National Toxicology Program

Exposure Panel

• Chair: Dr. Steve Olin, International Life Sciences Institute
• Dr. Gunnar Nordberg, University of Umea
• Dr. H. Vasken Aposhian, University of Arizone
• Dr. Fred Farris, Mercer University
• Dr. Bruce Fowler, University of Maryland
• Dr. Alan Stern, New Jersey Department of Environmental Protection

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WORKSHOP ON SCIENTIFIC ISSUES RELEVANT TO
ASSESSMENT OF HEALTH EFFECTS FROM
EXPOSURE TO METHYLMERCURY

November 18-20, 1998
Raleigh, NC

Report of the Exposure Panel

Introduction

Mercury occurs in the environment in elemental, inorganic and organic forms. In aquatic ecosystems, inorganic mercury can be converted to organic forms, and in particular the methylation of mercury is the major route by which mercury enters the aquatic food chain. Methylmercury (CH₃Hg⁺) is the predominant form of mercury in fish, and all fish contain some methylmercury. Globally, fish consumption is generally considered to be the most common source of human exposure to methylmercury.

Data were presented at this workshop for populations exposed to methylmercury in Iraq, the Seychelles, the Faroe Islands, and the Brazilian Amazon region. [It is assumed that the data presented by the various research groups during the workshop will be summarized elsewhere in this report, or at least a bibliography of the relevant papers will be included, so the Exposure Panel has not attempted to reiterate these data in its report.] Given the workshop objective ("To discuss and evaluate the major epidemiologic studies associating methylmercury exposure with an array of developmental measures in children"), the studies in the Seychelles and the Faroe Islands provided the major focus for our discussions.

The Exposure Panel was asked to consider two broad questions:

1. For each study, what are the relative exposures to organic or inorganic mercury?
2. What are the sources of exposure? Is the consumption of fish, shellfish and marine mammals the dominant source? Are dental amalgams, occupational exposures or other sources significant confounders?

A number of subquestions were included under Question #2, and the research groups responded to these to varying degrees in their presentations. Comments from the Exposure Panel are interwoven in the discussion that follows.

In addition, the Exposure Panel identified three important issues for the interpretation of exposure data from these studies:

• Measurements in umbilical cord blood vs. maternal hair
• Patterns of exposure
• Other potentially relevant exposures

These issues relate to currently available data from the studies, as well as data gaps, and will be discussed in turn below.

Workshop Questions

Methylmercury is of particular interest for several reasons. It is the principal form of mercury in fish, in human hair, and in whole blood of fish-consuming populations. It readily passes the blood-brain barrier and through the umbilical cord to the fetus. Human exposures to high levels have resulted in neurotoxicity.

In the Iraqi incident in 1971-72, it has been well-documented that the poisonings resulted primarily from consumption in rural areas of homemade bread prepared from wheat (seed wheat) treated with methylmercury fungicides. In wheat and flour samples analyzed during and after the outbreak, methylmercury was the predominant form of mercury. These samples also contained small amounts of ethylmercury but this was never more than 8% of the methylmercury level in analyzed samples, and no ethylmercury was detected in hair samples of patients. Barley treated with methylmercury and phenylmercury fungicides and fed to farm animals was also examined as a potential source, but levels of mercury in meat and dairy products were too low to be significant contributors.

In the Seychelles, where deep sea and reef fish are staples in the population's diet, mercury exposure is primarily in the form of methylmercury from fish consumption. Mothers in the Seychelles Child Development Study reported consuming an average of 12 fish meals a week during pregnancy. Methylmercury comprised more than 90% of the total mercury content in 20 species of fish eaten in the Seychelles. It is reported that the Seychellois do not eat marine mammals. Although not systematically studied, dental amalgams and occupational exposures do not appear to be significant sources, and exposure to inorganic mercury appears to be low.

