Ecological Research Series
METHYLMERCURY: FORMATION IN PLANT TISSUES
Environmental Monitoring and Support Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Las Vegas, Nevada 89114
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into five series. These five broad
categories were established to facilitate further development and application of
environmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal
species, and materials. Problems are assessed for their long- and short-term
influences. Investigations include formation, transport, and pathway studies to
determine the fate of pollutants and their effects. This work provides the technical
basis for setting standards to minimize undesirable changes in living organisms
in the aquatic, terrestrial, and atmospheric environments.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/3-76-049
May 1976
METHYLMERCURY: FORMATION IN PLANT TISSUES
by
Don D. Gay
Monitoring Systems Research and Development Division
Environmental Monitoring and Support Laboratory
Las Vegas, Nevada 89114
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
ENVIRONMENTAL MONITORING AND SUPPORT LABORATORY
LAS VEGAS, NEVADA 89114
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DISCLAIMER
This report has been reviewed by the Environmental Monitoring and
Support Laboratory-Las Vegas, U.S. Environmental Protection Agency, and
approved for publication. Mention of trade names or commercial products
does not constitute endorsement or recommendation for use.
ii
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CONTENTS
Page
List of Tables iv
Introduction 1
Microorganisms 1
Animals 1
Plants 2
Summary and Conclusions 3
Materials and Methods 4
Plants 4
Growing Media 4
Planting Method 4
Germination Methods 5
Tissue Extraction Procedure 5
Modification of Westoo's Cysteine Extraction Solution 5
Infiltration and Incubation Methods 5
Gas Chromatography 6
Chelating Resin 6
Mercury Trap System 6
Monitoring Mercury from Columns 7
Verification of Methylmercury from Gas Chromatography 7
Exposure of Mercury Compounds to Ultra-Violet Irradiation 7
Purification of Reagent Grade Mercuric Nitrate 9
Thin Layer Chromatography of Stock Organic Mercury
Compounds 9
Gas Chromatographic Analysis of Stock Organic Mercury
Compounds 9
Gas Chromatograph Uniform Injection Procedure 10
Results and Discussion 10
Germination Experiments 10
Foliar Application of Mercury 11
Mercury Addition to Soil Medium 11
Infiltration and Incubation Experiments with Mercuric
Nitrate 12
Infiltration and Incubation Experiments with
Phenylmercuric Acetate 15
Extraction of Plants from a Mercury Mine Area 17
References 18
Bibliography 25
iii
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LIST OF TABLES
Number Page
1. Germination studies of little marvel peas in various
concentrations of mercuric acetate or mercuric
nitrate 11
2. Methylmercury formation from little marvel peas grown
14 days in 10 and 100 yg/g mercuric nitrate and
phenylmercuric acetate contaminated soil 12
3. Weights of alaska pea internodes used in the incubation
experiment with Hg(N03)2 and the amount of CH3HgCl
formed per gram of tissue in 90 hours 13
4. Little marvel stems and apices infiltrated and
incubated 20 hours with 10 ug/g Hg(N03)2 and control
plants with no addition of mercury to incubation
solution ..... 13
5. Formation of methylmercury as influenced by surface
sterilization and gentamicin treatment 14
6. Little marvel pea stems 57 days old, lOg, were used
for comparison on incubation media upon CH3 Hg
formation 15
iv
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INTRODUCTION
With the continued increase in mercury pollution of the entire
ecosystem through natural and man-made sources, increased study of the
exposure of vegetation is warranted. Data on the effects of mercury
pollution, the uptake and distribution of mercury, the transformation
and identification of mercury species, and the cycling of mercury in
vegetation are vitally needed to complete the environmental picture.
MICROORGANISMS
Studies on the interactions of microorganisms with mercury show that
methylation of mercury occurs with cellular populations of Clostrid-ium
(Yamada and Tonomura, 1972) and with extracts of methanogenic bacterium
(Wood, Kennedy and Rosen, 1968). Biological methylation by aquatic
microorganisms has been shown to occur anaerobically (Jensen and
Jernelov, 1969; McKinney, 1972; Yamada, 1972). The kinetics of aerobic
and anaerobic mercury methylation have been shown by Bisogni and Lawrence
(1973). The uptake, biotransformation and biodegradation of phenylmercury
by microorganisms have been studied by various investigators (Fang, 1973;
Matsaumura, Gotch and Bousch, 1971; Nelson, Blair and Bunkman, 1973).
The microbial conversion of mercury in cell-free systems was found
to involve cytochrome c in a mercury resistant strain of Pseudomonas by
Furukawa and Tonomura (1973) and Tonomura et al. (1971). Kasahara and
Anraku (1972) have shown that mercury has an inhibitory effect in the
respiratory chain of EsoheT^ahia ooli. Landner (1971) has suggested a
possible relationship between the methylation of mercury and methionine
biosynthesis in Staphylococeus. Jensen and Jernelov (1969) suggest
methylation via a complex between inorganic bivalent mercury and
homocysteine. The deactivation and degradation of mercurial seed dressings
by soil microorganisms have been shown to occur (Balicka et al., 1973;
Balicka and Musial, 1972; Kressling, 1961; and Kimura and Miller, 1964).
Rose (1968) and Wedding and Kendrich (1959) have shown the mechanisms of
binding of soluble mercuric compounds by microorganisms. The volatilization
of mercury by certain bacteria was shown by Magos et al. (1964).
ANIMALS
The metabolism of methyl- and dimethylmercury has been studied in
animals (Ostlund, 1969). The biotransformation and biodegradation of
mercury and mercury compounds were studied in animals by LeFevre and
Daniel (1973) and Norseth and Clarkson (1970). The use of a liver
homogenate.was employed by Imura, Pan and Ukita (1972) to show methylation
of inorganic mercury.
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PLANTS
Most studies are concerned with the uptake of mercury by various
plant species (Aarkrog and Lippert, 1971; Gilmour and Miller, 1973;
John, 1972; Bache et al., 1973; Ross and Stewart, 1962; Gerdes, et al.,
1974; Haney and Lipsey, 1973; Tkachuk and Kuzina, 1972; Lee et al.,
1972; Smart, 1964, Fukunaga et al., 1972; Newsome, 1971; and Alvarez,
1974) . In addition to the translocation studies of phenylmercury
compounds in rice plants, Fukunaga et al. (1972) found traces of methyl-
mercury present in phenylmercury treated rice plants.
Clendenning and North (1958), working with the giant kelp Maeroeystis
pyrifera, showed that 100 nanograms/gram of mercuric chloride (HgCl,) in
water caused a 50% inactivation of photosynthesis. Harriss et al. (1970)
reported that concentrations of organomercurial fungicides as low as
0.1 nanograms/gram in water reduced photosynthesis and growth of plankton.
The fresh water planktonic diatom, Synecbfa ulna, was investigated by
Fujita and Hashizume (1972) for its ability to accumulate mercury. The
uptake of mercury from the medium occurred rapidly during the first 7
hours. The mercury was found mainly adsorbed to the surface of the cells.
