first value for this element to be reported for any lunar sample. The overall precision of our results is on the order of 10-40%, which is reasonable considering the fact that the method involves the determination of nanogram and subnanogram amounts of these elements. In addition to analytical errors, there is a strong possibility of sample inhomogeneiety for these elements. In favorable cases, such as the analysis of Allende where the concentrations of the noble metals are considerably higher, the precision expressed as relative standard deviation for a single determination varied from 0.7% for platinum to 14% for ruthenium. Thus, the proposed method for the determination of
noble metals in geological samples is capable of providing good analytical data for the platinum metals and gold. Although Ru and Os could not be determined in every terrestrial rock in this study because of their low concentrations, use of a higher neutron flux, longer irradiation time, and/or larger samples should make their determination possible using this method. The method is simple and rapid and provides data on all six elements in the same aliquot of sample. Received for review July 9, 1973. Accepted August 23, 1973. This work was supported in part by the National Aeronautics and Space Administration under Grant NGR-33-010-166.
Instrumental Neutron Activation Analysis for Mercury in Dogs Administered Methylmercury Chloride: Use of a Low Energy Photon Detector Melvin
H. Friedman, Eugene Miller,
and James T. Tanner
Bureau of Foods, Food and Drug Administration, Washington, D. C. 20204
Mercury has been determined by nondestructive neutron activation analysis in samples of brain tissue from beagles which had been fed methylmercury chloride. The mercury concentration was not uniformly distributed throughout the central nervous system and the fastest rise in concentration occurred in components of the visual system. The analytical procedure was capable of measuring mercury instrumentally and routinely in small samples of biological materials at approximately the 0.2-ppm level within a few days after irradiation with short counting times. Comparative measurements showed that mercury determination based on lg7Hg could be done with greater sensitivity by using a Ge(Li) low energy photon detector rather than a conventional high resolution, high efficiency coaxial Ge(Li) detector.
The Food and Drug Administration, as part of its continuing toxicological program, initiated a histopathological study of possible damage to the central nervous system of animals exposed to lethal and sublethal doses of methylmercury. T w o comprehensive reports have appeared ( I , 2) which pointed out the need for accurate studies of the toxicological effects of methylmercury compounds on the brain. Tissues from the central nervous system were chosen for these studies since these tissues were shown to be the ones most critically affected in methylmercury poisoning (2). Tissues from discrete parts of the central nervous system were used, rather than brain homogenates, since it was suspected that mercury might not be distributed uniformly in the brain. ( 1 ) "Mercury in the Environment-A Toxicoiogical and Epidemiological Appraisal," L. Friberg and J. Vostal, Ed., Environmental Protection Agency Report (Nov. 1971), Contract No. CPA 70-30. ( 2 ) G . Lofroth. "Methylmercury," Ecological Research Committee, Bulletin No. 4 . Swedish Naturai Science Research Council, 1970.
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The main emphasis in this report will be on the analytical technique used t o determine mercury in small brain tissue samples. In addition, a summary of the major biological implications will be given. The technique employs neutron activation analysis as the analytical method. Typically, neutron activation analysis measurements have been based on 197Hg or z03Hg. The 197Hg nuclide initially has a much greater activity than the 203Hg nuclide (3) and so offers the possibility of a more sensitive measurement. However, the electromagnetic radiation (X-ray or gamma-ray) associated with the decay of 197Hg is low in energy ( < l o 0 keV), and conventional measurements of low levels of mercury have been hampered by a large Compton continuum in this region and by a resolution inadequate to resolve possibly interfering X-rays from elements with adjacent atomic numbers. A Ge(Li) low energy photon detector (LEPD) was used in this work because of the smaller continuous background in the low energy region and better resolution, and so was expected to reduce these problems ( 4 ) . Instrumental neutron activation analyses for mercury have been reported with a sensitivity of approximately 0.02 ppm. In these analyses, a large volume Ge(Li) detector was used. These measurements ( 5 ) involved 3-hour irradiations, 2-gram samples, and counting times of approximately 2 hours per sample. In this work, a comparison was made between a conventional large volume Ge(Li) detector and the Ge(Li) LEPD for the determination of mercury based on 197Hg.
(3) F. Baumgartner, "Table of Neutron Activation Constants," Verlag Karlthiemig KG, Munchen, 1967, p 30. (4) J. Weaver, Amer. L a b . , Mar,ch, 1973, p 36. (5) V. P. Guinn and R. Kishore, "Proceedings of the Amerlcan Nuclear Society Topical Meeting on Nuclear Methods in Environmental Research, University of Missouri, Columbia, Mo., Aug. 23-24, 1971, p
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EXPERIMENTAL Apparatus. The 10-megawatt reactor [6 X 1013 neutrons/(cm* sec)] a t the National Bureau of Standards, Washington, D.C., was used. An ORTEC Ge(Li) low energy photon detector was used. I t has a resolution of 522 eV a t 122 keV; sensitive depth, 4.83 m m ; diameter of 16 m m ; and a 0.13-mm beryllium window which allows low energy electromagnetic radiation t o pass through with little attenuation. Signals from the detector were fed through a preamplifier, amplifier, and then into a biased amplifier and from there into a computer-based Nuclear Data 4410 analyzer. A biased amplifier was used to expand the region of interest over a few channels. Samples a n d standards were contained in cylindrical quartz vials, 7 x 50 m m . Standards. Mercbry standards, each consisting of 25 pl of a dilute mercury solution adsorbed onto Si02 powder (99.9999% SiOn) in a quartz vial and sealed with a n oxygen--methane torch, were prepared as follows: A weighed quantity of reagent grade mercuric acetate was dissolved in acetic acid a n d diluted to volume with distilled water. Standards were made several times during the course of this work and contained between 1 2 and 15 c(g, depending on the particular standard. Procedure. Sample Preparation. A daily dose of 3 mg/kg of methylmercury chloride in olive oil was administered orally by capsule to beagles for periods of 2, 4, 6, and 8 days. I n addition, there was a control dog which was not fed any methylmercury chloride. The animals were sacrificed after a predetermined number of doses, and their brains were perfused with physiological saline solution to avoid artifacts introduced by methylmercury in the blood. Discrete areas of the central nervous system were dissected and taken for analysis. Because of the small size of the central nervous system, the sample size was typically 50-200 mg. Samples in quartz vials were dried for 5-7 days in a vacuum desiccator t o prevent possible pressure buildup during subsequent irradiation. Later experiments showed that it was unnecessary to dry the tissue because pressure buildup was negligible, and additional samples were then analyzed directly without drying. The quartz vials containing the samples were sealed with a n oxygenmethane torch and scribed for identification. An empty quartz vial served as a blank. Irradiation. The 2 mercury standards and 5 samples were packaged together in an irradiation container and irradiated for 1 hr in the high flux position of the reactor. After irradiation, the samples and standards were allowed to stand for about 3 days to permit short-lived radionuclides time to decay. The irradiation container was opened, and the vials were prepared for counting by cleaning with aqua regia ( t o remove any surface contamination) and then rinsing with water. Counting. T h e samples and standards were analyzed with the Ge(Li) L E P D detector. Samples, standards, and blanks were counted approximately 3 cm away and with t h e vial's longitudinal axis parallel to the plane of the detector window. With this geometry, variations in distance between the active area of the detector and any point within the sample vial were minimized. Mercury was not detected in the blanks. A counting time of 20 min was adequate for these determinations. Data Reduction. T h e spectra obtained from the low energy photon detector were read out on a magnetic tape unit compatible with the UNIVAC 1108 computer a t the National Bureau of Standards. Under control of a d a t a reduction program, spectra for each sample were read from magnetic tape and parameters describing the individual sample were read from IBM cards. The concentration of mercury or an upper limit was calculated by comparing the activity of the sample with t h a t of the standard irradiated with it. Independent determinations of the activity of a sample or standard were obtained using the total peak area method (6) for the three most prominent Hg photo peaks (66.9, 68.8, and 7 7 . 3 keV) of the spectrum. T h e concentration of Hg computed from the peaks and the average were output by the computer.