In the Faroe Islands two main dietary sources of exposure to methylmercury have been identified: pilot whale and fish. Pilot whale is a traditional food in the Faroe Islands, but its availability varies substantially with location and over time. Pilot whale meat, on average, contains significantly higher concentrations of methylmercury than the local fish (mainly cod), although detailed data on levels in the several species of fish commonly consumed in the Faroe Islands were not available. In addition, it is reported that about half of the total mercury in pilot whale meat is inorganic. Dental amalgams and occupational exposures are not significant sources of mercury exposure in the Faroe Islands.

In the Amazon, the use of mercury to amalgamate gold in mining operations has led to the release of large quantities of elemental and inorganic mercury into the air and waterways. The "slash and burn" agricultural practices and subsequent deforestation also releases mercury from the soil into the waterways, where it is converted to methylmercury. In the studies reported at the workshop, for several villages along the Tapajós River at least 250 km downstream from the mining operations, it is believed that the principal exposure is to methylmercury from fish consumption, although it is difficult to exclude the possibility of some exposures to elemental and inorganic mercury. Dental amalgams do not appear to be a significant source of exposure for this population.

General Exposure Issues

The Exposure Panel focused on three key issues (identified above) that emerged from the presentations and subsequent discussions. These are addressed briefly in the following paragraphs.

Measurements in umbilical cord blood vs. maternal hair

In the two large studies (Faroe Islands and Seychelles), a principal objective was to evaluate the offspring of mothers exposed to methylmercury during pregnancy for possible developmental effects. In the Faroe Islands study umbilical cord blood, obtained by the midwife at delivery, was selected as the preferred biomarker of fetal exposure, although maternal hair was also collected and analyzed. In the Seychelles study maternal hair was used. The question is, What is the best biomarker (or measure) of fetal exposure to methylmercury: cord blood or maternal hair?

The Exposure Panel concluded that each of these surrogate measures has advantages and disadvantages, strengths and weaknesses, and declined to select either as the single ideal measure. Instead, the Panel recommended that in future studies, where possible, both cord blood and hair be collected until there are sufficient comparative data to better evaluate their relative merits.

Some of the advantages and disadvantages of cord blood noted by the Panel included:

Measurement of methylmercury in cord blood offers as near to a direct and noninvasive measure of fetal blood levels as can be obtained. However, it is only available at a single point in time (delivery) and is a sort of composite value for the range of exposures occurring mainly during the third trimester.

In the Faroe Islands study mercury exposure as a predictor of performance in neurobehavioral tests was qualitatively similar for both cord blood hair measures, but regression coefficients for cord blood were generally larger. For both measures changes were observed in most of the same tests, although there was some indication that hair measurements may provide a better correlation with tests for motor function.

Blood is a convenient matrix for measurement of other potentially relevant exposures (e.g., Se, Pb, markers of nutritional status), and cord tissue was a useful matrix for measurement of PCBs and other lipophilic substances.

For maternal hair as a surrogate measure, the Panel noted that:

Hair is a well-established matrix for measuring exposures of an individual to methylmercury and related substances, facilitating intercomparison of studies. The correlation of levels of methylmercury in maternal hair and newborn infant brain has been reported. However, there are a number of additional variables (e.g., hair growth rate, density, color; external sources of contamination) that must be taken into account or controlled when using hair measurements.

Analysis of sequential segments of hair or continuous single strand analysis, although somewhat costly and time consuming, allows a reconstruction of the time profile of exposures over the course of a pregnancy. Analysis of longer segments of hair can provide an integral of exposures over the entire pregnancy and before.

Hair is easier to take, store, and transport than blood.

If maternal exposures to methylmercury do not vary much during pregnancy, analysis of either maternal hair or cord blood should provide similar measures of fetal exposure. If peak exposures occur at some time during pregnancy and if they are important for an understanding of neurodevelopmental effects, segmental hair analyses may be required.

Patterns of exposure

The apparent differences in outcomes between the two major studies may be due to differences in the neurobehavioral tests used or their interpretation, or to a variety of other parameters (see reports from the other Panels). But there may also be differences in patterns of exposure between the cohorts that contribute to the disparity. Temporal exposure patterns may be important for at least three reasons: (1) we do not know if there are "windows" (periods) of enhanced sensitivity of the developing fetus to the neurotoxic effects of methylmercury, but if there are, the time course of maternal/fetal exposures may be a critical factor; (2) similarly, it may be that the neurotoxic effects are more a function of episodic high-level (peak) exposures than of average (continuous) exposures over the course of a pregnancy; and (3) the pattern of consumption of pilot whale (in the Faroe Islands) and fish may affect not only methylmercury intake but also other potentially relevant exposures.

To test the correlation of patterns of exposure with the observed effects, ideally one would like to have a detailed characterization of fetal brain levels of methylmercury and any effect modifiers over the entire gestational period. Since such a detailed characterization will never be available for the populations in question, what practical steps can be taken to improve our understanding of exposure patterns and to assess their significance? The following are some suggestions for consideration, subject to the usual (and important) constraints of cost, resources, time, and likelihood of success:

• Analyze hair samples segmentally or continuously.

  Although some segmental analyses have been done for both of the large cohorts, the number of cases is insufficient to examine the question of possible correlation of outcomes with patterns of exposure (e.g., peak level, frequency of excursions above a certain level, peaks within a hypothetical sensitive "window," etc.). Segmental analyses of hair, when performed, have been measured as approximate monthly averages (ca. 1.1 cm segments), generally by a cold-vapor atomic absorption (CVAA) technique. In some cases x-ray fluorescence (XRF) spectrometry, with a resolution of about 2 mm, has been used to provide "continuous" single strand mercury analyses and to validate the CVAA data. Continuous single strand analysis by XRF might be used to determine the time course of exposures more precisely.

• Conduct more complete dietary surveys to characterize intake of fish/pilot whale over time.

  Population-based dietary surveys are difficult, time-consuming, and expensive, particularly if data are needed on long-term patterns of intake for individuals. However, the data currently available from the Faroe Islands and Seychelles studies do not permit an evaluation of temporal patterns of exposure based on the type and amount of fish or pilot whale consumed. It is reported, for example, that consumption of pilot whale by the Faroese is episodic, regional, and dependent on occasional "catches," but also that stored (frozen) portions are consumed in small quantities over extended periods of time. More detailed data are needed to characterize the patterns of exposure and their possible correlation with neurobehavioral test outcomes. The currently available measures of fish consumption (frequency, amount, species) also are only crudely characterized in either study. Of course, these kinds of dietary data can no longer be collected for the original cohorts during the relevant periods of exposure. But the inclusion of such data-gathering in new studies, or even population-based survey data, could be helpful in evaluating the possible significance of peak exposures, variability as a function of type of fish consumed, and exposures to other potentially relevant substances (e.g., PCBs in pilot whale).

• Examine the "spiking effect" of whale dinners in the Faroe Islands.

  Because pilot whale, on average, has substantially higher concentrations of methylmercury, traditional Faroese whale dinners may result in a "spike" (short-term peak) in blood methylmercury levels. The magnitude of this peak has not been measured directly, but special studies could be designed for this purpose.

• Compare full exposure distributions for the populations of interest in the Faroe Islands and the Seychelles.

  Ultimately, an adequate evaluation of the factors potentially influencing the differences in outcomes in the two large cohorts may require the characterization and comparison of the distributions of methylmercury (and other pertinent) exposures for these populations. The data currently available are not collected, presented or analyzed consistently between the studies. It seems unlikely that actual exposures during pregnancy can be reconstructed in detail at this time for the individuals in the cohorts. However, population-based data such as described above could help in modeling these exposures retrospectively and in developing realistic distributions for comparison.

• Determine historical exposures in the Amazon populations.

  The Amazon studies described at the workshop provided an interesting and credible rationale for the seasonal variation in methylmercury exposures (based on segmental analysis of hair samples), in the parallel seasonal variation in consumption of fish with high levels (piscivorous or omnivorous) or low levels (herbivorous) of methylmercury. The Exposure Panel supported the planned follow-up studies of historical exposures in the region (e.g., from sediment core samples, other populations), in view of the unique processes thought to result in these exposures and the need to confirm the correlation of effects with methylmercury.

Pattern of exposure is an important parameter that has not been fully examined as yet in any of the studies. On the other hand, it is by no means certain that the differences in outcomes of the studies will be solely, or even partially, attributable to differences in this parameter. Nevertheless, it seems prudent to consider the possible significance of this parameter to the extent that it is feasible.

Other potentially relevant exposures

Based on the available data, it is possible that other exposures are playing a role in the effects (or lack of effects) associated with methylmercury in these studies. Exposures to elemental, inorganic, and other organic forms of mercury have been considered in each case. For example, it is reported that about half of the mercury in pilot whale meat is inorganic. Inorganic mercury, however, does not readily pass the placental barrier, so with the possible exception of the Amazon studies where the effects are being studied in adults, it seems unlikely that other forms than methylmercury are significant contributors to the neurodevelopmental effects being evaluated in these studies.

Three other exposures were considered by the Exposure Panel: PCBs (and chlorinated pesticides), selenium, and omega-3-fatty acids. Specific recommendations include:

• PCBs - Extend quantitative assessment of exposures.

  Pilot whale blubber contains substantial concentrations of PCBs and other organochlorine compounds. In the Faroe Islands study PCBs were analyzed in samples of cord tissue, rather than the more traditional matrices of serum or breast milk. It was reported that a follow-up study is underway to determine the partitioning of PCBs between mother, cord tissue, and fetus. From the cord tissue data, there is already some suggestion of a PCB effect on neurological test outcomes, and it seems that the exposure data needs to be placed on a firmer basis. One possibility would be to conduct additional studies to determine the correlation of PCB levels in cord tissue (lipid-adjusted and unadjusted) with those in serum and breast milk. The possibility that PCBs may modify the effects of methylmercury, as suggested by another Panel at this workshop, clearly needs to be thoroughly examined.

  Analysis of serum from study subjects in the Seychelles showed no detectable levels of any PCB congeners. It was speculated that exposures to organochlorine pesticides may be higher in the Seychelles than in the Faroe Islands, and for completeness it would be helpful to have comparable information on exposures for the two populations.

• Selenium - Compare levels in Faroe Islands and Seychelles.

  Selenium was measured in cord tissue in the Faroe Islands study and evaluated as a possible confounder, but failed to meet the criterion for inclusion in the data analysis. No data were available from the Seychelles study. It may be useful to develop comparative data on selenium levels for the two studies, if possible. (Another Panel noted that the nature and direction of the effect of selenium on methylmercury neurotoxicity is not well understood in experimental animals, and presumably also in humans.)

• Omega-3-fatty acids - Compare existing data for the two populations.

  Omega-3- fatty acids have been shown to have beneficial effects on brain development in infants, so if there were differences in such levels within or between populations, they might help to explain the disparity in the outcomes. Plasma levels of omega-3-fatty acids parallel fish intake. Pilot whale blubber is especially rich in essential fatty acids, and in the Faroe Islands study the frequency of whale blubber dinners during pregnancy was a significant predictor of increased serum levels of essential fatty acids. It may not be possible to compare the two cohorts directly, but it may be of interest to compare the available data for the two populations as a whole to see if there are significant differences.

In addition to these three potentially relevant exposure factors, it was noted that there is an increasing awareness of the effects of general nutritional status on toxicity and of the need to fully characterize the status of study subjects. Similarly, the presence of intermittent or chronic disease in the study population may be an important variable (e.g., in the Amazon studies).

Final Thoughts

The epidemiologic studies presented at this workshop are unusually rich in exposure data. Even in occupational epidemiology, only rarely does the epidemiologist have comprehensive biological monitoring data on the study subjects themselves. The quality of the analytical methods and measurements in these studies also is undisputed. It is only because we are presented with such a rich data set that we can ask the important dose-response questions for risk assessment and risk management.

Nonetheless, the issues and recommendations that were discussed at this workshop are important and should be carefully considered and addressed to the extent possible. The risk assessment of methylmercury must be placed on the firmest scientific basis possible, and the existing apparent disparity between the only two large cohort studies is an impediment and a puzzle that, one senses, may yield to further investigation.

Stephen S. Olin, Ph.D.
Chair, Exposure Panel