An inhibition of photophosphorylation by isolated chloroplasts was
found by Bradeen et al. (1973) to be inhibited by the addition of mercuric
chloride. Only one of the two functionally isolated sites of photo-
phosphorylation coupled to electron transport, the site located between
the oxidation of plastoquinone and the reduction of cytochrome f was found
to be sensitive to mercuric chloride. Watling-Payne and Selwyn (1974)
found that phenylmercuric acetate was a poor inhibitor of photophosphory-
lation. Radmer and Kok (1974) showed that mercuric chloride, when added
to isolated chloroplasts, inhibited the electron flow between Photosys terns
I and II. This was due to the inactivation of plastocyanin, an electron
carrier close to
Ahmed and Grant (1972) showed cytological abnormalities occurring in
root tips of Tradescantia and Vioia faba following treatment of the tips
with 1 to 5 micrograms/gram of Panogen 15, a mercurial fungicide. Rao
et al. (1966), working with pea roots in mercuric and phenylmercuric
acetate solutions showed a cellular distribution of mercury based on
differential centrifugation of homogenized root tips into nuclear,
mitochondrial, microsomal, and soluble fractions. Spraying of phenyl-
mercuric acetate onto the leaves of Coffea ardbiaa greatly reduced the
zinc content of leaves at the distal ends of the shoots. Bock et al.
(1958) measured the zinc content of leaves towards the end of the dry
season and eight weeks later after a rainy period and obtained the same
reduction in zinc content of leaves sprayed with phenylmercuric acetate
as compared to nonsprayed control leaves. Puerner and Siegel (1972)
showed that mercuric chloride alone, in admixture with fluorescein or
chemically combined as mercurochrome , inhibited cucumber growth and induced
dis orientation of root and shoot. The inhibitory effects of mercury-ion
were reduced but the disorienting (ageotropic) effects enhanced by the
presence of fluorescein.
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Anelli et al. (1973) investigated the influence of metallic mercury
vapor on the amino acid content of tobacco leaves. Their selection of
tobacco is based on the work of Zimmerman and Crocker (1934) which showed
that tobacco is one of the most resistant plants to mercury poisoning
since it can accumulate more than 3,000 micrograms/gram of mercury in
leaves and still be viable at maturity. Anelli et al. (1973) showed that
the insoluble proteic amino acid content increased throughout the experi-
ment (after a slight decrease due to initial poisoning of mercury). The
amounts of cysteine, glutamic acid and glycine appeared exceptionally
high. The level of cysteine in mercury vapor treated plants was over
six times as great as the level in control plants.
Through the use of x-ray crystallography, Wong et al. (1973a)
determined that the binding of methylmercury to the sulphur-containing
amino acid penicillamine in a 1:1 ratio occurs by the deprotonation of
the sulfhydryl group. Simpson et al. (1973), using a model peptide,
N-acetyl-L-cysteine, showed binding of methylmercury to the peptide via
proton magnetic resonance studies.
The possibility of mercury as a trace element for plant growth and
development was suggested by Dobrolyubskii (1959). In his work with
grapevines sprayed twice with mercuric sulfate (HgSO^), he noted an
increase in the weight of 100 berries sprayed vs. control, an increased
sugar content, decreased berry acidity as tartaric acid and an acceleration
of grape ripening as shown by an increase in its glucoacidometric index.
The chlorophyll content in treated leaves increased over the control.
The carbohydrate metabolism was changed after the addition of mercuric
sulfate. The sugar content increased -at the expense of sucrose which was
all converted to glucose and fructose. This conversion is due to
invertase activity which likewise increased in the treated plants.
Siegel et al. (1974) noted analytical inconsistencies in mercury
content of plant parts from a single collection of plants when assayed
over a period of several hours. They have found an unknown volatile,
organic-solvent soluble mercury compound that is not methyl- or
dimethylmercury as determined by gas chromatography. The loss of the
volatile compounds from hexane, methanol, and water follows the volatility
series hexane>methanol>water.
The areas of metabolism and biotransformations of mercury in plants
need further study. The purpose of this study was to determine the
chemical forms of mercury in plants. Subsequent to this determination
was the elucidation of the role of the plant in transforming one chemical
form of mercury to another.
SUMMARY AND CONCLUSIONS
This investigation identified methylmercury (CH3Hg) as one of the
mercury species present in peas. The study also showed that methylmercury
was formed by the plants (a) when grown in vermiculite and the leaves
sprayed with as little as 90 milliliters of 10 micrograms/gram of mercuric
nitrate (Hg(N03)2), (b) when grown in soil with mercuric nitrate or
phenylmercury added, or (c) when sections of the pea plants were surface
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sterilized with 5% Chlorox or Gentamicin added to the incubation medium
and incubated 20 hours in 10 micrograms/gram phenylmercury acetate. The
age of the peas plays a role in the amount of methylmercury formed.
Older pea tissues produce less methylmercury than young tissues when
these tissues are infiltrated and incubated with 10 micrograms/gram of
mercuric nitrate.
To check for the presence of methyLnercury in plants grown in a
natural environment but near a mercury source, samples of three species
of plants were collected around an abandoned mercury mine. All three of
the species show the presence of methylmercury but the influcrescence of
Brcmus riibens was especially high in methylmercury. These plants were
collected in early May 1975, before the summer heat had dried all aerial
portions.
MATERIALS AND METHODS
PLANTS
Seeds of Pisum sativwn L. var. Alaska and Little Marvel were purchased
from Burpee Seed Company in Riverside, California. The supplier stated
that the expected germination was 92%.
Plants collected from the mercury mine area were (a) Bromus nJyens,
(b) SphopalQea arribigua, and (c) a Bovaginaceae.
GROWING MEDIA
Vermiculite and soil which was collected from the University of
Nevada Agricultural Experiment Station at Logandale, Nevada, were used.
The soil, a fine sandy loam, has the following characteristics:
(a) 54% sand
(b) 11% clay
(c) 35% silt
(d) 1.3% organic carbon
(e) pH 8.6
PLANTING METHOD
Plastic trays, 29.16 x 36.78 x 8.84 cm (11.5 x 14.5 x 3.5 inches) were
filled to within 1.22 cm (ig inch) of the top with a medium of vermiculite
or soil. Pea seeds were placed 2.54 to 3.76 cm (1 to 1.5 inches) apart and
pushed down into the medium even with the surface. The trays were then
filled to the top with medium, lightly tamped, and watered thoroughly.
Additional watering was done via an automatic system twice a day at 8 a.m.
and 4 p.m. If peas were grown longer than two weeks, complete nutrient
solution was added weekly.
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GERMINATION METHODS
Pea seeds were placed in a beaker of water or mercury solution,
depending on the experiment. A stream of air was bubbled through the
solution for the entire imbibing period. The swollen seeds were washed
thoroughly with distilled water and placed in pans lined with moistened
paper towels. The pans were covered with plastic-coated paper and the
paper secured with the aid of rubber bands. The pans with seeds were
then placed inside an oven set at 25° C. At the end of the germination
period, the young pea plants were separated into epicotyls, roots, and
cotyledons, and each weighed. The lengths of epicotyls and roots were
also measured and recorded.
TISSUE EXTRACTION PROCEDURE
All tissue was washed with distilled water prior to use. The tissue
was homogenized in Waring blender with 2.2N. HC1 (1 milliliter HCl/gram
tissue) for two minutes. The resultant brei was centrifuged at 10,000 rpm
for 30 to 45 minutes. The supernatant solution was decanted and filtered,
and extracted with three volumes of nanograde quality benzene, each equal
to the volume of supernate. All benzene fractions were passed through
Whatman Phase Separating paper and combined. A freshly prepared 1%
cysteine solution (toestoo 1966, 1967, 1968, 1973) was titrated to pH 8.3
to 8.4 with 5N sodium hydroxide immediately before use; 10 milliliters of
the cysteine solution per 100 milliliters of benzene was added to the
benzene and vigorously shaken. After separation of the aqueous and organic
layers, the cysteine layer was drawn off and acidified to pH 0.5 to 0.7
with 511 HC1. This acidified solution was allowed to stand for 15 minutes
after which 10 milliliters of benzene was added and vigorously shaken.
After separation, the benzene layer was drawn off into a glass vial with
anhydrous sodium sulfate covering the bottom.
MODIFICATION OF WESTOO'S CYSTEINE EXTRACTION SOLUTION
Westoo's cysteine extraction solution consists of cysteine* HC1,
sodium acetate, and anhydrous sodium sulfate. The pH of the resulting
solution is ^3.8. Using a freshly prepared methylmercury solution, only
50 to 60% recovery of the methylmercury could be obtained. Wong et al.
(1973b) found that binding of methylmercury to another sulfur-containing
amino acid, penicillamine, occurred via deprotonation of the sulfhydryl
group. The ionization of cysteine at pH 7 is only 8%. Raising the pH
to 8.3 results in 50% ionization of the sulfhydryl group of cysteine.
Westoo's cysteine solution pH of 3.8 was adjusted to pH 8.3 to 8.4 with
5% sodium hydroxide and used immediately. The recovery of methylmercury
increased to 86%.
INFILTRATION AND INCUBATION METHODS
Selected plant sections were placed in a flask containing the mercury
solution. This flask was placed inside a plastic desiccator fitted with a
stopcock. The clear plastic lid was covered with aluminum foil to make
the chamber light-free. An in-line activated charcoal filter was placed
in the pressure tubing between a vacuum pump and the desiccator. The
tissue was placed under a vacuum for 15 minutes, the vacuum pump turned
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off, and the inside of the desiccator allowed to return to an equilibrium
with the ambient pressure. The stopcock was then closed and the incubation
allowed to proceed inside the darkened chamber.
GAS CHROMATOGRAPHY
A Hewlett-Packard1 Gas Chromatograph Model 5713 with a nickel-63
linearizing electron-capture detector and a 6-foot column packed with
5% HIEFF was used to separate organic mercurial halides. The carrier
gas was 95% argon/5% methane obtained from Matheson2 and used at a flow
rate of 60 milliliters/minute. The oven temperature was 170 C. and
the detector and injection port temperatures were 200° C. The column
was on-line in the injection port.
CHELATING RESIN
To remove ionic mercury from samples before injection into the gas
chromatograph, Srafion NMRR3 chelating resin was used. Preliminary work
by Law (1971) indicated the possibility of separating ionic mercury from
methylmercury as a function of pH. Experiments at the Environmental
Monitoring and Support Laboratory-Las Vegas confirm this separation. The
resin chelates ionic mercury at low pH (pH M),5) and chelates methylmercury
at pH 6.4. The methodology of utilization of the resin is as follows.
The resin is washed repeatedly with glass distilled water, allowed to
settle for 10 minutes and the liquid decanted. After washing, 10 to 20
grams of the resin is poured into a column. A glass fiber filter is cut
to fit the inside diameter of the column. With the stopcock open, the
filter is gently pushed down on top of the resin bed. The pH of the
solution to be placed on the column is critical for chelation of the
desired mercury species.
In this case, the chelation of ionic mercury was desired. The sample
(10 to 20 milliliters) was placed on the column and flow rate adjusted to
0.5 to 0.6 milliliters/minute. After the void volume of the column had
passed through, the volume collected was equal to the volume of sample
plus 10 milliliters of the glass distilled water. Elution of the ionic
species of mercury from the column was accomplished by the addition of a
5% thiourea in 0.061* HC1. When this elution was complete, the column was
flushed with 300 milliliters of glass distilled water and regenerated for
future use.
MERCURY TRAP SYSTEM
To determine whether ethyl- or methylmercuric chloride was released
during the incubation of tissue in mercuric nitrate solution, a modification
of the Kimura and Miller (1960) mercury entrapment procedure was used.
1 Hewlett-Packard, 11300 Lomas Boulevard, Albuquerque, New Mexico 87123
2 Matheson Gas Products, 8300 Utica Avenue, Cucamonga, California 91730
3 Ayalon Water Conditioning Company, P.O. Box 586, Haifa, Israel
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Nitrogen gas was bubbled through the incubation solution and through
two traps. The first was a carbonate-phosphate solution followed by
a 1% cysteine solution at pK 8.3. The flow rate of nitrogen was 112
ailliliters/minute for 20 hours of incubation. The carbonate-phosphate
trap removes volatile ethyl- and methylmercuric chloride. The cysteine
trap is used as a check on the primary carbonate-phosphate trap. Figure
1 depicts the flow system.
MONITORING MERCURY FROM COLUMNS
A Gilson UV Monitor1* was attached to the column by 1.59-centimeter
(1-1/16-inch) tubing. By selecting the 254-nanometer filter, mercury
compounds separated by column chromatography can be easily monitored.
The small volume of the cell, 1 milliliter coupled with a reference cell,
allows for the detection of mercury down to 0.5 micrograms/gram.
VERIFICATION OF METHYLMERCURY FROM GAS CHROMATOGRAPHY
To verify that mercuric nitrate and/or the concentration of mercuric
nitrate used in the experiments was not producing a peak on the chromatogram
at the same time as methylmercuric chloride, mercuric nitrate at the
concentration of 10 micrograms/gram was dissolved in distilled water and
90 milliliters of this solution was taken through the extraction procedure.
A 5-microliter aliquot of the extract was injected into the gas chromatograph.
No methylmercuric chloride peak appeared.
With the formation of the chloride species of mercury in the
extraction procedure and the possibility of forming mercuric chloride
from the mercuric nitrate, a mercuric chloride solution at the concentra-
tion of 170 micrograms/gram was made in benzene. When 5 milliliters
of this solution was injected onto the gas chromatographic column, the
mercuric chloride peak appeared the same time as the reference methylmercuric
chloride peak. This 170-microgram/gram mercuric chloride benzene solution
was extracted via the cysteine procedure and 5 microliters of the final
10-milliliter benzene layer was injected onto the gas chromatograph
column. In this case, no peak corresponding to the methylmercuric chloride
peak appeared.
EXPOSURE OF MERCURY COMPOUNDS TO ULTRA-VIOLET IRRADIATION
Quartz cuvettes were filled with 50 or 100 micrograms/gram of mercuric
acetate or mercuric nitrate dissolved in glass distilled water. Each was
placed inside a fluorescent chamber and irradiated with ultra-violet (UV)
wave lengths for 5 to 6 hours. At the end of the irradiation period,
each was extracted with benzene, cysteine, and back into benzene. Gas
chromatographic analysis showed the formation of methylmercury by mercuric
acetate but not by mercuric nitrate. These results are in agreement with
those of Akagi and Takabatake (1973). Mercuric nitrate was chosen for use
in further experiments.
Gilson Medical Electronics, Middleton, Wisconsin
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00
— Dessicant
N
Activated Charcoal
Tinfoil Covering
Incubation
System
co3-po4
Trap
1%Cysteine
pH 8.3
Figure 1. Mercury trapping system modification, of Kimura and Miller (1960)
procedure.
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PURIFICATION OF REAGENT GRADE MERCURIC NITRATE
Reagent grade mercuric nitrate was found to contain mercuric chloride
impurity. To remove this impurity, a small portion of the mercuric
nitrate was poured into a flask and washed three times with benzene.
The mixture was shaken, allowed to settle, and the benzene was carefully
decanted. After three such treatments, diethylether was added and
carefully swirled. After allowing to settle and decanting the ether,
acetone was added and the same procedure employed except that the acetone/
mercuric nitrate was in the filter. The mercury was stirred while under
vacuum. The mercuric nitrate was transferred to a dark bottle and placed
in the freezer for future use.
THIN LAYER CHROMATOGRAPHY OF STOCK ORGANIC MERCURY COMPOUNDS
The purities of ethyl-, methyl-, and phenylmercuric compounds were
tested by thin layer chromatography with 500-microgram/gram solutions of
each made in benzene. Silica gel GF-254^ was used as the stationary
phase. Glass plates 20- x 20-centimeters and 10- x 20-centimeters were
prepared by mixing 22 grams of silica gel with 70 milliliters of boiling
water for 2 minutes in a blender. The slurry was poured into a Brinkman-
Desaga6 adjustable spreader. The slurry was spread over 100 centimeters
of glass plates at 0.25-millimeter thickness. The plates were air-dried
and stored until use. Before use the plates were dried in an oven for
30 minutes at 105° C.
Twenty-five microliters of each mercury solution was spotted on the
thin layer chromatography plates. The developing solvent systems used
to separate the organic mercury compounds were
(a) carbon tetrachloride:methylene dichloride (2:1)
(b) hexane:acetone (93:7)
(c) pentane:ethylacetate (10:1)
(d) hexane:ether (9:1)
After developing and drying, the mercury compounds were visualized on
the plate in a fluorescence chamber under short UV irradiation. Silica
gel GF-254 has a flucrescein dye added that appears green under short UV
light. Compounds on the thin layer chromatography plate mask this colora-
tion leaving a dark spot. Phenyl- and methylmercury showed one spot each;
ethylmercuric chloride showed a methylmercury spot as well as an ethyl-
mercury spot.
GAS CHROMATOGRAPHIC ANALYSIS OF STOCK ORGANIC MERCURY COMPOUNDS
Solutions of 50 micrograms/gram were made from the stock 500-microgram
gram benzene solutions of ethyl-, methyl-, and phenylmercury chlorides.
Five microliters of each was injected onto the gas chromatograph column.
The contamination of ethylmercuric chloride with methylmercuric chloride
was confirmed. The phenylmercury chloride showed no contamination of
methyl- or ethylmercury compounds.
Brinkman, 110 River Road, Des Plaines, Illinois
9
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GAS CKROMATOGRAPH UNIFORM INJECTION PROCEDURE
To ensure consistent and known volumes injected onto a gas chromato-
graphic column, a 0- to 10-^microllter syringe was used. The procedure
Is as follows:
(a) Rinse the syringe twice with solution to be injected
and flush.
(b) Draw 0.6 microliters of air into the syringe.
(c) Draw 5.0 microliters of solution to be injected into
the syringe after the 0.6 microliters of air and then
inject into the gas chromatograph.
Peak heights of standard methylmercury-benzene solutions are
reproducible to within 1%.
RESULTS AND DISCUSSION
GERMINATION EXPERIMENTS
The sensitivity of Little Mervel pea seeds to concentrations of ionic
mercury compounds was determined by germinating pea seeds in 1-, 10-, and
25-micrograms/gram distilled water solutions of mercuric acetate and
mercuric nitrate for 6 hours with aeration. They were placed in separate
germination pans after extensive washing with distilled water. The seeds
were germinated at 25°« C for 4 days completely in the dark. At the end
of this time, the young pea plants were separated into three sections—
epicotyls, roots, and cotyledons. The epicotyls and roots were measured
and the combined tissues of each section were weighed. The percent
germination of each lot of peas was recorded. Burpee Seeds states a 92%
germination for this batch of peas. Each section for each condition was
extracted according to the modified Westoo procedure to determine if any
detectable organic mercury halides were present.
The results are given in Table 1. No organic mercury was detected.
The data show that the nitrate and acetate forms of ionic mercury do not
elicit comparable responses in the same variety of peas. Only 52%
germination occurred for peas in 25 microgram/gram mercuric nitrate while
68% was noted for 25 microgram/gram mercuric acetate. The total weights
of the epicotyls and roots in the 25 microgram/gram mercuric nitrate were
100% and 73% greater, respectively, than those in 25 microgram/gram
mercuric acetate. The overall response of the peas to the mercuric acetate
was a diminution in growth and weight with an increase in mercuric acetate
concentration. The response to mercuric nitrate definitely did not
follow a diminution pattern as concentration of mercuric nitrate increased.
Based on these experiments, mercuric nitrate was chosen as the ionic form
of mercury to be used in subsequent investigations and 10 micrograms/gram
was chosen for the preliminary concentration with which to work.
10
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TABLE 1. GERMINATION STUDIES OF LITTLE MARVEL PEAS IN VARIOUS
CONCENTRATIONS OF MERCURIC ACETATE OR MERCURIC NITRATE
HgOAc yg/g
1 •
% Germination 76%
Epicotyl length-av. 1.7 cm
Root length-av. 3.4 cm
Epicotyl total wt. 0.93 g
Root total wt. 0.96 g
Cotyledon total wt. 12.35 g
CHsHg present NDa
10
76%
1.6 cm
2.8 cm
0.96 g
0.90 g
15.87 g
ND&
25
68%
1.4 cm
2.1 cm
0.62 g
0.52 g
16.6 g
NDa
Hg(N03)2 yg/g
1
92%
2.2 cm
3.4 cm
1.62 g
1.43 g
14.1 g
NDa
10
88%
1.3 cm
2.6 cm
0.84 g
1.7 g
16.13 g
NDa
25
25%
1.6 cm
2.7 cm
6.0 g
7.1 g
15.8 g
NDa
Not detectable
FOLIAR APPLICATION OF MERCURY
Little Marvel peas were grown in vermiculite in exposure chambers for
34 days with weekly addition of a complete nutrient solution. On the 34th
day after planting, the leaves of the peas were sprayed by an atomizer
over an 8—hour period with a total of 90 milliliters of 10 micrograms/gram
mercuric nitrate solution made up in distilled water. Watering was done
at the vermiculite level, so little if any of the mercuric nitrate was on
the vermiculite available to root absorption. One flat of peas was used
and the dense canopy of leaves coupled with the small volume sprayed at
any one time and the fineness of the mist eliminated any runoff to the
vermiculite. The peas were grown an additional 5 days with no further
addition of mercury. On the 39th day after planting, all tissue above
the third node was harvested and extracted according to the modified
Westoo cysteine procedure. A total of 125.53 grams of tissue fresh weight
was obtained. When 5 microliters of the final benzene layer was analyzed
by gas chromatography, a peak at the time of the methylmercury chloride
reference appeared. Relating the peak height to standards gave a concentra-
tion of 0.16 nanograms _+ 0.06 nanograms of methylmercury chloride/gram of
tissue fresh weight based on a conservative 60% efficiency for the entire
extraction procedure.
MERCURY ADDITION TO SOIL MEDIUM
Mercuric nitrate and phenylmercuric acetate in powder forms were each
mixed with 6 kilograms of sandy-loam soil, pH 8.6, with characteristics
given previously, to obtain 10 and 100 micrograms/gram soil/mercury mixture
of each. This contaminated soil was placed in trays and Little Marvel
seeds were planted in each 2.54 centimeters (1-inch) apart. After 14 days,
the aerial portions were harvested and rinsed thoroughly. The modified
cysteine extraction procedure was used to extract the tissue.
11
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Table 2 gives the amount of methylmercury found per gram of aerial
tissue from peas grown in the mercuric nitrate of phenylmercuric acetate
contaminated soil. The amount of methylmercury detected in the tissue
increased 100% from 10 micrograms/gram to 100 micrograms/gram mercuric
nitrate in the soil. The amount of methylmercury decreased with corres-
ponding concentrations of phenylmercuric acetate in the soil, indicating
that the two forms of mercury have different effects or sites of activity
within the plant.
TABLE 2. METHYLMERCURY FORMATION FROM LITTLE MARVEL PEAS GROWN 14 DAYS
IN 10 AND 100 ug/g MERCURIC NITRATE AND PHENYLMERCURIC ACETATE
CONTAMINATED SOIL
Mercury
contaminated
Hg(N03)2
Hg(N03)2
Ph-Hg-OAc
Ph-Hg-OAc
Concentration
in soil
10 yg/g
100 yg/g
10 yg/g
100 yg/g
Weight of
aerial tissue
12.9 g
23.6 g
29.1 g
24.1 g
Methylmercury present
ng/g fresh weight
3.1 +0.9 ng/g
7.6 + 2.3 ng/g
3.8+1.1 ng/g
2.1 + 0.6 ng/g
Various investigators (D'ltri, 1972; Hale and Wallace, 1970; and
Van Loon, 1974), working with soil types and soil pH have found the root
uptake of mercury into plants to decrease with neutral or alkaline soils.
The use of an alkaline soil was to determine if mercury uptake would
occur and, if so, if the small amount taken up would be transformed to
methylmercury in concentrations large enough to be detected with the
system at hand.
INFILTRATION AND INCUBATION EXPERIMENTS WITH MERCURIC NITRATE
The detection and identification of methylmercury in pea plants
after the addition of mercuric nitrate to leaves or applied to the soil
could be the result of microbial conversion of the ionic to an organic
mercury species with the concomitant uptake of the methylmercury compound
by the plant. An experimental procedure was devised to determine if the
plant tissue was capable of transforming ionic mercury to methylmercury.
Alaska peas were grown in vermiculite and after 20 days from planting
various internodes were harvested, weighed, thoroughly rinsed with dis-
tilled water, and placed in flasks with 100 milliliters of 10 micrograms/
gram mercuric nitrate in glass distilled water. The flasks were placed
under vacuum from a water aspirator for 30 minutes. The flasks were
sealed and incubated for 89% hours. The tissue in each flask was
thoroughly rinsed with distilled water (a minimum of five complete rinses
was used) and extracted via the modified Westoo procedure.
12
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Gas chromatographic analysis of TABLE 3. WEIGHTS OF ALASKA PEA
5 microliters of each final benzene INTERNODES "USED IN THE
extract revealed the presence of INCUBATION EXPERIMENT WITH Hg(N03)2
methylmercury chloride from all stem AND THE AMOUNT OF CH3HgCl FOEMED
sections. Table 3 gives the weight PER GRAM OF TISSUE IN 90 HOURS.
of tissue in each fraction and the
amount of methylmercury chloride
detected. The amount of methyl-
mercury in internode sections up to
the apical segments is three times
as much as the subtending internodes.
Little Marvel peas were har-
vested after 17 days growth into
stems and apical region. No leaves
or laterals were used. The same
infiltration procedure was used.
The incubation period was only 20
hours. After thoroughly rinsing
the tissue, each was extracted according to the modified Westoo procedure.
A comparable amount of tissue was used for a control. The stems plus
apices were thoroughly washed and infiltrated the same length of time in
glass distilled water in separate chambers with in-line activated charcoal
to eliminate the possibility of mercury contamination. The control tissue
was incubated 20 hours after which it was washed and homogenized in
2.2N HC1 in a blender and centrifuged. The supernatant solution was
extracted via the modified Westoo procedure. The pellet was resuspended
in glass distilled water by magnetically stirring for 15 minutes. The
suspension was extracted via the modified Westoo procedure.
Stem
internode
0-3
4
5
6
Apical
Total
weight
14.6 g
15.0 g
17.8 g
14.2 g
8.1 g
CH3HgCl
ng/g tissue
3.4 + 1
3.3+1
2.5 + 0.7
2.3 + 0.7
10.9 + 3.3
TABLE 4. LITTLE MARVEL STEMS AND
APICES INFILTRATED AND
INCUBATED 20 HOURS WITH 10 yg/g
HRCN03)2 AND CONTROL PLANTS WITH
NO ADDITION OF MERCURY TO INCUBA-
TION SOLUTION
The results of the tissue
incubated in 10 micrograms/gram
mercuric nitrate and the control
tissue are given in Table 4.
After 20 hours of incubation in
mercuric nitrate, steins and apices
from methylmercury with the stems
forming more per gram than apical
region. The control plants with no
addition of mercuric nitrate show
no methylmercury chloride in either
the supernate or the pellet.
The formation of methyl-
mercury from mercuric nitrate by
plant tissue within 20 hours would
seem to rule out bacterial contam-
ination as causing the transforma-
tion because the time involved is so
short. However, to determine if
bacteria associated with the pea stems are causing this transformation, two
experiments were performed using two different sterilization procedures.
Stems from Little Marvel peas grown 18 days were harvested and divided into
Tissue
Stems
Apices
Control
Total
weight
22.3 g
3.6 g
27.8 g
CH HgCl
ng/g tissue
7.3 + 2.2
4.1 + 1.2
*
* Supernatant = none detected
Pellet = none detected
13
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TABLE 5. FORMATION OF METHYLMERCURY
AS INFLUENCED BY SURFACE
STERILIZATION AND GENTAMICIN TREATMENT
two 10-gram fractions. Both, fractions were washed thoroughly with dis-
tilled water and one fraction was washed again for 5 minutes in 5%
Chlorox solution. The other fraction was the control. The Chlorox was
rinsed off thoroughly and both were infiltrated under vacuum in separate
chambers for 15 minutes and incubated for 20 hours in 100 milliliters of
10 microgram/gram mercuric nitrate. The stems were thoroughly rinsed
again to remove any mercury on the surfaces and extracted via the modified
Westoo procedure. The second experiment involved stems from Little Marvel
peas grown 38 days. Two 10-gram
fractions were obtained. Both
fractions were washed, one
placed in 100 milliliters of 10
microgram/gram mercuric nitrate
to which Gentamicin, an anti-
bacterial agent, was added
(2 milligrams/milliliter). Both
were infiltrated and incubated
in separate chambers for 20 hours,
The stems were again rinsed and
extracted via the modified
Westoo procedure. Also via the
modified Westoo procedure, 50
milliliters of concentrated
Chlorox was extracted to deter-
mine if any methylmercury was
present as a contaminant in the
Chlorox. The results of both
experiments are given in Table 5.
Treatment
5% Chlorox
No Chlorox
Gentamicin
No Gentamicin
Concentrated
Chlorox
Age of
peas
(days)
18
18
38
38
Weight
of
stems
(grams)
10
10
10
10
CH HgCl
formed
ng/g
tissue
7.6 + 2.3
5.6 + 1.7
4.0 + 1.2
4.0 + 1.2
None present
There are no significant differences between comparable experiments.
The age of tissue seems to have an effect on the ability to transform ionic
mercury into methylmercury. Stems 38 days old produced only 61% as much
methylmercury as did the stems 18 days old (using the average of both
treatments).
The incubation medium used so far has been glass distilled water.
To determine if different media would have an effect on the transformation
of ionic mercury to methylmercury, the infiltration/incubation procedure
was utilized. Comparisons were made with glass distilled water, 0.01M
dibasic potassium phosphate/monobasic potassium phosphate (K^HPO^/KI^PO^)
at pH 7.0 and K2HP0lt/KH2POJf at pH 7.0 with 2% sucrose. Little Marvel
stems 57 days old were harvested and separated into 6 fractions, 10 grams
each. A control with no mercury added was used for each incubation
medium because the stems were much older than those previously used. The
fractions of tissue containing 10 micrograms/gram of mercuric nitrate
were infiltrated and incubated in separate chambers. All the control
tissue was infiltrated and incubated in one chamber. The incubation time
was 20 hours. At the completion of the incubation period each fraction of
tissue was rinsed and extracted via the modified Westoo procedure.
The results are given in Table 6. Incubation in glass distilled water
produces the least amount of methylmercury but within an overall efficiency
estimate of 70%; the formation of methylmercury is comparable in all three
media.
14
-------
Control in glass
distilled water
10 yg/g Hg(N03)2 in
glass distilled
water
Control in PO^ buffer
10 yg/g Hg(N03)2 in
PO,
Control in P04 buffer
+ 2% sucrose
10 yg/g Hg(N03)2 in
P0[j buffer + 2% sucrose
Peak
3.
12
0
12
2.
14
Height
5 units
units
units
units
5 units
units
The influence of pH on the trans- TABLE 6. LITTLE MARVEL PEA STEMS
formation of ionic mercury to methyl- 57 DAYS OLD, 10G, WERE
mercury was determined by adjusting USED TOR COMPARISON ON INCUBATION
the pH of the incubation solutions to MEDIA "UPON CH3 Hg FORMATION
3.5, 4.5, 5.5, 6.5, 7.5 or 8.5. A
physiologic concentration of 0.01M
was used for dibasic potassium phos-
phate and monobasic potassium phos-
phate. The lowest pH (3.5) was
obtained by adding phosphoric acid.
Little Marvel peas, stems and apices
13 and 35 days old were used. The
tissue from each age group of peas
was divided into 6 10-gram frac-
tions, infiltrated and incubated
20 hours. The results as given in
Figure 2 show that the age of the
tissue has some effect on the
transformation of ionic mercury to
methylmercury. It is interesting
to note that the peak heights of
methylmercury at the two extremes
of pH comparing age of tissue are
almost the same. Obviously, other
factors that are not present or
suppressed in the physiological pH
range are influencing the formation
of methylmercury at these extremes.
With the possibility of the loss of some volatile organic mercury com-
pounds from the incubation flask during incubation, an experiment was per-
formed to trap these volatile organic mercury compounds. Little Marvel
pea stems 13 days old were harvested and 21.9 grams obtained. The stems
were thoroughly rinsed and infiltrated in a glass distilled water incubation
medium with 10 micrograms/gram mercuric nitrate. The incubation flask
with steins was placed in a nitrogen gas train as described in the methods.
The incubation period was 20 hours after which the tissue was rinsed
thoroughly and extracted via the modified Westoo procedure. The carbonate-
phosphate solution and cysteine solution were removed from the traps and also
extracted via the modified Westoo procedure. Gas chromatographic analysis
of the traps revealed the possibility of a very small amount of methyl-
mercury present. If such peaks are indeed present on the chromatograms,
they are almost at the level of background noise. The stems showed 8.3 ±
2.5 nanograms/gram tissue methylmercury.
INFILTRATION AND INCUBATION EXPERIMENTS WITH PHENYLMERCURIC ACETATE
Rice plants treated with phenylmercurie acetate reportedly produce
small quantities of methylmercury (Fukunaga et al., 1972). To determine
if phenylmercury is transformed to methylmercury, Little Marvel stems
with apices and leaves were harvested and 10 grams of each tissue was
infiltrated and incubated in separate chambers with phenylmercuric
acetate. Two 10-microgram/gram aqueous solutions were attempted but
15
-------
24
co 22
CO
CO
ca
20
18
16
14
12
10
3.5
4.5 5.5 6.5 7.5
pH OF INCUBATING SOLUTION
8.5
Figure 2. Effect of pH of the phosphate buffer incubation medium
on the transformation of ionic mercury to methyliaercury
in Little llarvel Peas as determined by methylmercury peak
height.
A = Lit peas 13 days old; 0 = U! peas 35 days old.
16
-------
after stirring for 48 hours an estimated 20% of the phenylmercury in
each may have gone into solution. The solutions were allowed to settle
and the aqueous solution decanted without carrying any of the undissolved
material over into the incubation flasks. After 20 hours incubation,
the tissues were thoroughly rinsed and extracted via the modified Westoo
procedure. Gas chromatographic analysis of the final benzene extracts
shows the presence of methylmercury in stems with apices at 10 +_ 3 nano-
grams/gram and leaves at 8+2.4 nanograms/gram. This level of trans-
formation occurred with an estimated 2-microgram/gram solution of phenyl-
mercuric acetate.
EXTRACTION OF PLANTS FROM A MERCURY MINE AREA
Methylmercury has been shown to be present in plant tissue from
foliar application of ionic mercury, from root uptake of ionic and phenyl-
mercury and from tissues incubated in ionic and phenylmercury solutions.
Is methylmercury present in plants which are growing naturally in an area
with elevated levels of mercury? To find out, several plants were
collected from the mercury mine area at the Nevada Test Site in mid-May
1975. Bromus rubens and Spharaleea ambigua were two of the species
collected. A Boraginaoeae was also collected.
In plant samples collected in November-December 1974 from the Four
Corners area around the coal-burning power station, no appreciable high
levels of mercury were noted. The physiology of the plants during this
collection period is far different from that of the same plants in the
Spring. To survive the long, hot, dry summers in the desert, the plants
enter a dormant state. In the Spring after a few showers, the physiology
changes to an active growth period. Mid-May was selected for collection
to obtain plants still actively growing and also so that small leafy plants
could be obtained.
The plants were extracted in 2.2IJ HC1 which was extracted with
benzene. The modified Westoo procedure was used for the extraction and
concentration of organic mercury compounds from the benzene. A 5-micro-
liter aliquot of the final 10-milliliter benzene layer was injected onto
the gas chromatographic column. All three plants showed a methylmercury
peak, but that from Bromus rubens was exceptionally large. To determine
that the peak was, in fact, methylmercury, 6 milliliters of the final
benzene layer was extracted with 5 milliliters of the 1% cysteine
solution at pH 8.4. The cysteine/mercury complex was broken by the
addition of 5N HC1 to a pH 0.7. This acidified fraction was placed on a
column of Srafion NMRR resin. The eluate was extracted into benzene
(6 milliliters) and a 5-microliter sample injected on the gas chromato-
graphic column. Again the methylmercury peak showed up almost as large as
before. The amount of methylmercury present in Brormis rubens was 10 + 3.3
nanograms/gram, for the Boraginaoeae^ 7.5 +_ 2.3 nanograms/gram, and for
Sphapolcea ambigua,, 2.5 + 0.8 nanograms/gram.
Since the purple influcrescence of Bromus rubens accounted for 80%
of the fresh weight of tissue extracted, methylmercury had concentrated
in the developing seeds.
17
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REFERENCES
Aomine, S., Kawasaki, H., and Inoue, K., "Retention of Mercury by Soils,"
Soil Sci. Plant Nutrition, p 13 (1967).
Bache, C. A., and Lisk, D. J., "Gas Chromatographic Determination of
Organic Compounds by Emission Spectrometry in a Helium Plasma,"
Anal. Chem.. 43, No. 7, pp 950-952 (1971).
Barker, W. G., "Toxicity Levels of Mercury, Lead, Copper, and Zinc in
Tissue Culture Systems of Cauliflower, Lettuce, Potato, and Carrot,"
Can. J. Bot., 50, pp 973-976 (1972).
Baughman, G. L., Carter, M. H., et al., "Gas-liquid Chromatography-mass
Spectrometry of Organomercury Compounds," J. Chromatog., 76, No. 2,
pp 471-476 (1973).
Beauchamp, I. L., and Tebbins, B. D., "Mercury Vapor Hazards in University
Laboratories," Amer. Ind. Hyg. Assoc. Quart., 12, pp 171-174 (1951).
Bertilsson, L., and Neujahr, H. Y., "Methylation of Mercury Compounds by
Methylcobalamin." Biochemistry, 10, No. 14, pp 2805-2808 (1971).
Booer, J. R., "The Behavior of Mercury Compounds in Soil," Ann. Appl.
Biol., 31, pp 340-359 (1944).
Booer, J. R., "Action of Mercury as a Soil Fungicide," Ann. Appl. Biol.,
38, pp 334-347 (1951).
Bowen, B. E., and Cran, S. P., "Effects of Sample Size on Chromatographic
Behavior." J. Chromatogr. Sci., 12, No. 10, p 579 (1974).
Brown, K. W., McFarlane, J. C., and Bernhardt, D. E., "Accidental
Inhalation of Mercury-203," Health Phys., 28, pp 1-4 (1975).
Bryant, W. F., and Kinstle, T. H., "Mass Spectroscopy of Organic Mercury
Compounds," J. Org. Chem., 24, pp 573-587 (1970).
Clarkson, T. W., and Greenwood, M. R., "Selective Determination of
Inorganic Mercury in the Presence of Organomercurial Compounds in
Biological Material." Anal. Biochem.. 37, pp 236-243 (1970).
D'ltri, F. M., The Environmental Mercury Problem, CRC Press, Cleveland,
Ohio, pp 63-67 (1972).
Estes, G. 0., Knoop, W. E., Houghton, F. D., "Soil-plant Response to
Surface Applied Mercury," J. Environ. Qual., 2, No. 4, pp 451-452
(1973).
18
-------
Feldman, C., "Preservation of Dilute Mercury Solutions," Anal. Chem.,
46, No. 1, pp 99-102 0-974).
Frissel, M. J., Poelstra, P., Reiniger, P., Das, H. A., "Contamination
of Soil with Mercury," Prbc. Int. Symp., Radioecol. Appl. Prot.
Man And His Environ., 2, pp 941-955 (1971).
Frissel, M. J., Poelstra, P. and Van der Klugt, N., "The Contamination
of Dutch Soils with Mercury and a Few Other Heavy Metals,"
ftp.nl ng-f ft pn Mijnhnirer, 53, No. 4, pp 163-170 (1974).
Furukawa, K., and Tonomura, K., "Enzyme System Involved in the Decom-
position of Phenylmercuric Acetate by Mercury-resistant Pseudomonas3 "
Agr. Biol. Chem.. 35, pp 604-610 (1971).
Gage, J. C., "Trace Determination of Phenyl- and Methylmercury Salts in
Biological Material." Analyst, 86, P 457 (1961).
Gage, J. C., and Warren, J. M., "Determination of Mercury and Organic
Materials in Biological Samples," Ann. Occup. Hyg., 13, No. 2,
pp 115-123 (1970).
Glooschenko, W. A., "Accumulation of 203Hg by the Marine Diatom
Chaetooeyos costatim," J. of Phycology, 5, No. 3, pp 224-226 (1969).
Goldwater, L. J., "Mercury in the Environment," Sci. Amer., 224, No. 5,
pp 15-21 (1971).
Goldwater, L. J., "Toxicology of Inorganic Mercury," Ann. N. Y. Acad.
Sci.. 65, pp 498-503 (1957).
Greenway, W., "Permeability of Phenyl-Hg -resistant and Phenyl-Hg -
susceptible Isolates of Pyrenophora avenae to the Phenyl-Hg Ion,"
J. Gen. Microbiol., 73, No. 2, pp 251-255 (1972).
Hale, V. Q., and Wallace, A., "Effect of Chelates on Uptake of Some
Heavy Metal Radionuclides from Soil by Bush Beans," Soil Sci., 109
No. 4, pp 262-263 (1970).
Hardcastle, J. E., and Mavichakana, N., "Uptake of Mercuric Chloride and
Methylmercury Chloride from Liquid Media by Aspergillus nigeic and
Penioilti^ian notatwn," Bull. Environ. Contain, and Toxicol., 11, No. 5,
pp 456-460 (1974).
Hardcastle, J. E., and Mavichakana, N., "Effect of Mercuric Chloride
and Methylmercuric Chloride on Spore Germination and Colony Growth
Rate of Penic-ilHum notation and Aspergillus nigev, " Chemosphere,
No. 3, pp 107-114 (1974).
Hitchcock, A. E., and Zimmerman, P. W., "Pharmacology and Toxicology.
Toxic Effects of Vapors of Mercury and of Compounds of Mercury on
Plants," Acad. Sci., 65. pp 498-503 (1957).
19
-------
Hosli, E., "Effects of Methylmercury on the Morphological and Histo-
chemical Properties of Human and Rat Spinal Cord," Expeferitia,
30, No. 11, p 1300 (1974).
Huisingh, D., "Heavy Metals: Implications for Agriculture," Ann. Rev.
Phytopathology. 12, pp 375-388 0-974).
Hunter, D., Bomford, R. R., and Russell, D. S., "Poisoning by Methyl-
mercury Compounds," Quart. J. Med.. 9, pp 193-213 (1940).
Imura, N., Sukegawa, E., Pan, S., et al., "Chemical Methylation of
Inorganic Mercury with Methylcobalamin, a Vitamin B12 Analog,"
Science, 172, pp 1248-1249 (1971).
Ingraham, L. L., "Bj^ Coenzymes: Biological Grignard Reagents," Ann.
N. Y. Acad. Sci., 112, p 713 (1964).
James, P. E., Lagerwerf, J. V., and Dudly, R. F., "Translocation of
Mercury from Seed Treatment," Int. Symp. Ident. Meas. Envir. Follut.
Proc.. pp 213-215 (1971).
Jernelov, A., "Conversion of Mercury Compounds," Chap. 4, pp 68-74,
In M. W. Miller, and G. C. Berg (Eds.), Chemical Fallout, C. C.
Thomas, Springfield, Illinois, p 531 (1969).
Jernelov, A., "Mercury Conversions in Nature and What We can Do to Affect
Them." Norwegian J. Hyg.. 50, pp 174-178 (1969).
Jernelov, A., "Release of Methylmercury from Sediments with Layers
Containing Inorganic Mercury at Different Depths," Limnol. Oceanogr.,
15, No. 6, pp 958-960 (1970).
Kamp-Nielsen, L., "The Effect of Deleterious Concentrations of Mercury
on the Photosynthesis and Growth of Chlovella pyrenoidosa3" Physiol.
Plant., 24, No. 3, pp 556-561 (1971).
Kapoor, R. C., Doughty, G., and Gorin, G., "The Reaction of Assay of
Glutathione with Hg2+ and Alkali," Biochim. Biophys. Acta. 10,
p 376, (1965).
Kimura, Y., and Miller, V. L., "Mercury Determination at the Microgram
Level by a Reduction-aeration Method of Concentration," Anal. Chim.
Acta. 27, p 325 (1962).
Klein, D. H., and Goldberg, E. D., "Mercury in the Marine Environment,"
Environ. Sci. Technol., 4, pp 765-768 (1970).
Kleine, R., and Meisel, P., "Occurrence and Some Properties of Mercury-
activatable Amino Acid Esterase from Rat Kidney Microsomes," Febs
(Fed. Eur. Biochem. Sdc.) Lett., 37, No. 2, pp 120-123 (1973).
Komura, I., "Vaporization of Inorganic Mercury by Cell-free Extracts of
Drug Resistant E* co££," Agr. Biol. Ghent., 34, No. 3, pp 480-482 (1970)
20
-------
Kothny, E. L., "Micro-method for Mercury," J. Amer. Ind. Hyg. Assn.,
31, No. 4, pp 466-471 (1970).
Kressin, I., "Rapid Separation and Determination of Mercury," Talanta,
19, No. 2, pp 197-202 (1972).
LaFleur, P. D., "Retention of Mercury When Freeze-drying Biological
Materials," Anal. Chem., 45. No. 8, pp 1534-1536 (1973).
LaHue, R., "Sensitive Biobehavioral Assay for Methylmercury," Buj.1.
Envif. Contain. Toxicol., 10, No. 3, pp 166-169 (1973).
Langley, D. G., "Mercury Methylation in an Aquatic Environment,"
J. Water Pollut. Contr. Fed.. 45, No. 1, pp 44-51 (1973).
Larock, R. C., "Mercury in Organic Chemistry. V. Direct-Esterification
of Alkyl Halides," J. Org. Chem.. 39, No. 25, p 3721 (1974).
Law, S. L., "Resin-loaded Papers for Methylmercury and Inorganic Mercury
Determination," Amer. Lab., July, pp 91-97 (1973).
Lawrence, J. B., "How Poisonous is Mercury?," Chem. Engn. News, 29,
No. 35, pp 3529-3531 (1951).
Lee, S. Y., and Richardson, T., "Use of Thiolated Amino Ethyl Cellulose
to Remove Mercury Bound to Solubilized Protein." J. Milk Food
Technol., 36, No. 5, pp 267-271 (1973).
Li, M. F., "Effect of Mercuric Chloride on Cellular Morphology and Acid
Phosphatase of Tissue Culture Cells Cultivated in Suspension,"
Environ. Physiol. Biochem., 4, No. 6, p 263 (1974).
Lindstrand, K., "Isolation of Methylcobalamin from Natural Source
Material," Nature. 204, p 188 (1964).
Mansy, S., Wood, T. E., et al., "Heavy Metal Nucleotide Interactions.
Binding of Methylmercury to Pyrimidine Nucleosides and Nucleotides,"
J. Amer. Chem. Soc.. 96, No. 6, pp 1762-1770 (1974).
Marchant, W. N., "Modified Cellulose Adsorbent for Removal of Mercury
from Aqueous Solutions," Environ. Sci. Technol., 8, No. 12, pp 993-996
(1974).
Matsen, R. S., Mustoe, G. E., and Chang, S. B., "Mercury Inhibition on
Lipid Biosynthesis in Freshwater Algae," Environ. Sci. Technol., 6
No. 2, pp 158-160 (1972).
Matsumura, F., Gotch, Y., and Bousch, G. M., "Phenylmercurie Acetate
Metabolic Conversion by Microorganisms." Science, 173 (1971).
Mazurski, M. A. J., Chow, A., and Gesser, H. D., "The Extraction of
Mercury from Aqueous Solution with Sulfide-treated Polyurethane
Foam." Anal. Chim. Acta, 65, pp 99-104 (1973).
21
-------
Neville, G. A., and Drakenberg, T., "Mercuric Mercury and Methylmercury
Complexes of Glutathione," Acta Chemica Scarid., B 28, pp 473-477
(1974).
Neville, G. A., and Drakenberg, T., "Mercury II Complexation of Cysteine,
Methyl Cysteinate in Acidic Media." Can. J. Chem.t 52, No. 4, pp
616-622 (1974).
Neville, G. A., and Berlin, M., "Mercury (II) Complexes of L-cysteine
Methyl Ester." Can. J. Chem., 51, p 3970 (1973).
Ohmori, H., Nakatani, K., Shkmizu, S., and Fukui, S., "Correlation
Between the Level of Vitamin-B12-dependent Methionine Synthetase
and Intracellular Concentration of Vitamin B12 in Some Bacteria,"
Eur. J. Biochem., 47 pp 207-218 (1974).
Peakall, D. B., and Lovett, R. J., "Mercury: Its Occurrence and Effects
in the Ecosystem," Biosci., 22-j No. 1, pp 20-25 (1972).
Pellizarri, E. D., "Electron Capture Detection in Gas Chromatography,"
J. Chromatog.. 98, pp 323-361 (1974).
Poelstra, P., Frissel, M. J., Van Der Klugt, N., and Bannink, D. W.,
"Accumulation and Distribution of Mercury in Dutch Soils," Neth. J.
Agr. Sci.. 21, pp 77-85 (1973).
Prosperi, T., and Lederer, M., "Comparison of Some Ready for Use Thin
Layers for Separation of Heavy Metals with Butanol and 3N HC1,"
J. Chromatogr., 65, No. 2, pp 460-462 (1972).
Rawlinson, C. J., "Effect of Combined Seed Treatments of Chlormequat
and Organo-mercury on Foot Rot and Winter Growth of Oats," Proc. 5th
Br. Insectic. Fungic. Conf., pp 78-88 (1969).
Rawlinson, C. J., "Effect of an Organo-mercurial Fungicide, Chlormequat
and Decenyl-succinic Acid on Frost Hardiness and Seedling Diseases
of Oats," Ann. Appl. Biol.. 67, pp 223-234 (1971).
Rawlinson, C. J., and Colhoun, J., "Chemical Treatment of Cereal Seed
in Relation to Plant Vigor and Control of Soil Fungi," Ann. Appl.
Biol.. 65, pp 459-472 (1970).
Rawlinson, C. J., and Davies, J. M. L., "Effect of Eyespot (Cevcospore'ila
herpotrichoides) and Seed Treatments of Benomyl, Chlormequat and
Organomercury on Rye," Plant Path., 20, pp 131-136 (1971).
Reimers, R. S., and Krenkel, P. A., "Kinetics of Mercury Absorption and
Desorption in Sediment," J. Water. Pbllut. Contr. Fed., 46, No. 2,
pp 352-365 (1974).
Rissanen, K., and Mieltinen, J. K., "TLC of Alkyl and Alkoxy Mercury
Derivatives and Location of Mercury in Yolk of Hen Eggs," Ann. Agr.
Fenn. Suppl.. 7, p 22 (1968).
22
-------
Rothstein, A., "Cell Membrane as Site of Action of Heavy Metals,"
Fed. Proceed.. 18, pp 1026-1035 (1959).
Shacklette, H. T., "Bryophytes Associated with. Mineral Deposits and
Solutions in Alaska," U.S. Geol. Survey Prof.Paper, 1198C (1965).
Simpson, R. B., "Kinetics of Anion Exchange Between Methylmercuric
Complexes," J. Chem. Phys.. 46, No. 12, pp 4775-4783 (1967).
Smart, N. A., "Use of Residues of Mercury Compounds in Agriculture,"
Residue Rev., 23, pp 1-36 (1968).
Smith, R. A. H., and Bradshaw, A. D., "Reclamation of Toxic Metalliferous
Wastes Using Tolerant Populations of Grass," Nature, 227, pp 376-377
(1970).
Smith, W. H., "Lead and Mercury Burden of Woody Plants," Science, 176,
No. 4040, p 1237 (1972).
Smith, W. H., "Metal Contamination of Urban Woody Plants," Environ.
Sci. Technol., 7, pp 631-636 (1973).
Southard, J., Nitisewojo, P., and Green, D. E., "Mercurial Toxicity and
the Perturbation of the Mitochondrial System," Fed. Proceed., 33,
No. 10, pp 2147-2153 (1974).
Spangler, W. J., Spigarette, J. L., Rose, J. M., and Miller, H. M.,
"Methylmercury: Bacterial Degradation in Lake Sediments," Science,
180, pp 192-193 (1973).
Spanis, W. C., Munneche, D. E., and Solberg, R. A., "Biological Breakdown
of Two Organic Mercurial Fungicides," Phytopath., 52 p 455 (1962).
Stegnar, P. