to the diet.

         Similar effects of methylmercury were observed in four-year
    studies in non-human primates, in which the techniques used to detect
    neuronal damage included pathological and behavioural tests and
    investigations of the visual and auditory systems. Although the number
    of animals included in these experiments was small, the NOEL was 10
    µg/kg bw per day (expressed as mercury and corresponding to a
    steady-state blood concentration of 0.4 µg/L).The clearance, half-life
    and blood concentrations of methylmercury at steady-state depend on
    the body surface area. On the basis of body weight, small animals are
    much less sensitive to methylmercury than are humans, while the
    sensitivity of non-human primates is similar to that of humans.

         The two biomarkers used most frequently for quantifying the
    burden of methylmercury in the human body are blood and hair
    concentrations. Establishment of a quantitative relationship between
    exposure (daily intake) and concentrations in blood and hair began
    with a study of accidental consumption of grain treated with
    methylmercury fungicide in Iraq. Although the weight of evidence
    suggested that every microgram per litre increase in blood
    concentration results in an increase of 140-370 µg/kg of hair, in six
    of ten studies, the ratio of hair:blood concentration was 230-280. The
    Committee concluded that a ratio of 250 is a reasonable central
    estimate of the ratio of hair:blood concentration. The approximate
    relationships between weekly intake and blood concentration of mercury
    at steady state indicate that intake of 1 µg of mercury per kg bw per
    week in the form of methylmercury corresponds to a concentration of
    mercury of 10 µg/L of blood and 2.5 mg/kg of hair.

         Since the Committee's previous consideration of methylmercury, a
    considerable amount of data have become available on the possible
    neurobehavioural effects of prenatal and postnatal exposure. The most

    relevant data are from two large prospective epidemiological studies
    of cohorts assembled from the populations of the Faroe Islands and the
    Seychelles, who eat large amounts of seafood. The prenatal exposure of
    the two cohorts to mercury appears to have been similar. The geometric
    mean concentration of mercury in the hair of mothers during pregnancy
    was 4.3 µg/g (interquartile range, 3-8 µg/g) in the Faroe Islands and
    6.8 µg/g (range, 0.5-27 µg/g) in the Seychelles. In the Faroe Islands,
    the geometric mean concentration in umbilical cord blood was 23 µg/L
    (interquartile range, 13-41 µg/L). In the Faroes, no association was
    seen between the extent of prenatal exposure to mercury and
    performance in clinical or neurophysiological tests, although
    significant decrements were observed in the children's scores in tests
    of functions such as fine motor skill, attention, language,
    visual-spatial skills, and memory. When the 15% of the children whose
    mothers had had hair concentrations of mercury greater than 10 µg/g
    were excluded from the analyses, most of the associations were still
    apparent. In the study in the Seychelles, no adverse effects
    associated with exposure to mercury were reported.

         Several differences between the studies may have contributed to
    the apparent discrepancy in the findings. First, the children were
    evaluated for neurobehavioural end-points at different ages and with
    different tests. In the Faroe Islands, the first neurobehavioural
    evaluation was conducted when the children were 84 months (seven
    years) of age, whereas in the Seychelles, the children were assessed
    at 6, 19, 29, and 66 months of age. As the capabilities of young
    children change rapidly, the scores at different ages may reflect
    performance in qualitatively different types of tasks, and scores
    achieved by children of different ages cannot be compared easily. In
    addition, although early childhood development was assessed in both
    studies, different batteries of tests were used. In the Faroes, the
    battery consisted of tests that focus on specific aspects of language,
    memory, fine motor function, attention, and visual-spatial skills. In
    the Seychelles, the main test was a general test of development that
    includes performance in many aspects of neurological function,
    although general tests of language, visual-spatial skills and academic
    achievement were also used. Even though some types of neurological
    function were assessed in both studies (e.g. language and memory), the
    differences in the specific tests used make the findings difficult to
    compare.

         Second, the two study cohorts may also differ with regard to
    exposure to other factors that can affect the neurobehavioural
    development of children. In the Faroes, many potential confounding
    factors were addressed in the analysis, including exposure to
    polychlorinated biphenyls (PCBs). Pilot whale is the major source of
    both methylmercury and PCBs in this population, and PCBs are thought
    to adversely affect the neurodevelopment of children exposed
    prenatally. When PCBs were measured in samples of umbilical cord
    tissue (blood and plasma were not available) from one-half of the
    Faroe Islands cohort, the average PCB concentration in cord tissue

    lipids was lower than that previously reported in breast milk lipids
    in the same population, indicating that cord tissue concentration may
    not be an appropriate indicator of the burden of PCBs. In the
    Seychelles, potential confounding exposures were not addressed, but it
    has been suggested that the finding that a higher intake of mercury
    was associated with higher scores in some tests of development is a
    result of nutritional factors or mitigating substances present in
    fish.

         Third, the intake patterns of the two cohorts may have differed.
    Most of the methylmercury consumed in the Faroes is from pilot whale,
    which is eaten less frequently than fish but contains more mercury per
    serving. In contrast, the source of methylmercury in the Seychelles is
    fish, which is consumed almost daily. Therefore, the intake of
    methylmercury in the Faroes may be episodic, with high peak
    concentrations of intake. Although the effect of methylmercury on
    neurobehavioural development has generally been presumed to be a
    function of cumulative intake, short-term peak intake may also be
    important.

         Further follow-up of these cohorts, with greater coordination
    between the study organizers, would be helpful for addressing some of
    the issues of assessment. For example, the cohort in the Seychelles
    was evaluated at 96 months with many of the same tests as were used in
    the Faroe Islands, and the results are expected to become available in
    the near future.

         The Environmental Health Criteria monograph on methylmercury
    (WHO, 1990) cited the need 'for epidemiological studies on children
    exposed in utero to concentrations of methylmercury that result in
    peak concentrations of mercury in maternal hair below 20 µg/g, in
    order to screen for those effects only detectable by available
    psychological and behavioural tests'. This proposal arose from an
    evaluation of data from a study in Iraq, which implied that adverse
    effects were seen with peak concentrations of 10-20 µg/g of maternal
    hair.

    5.  EVALUATION

         The studies in the Faroe Islands and the Seychelles that were
    evaluated by the Committee did not provide consistent evidence of
    neurodevelopmental effects in children of mothers whose intake of
    methylmercury yielded hair burdens of 20 µg/g or less. The Committee
    could not evaluate the risks for the complex and subtle neurological
    end-points used in these studies that would be associated with lower
    intakes. In the absence of any clear indication of a consistent risk
    in these recent studies, the Committee recommended that methylmercury
    be re-evaluated in 2002, when the 96-month evaluation of the
    Seychelles cohort and other relevant data that may become available
    can be considered. The Committee noted that fish contribute

    importantly to nutrition, especially in certain regional and ethnic
    diets, and recommended that nutritional benefits be weighed against
    the possibility of harm when limits on the methylmercury
    concentrations in fish or on fish consumption are being considered.

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    See Also:
       Toxicological Abbreviations
       Methylmercury (EHC 101, 1990)
       Methylmercury (WHO Food Additives Series 52)
       Methylmercury (WHO Food Additives Series 24)
       METHYLMERCURY (JECFA Evaluation)