Figure 1. Comparison of a l g 7 H g spectrum taken with a LEPD; a 16%, 2.1 k e V Ge(Li) detector: and a 7%, 5 k e V Ge(Li) detector To prevent the curves from intersecting, the spectrum measured with the 7% detector was counted for the longest length of time and the spectrum measured with the LEPD was counted for the shortest length of time
Validation and Sensitivity. Available NBS standard reference materials were used to validate the procedure, namely, tuna fish (SRM No 1578) and 'orchard leaves (SRM No. 1571). Four determinations were made on the tuna fish samples, and the results averaged 0.9 f 0.1 ppm
Hg, as compared with the preliminary NBS value (7) of 1.00 f 0.04 ppm Hg. Two different determinations were made on the orchard leaves. However, a longer counting time (16 hr) and irradiation time (4 hr) were used because of the low level of mercury and the comparatively high value of the continuous background in the orchard leaf spectrum. The results obtained were 0.13 i 0.01 and 0.15 f 0.01 ppm Hg, as compared with the NBS certified value of 0.155 f 0.015 ppm Hg. The results from the orchard leaves indicate that the instrumental technique using a low energy photon detector and the 197Hg nuclide can measure mercury instrumentally a t approximately the 0.2-ppm level. Evaluation of Low Energy Photon Detector. In order to determine if a LEPD had advantages over a conventional Ge(Li) detector, a spectrum of the sample was taken with each type of detector under the best conditions for that detector. A 197Hg spectrum was counted: 1) with a Ge(Li) LEPD, 2) with a Ge(Li) detector of 16% efficiency [with respect to NaI(Tl)], 2.1 keV FWHM resolution a t 1.33 MeV, and 3) with an older 7% Ge(Li), 5 keV FWHM detector for the purpose of comparing resolution in the 70 keV region. These results are shown in Figure 1. With the 7% detector the peaks are not resolved, while with the 16% detector the peaks are only partially resolved; this is to be compared with the resolution of the low energy photon detector where the photo peaks are completely resolved. To determine the relative sensitivity of the 16% detector and the Ge(Li) LEPD, a mercury spectrum from a brain sample was taken with each detector. The comparison is shown in Figure 2. A counting time of 20 min was used for both detectors. T o avoid having an excessive count rate with the 16% Ge(Li), it was necessary to count the sample further away with this detector than with the LEPD. The continuous background was approximately the same with both detectors. It appears from Figure 2 that for analyses based on 197Hg, the LEPD can analyze for mercury instrumentally a t lower levels than a conventional Ge(Li) detector. The improved sensitivity comes about because of an improved peak to adjacent Compton background ratio.
(6) P. A. Baedecker,AnaL Chern.. 4 3 , 4 0 5 (1971).
(7) P. D. LaFleur, National Bureau personal communication, 1973.
RESULTS AND DISCUSSION
of
Standards, Washington, D. C..
ANALYTICAL CHEMISTRY, VOL. 46, NO. 2, F E B R U A R Y 1974
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COUNTED W I T H L E P O
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Figure 3.
Probability of detecting a photon vs. detector thick-
ness
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Figure 2. Comparison of a mercury spectrum from a brain tissue sample measured with a Ge(Li) detector (16% efficiency) and with a Ge(Li) LEPD The mechanism by which the LEPD obtains a reduced Compton background in the low energy region without reducing the photopeak in this region can be understood by realizing that in going from 200 keV to 50 keV, the photoelectric cross section for germanium increases by almost two orders of magnitude (8). Thus, low energy electromagnetic radiation (-70 keV) will be detected with almost 100% probability after it traverses a short distance ( - 5 m m ) through germanium. A thicker detector could not substantially increase the number of photoelectric events at this energy but it does increase the number of Compton events observed (9). This is shown graphically in Figure 3, where the probability for detecting a 69-keV photon (this corresponds to the strongest photo peak of 197Hg) and a Compton event from a 660 keV gamma ray are plotted us. the thickness of a Ge(Li) detector. Interpretation of the Spectra. The two prominent low energy photo peaks and the highest energy photo peak in the LEPD spectrum (see Figure 2 , first, second, and fourth peaks) correspond to the Ka2, K a 1 , and KP'2 gold X-rays produced when 197Hg undergoes electron capture. The third peak was interpreted as being an unresolved doublet which consisted of the KP'1 X-ray from gold and 77.3-keV gamma ray associated with the decay of197Hg. There are two pieces of experimental data which indicated that the third peak (at 77 keV) was a doublet. First, close examination showed that there was a shoulder on the right side of the peak and second, the four peaks were found to have relative peak areas in the proportions of 58: 100:82:8.3. The relative experimentally determined and Kp'2 peaks quoted strengths for gold Ka2, KCQ, by Wapstra et al. (IO, I I ) are 55:100:35:9. Thus, the mea(8) J. H . Hubbell, "Photon Cross Sections, Attenuation Coefficients, and Energy Absorption Coefficients from 10 keV to 100 GeV," National Bureau of Standards Report NSRDS-NBS 29, 1969. ( 9 ) D. M . Walker, M . E. McLain, and G. W. Leddicotte, /E€ Trans. Nucl. Sci., 19, 186 (1972). (10) A . H. Wapstra, G. J. Nijgh. and R . van Lieshout, "Nucl. Spect. Tables," North Holland Publishing co., Amsterdam, 1959. (11) C. M . Lederer, J. M . Hollander, and I. Perlman, "Table of Isotopes," Sixth ed., John Wiley and Sons, New York, N . Y . , 1967, p 571.
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sured relative areas of the Ka2, K u l , and Kp'2 peaks are in fairly good agreement with those determined by Wapstra et al., but the third peak at 77 keV has more than twice the strength expected for a KP'1 X-ray. This additional strength was attributed to the 77.3-keV gamma ray Retention of Mercury d u r i n g Drying. The technique reported here, which involved drying the sample by vacuum desiccation, and the method of flameless atomic absorption spectrophotometry of Magos (12), for which no drying was necessary, were compared on several homogenized spiked and unspiked samples. The results determined were equivalent within experimental errors in all cases, indicating that losses of mercury were not a problem in this work. In addition, recent work by LaFleur (13) has also shown that methylmercury was not lost from brain tissues during freeze-drying. Biological Implications. Table I shows the various concentrations of mercury in the central nervous system of beagles following daily administration of 3 mg/kg of methylmercury for a period of 2 , 4, 6, and 8 days. Also shown are results for a control dog which received no mercury, Mercury was not distributed evenly throughout the central nervous system. The fastest rise in levels of mercury occurred in the components of the visual system (occipital and calcarine cortex), This was consistent with the observation that alkylmercury poisoning almost always results in impaired vision or blindness. Low concentrations of mercury were observed in the spinal cord. The dose of CH3HgC1 was administered orally and the mercury in the olive oil sometimes induced vomiting at various intervals following the administration. This fact plus the biological variations between animals contributed the greatest uncertainty to the measurements. The accuracy of the analysis, apart from biological variations, was determined to be better than 10%. Gamma and X-ray peaks associated with the decay of 197Hg were not seen in the spectra of the control dog tissues. The values quoted in the first column of Table I (control dog A) are upper limits equal to 2.33 times the standard deviation of the background and correspond to the critical limit defined by Currie (14).
CONCLUSIONS Perhaps the most significant finding with regard to methodology was that neutron activation analysis affords a specific, rapid, convenient method for mapping mercury accumulation in the central nervous system. Low concen(12) L. Magos, Analyst (London), 96, 847 (1971). (13) P. D. LaFleur, Anal. Chem., 45, 1534 (1973). (14) L. A. Currie, Anal. Chsm.. 40, 586 (1968).
ANALYTICAL CHEMISTRY, VOL. 46, NO. 2, FEBRUARY 1974
Table I. Mercury Concentrations i n Discrete Areas of the Central Nervous System of Beaglesn (ppm Hg) Control Dog A (0 doses)
StNCtUP2
Motor cortex Prefrontal cortex Occipital cortex Superior collicuius Inferior colliculus Caudate nucleus Pons Medulla Vermis (of the cerebellum) Cerebellum hemisphere Spinal cord (cervical) Spinal cord (lumbar) Sciatic nerve Optic nerve Calcarine cortex a
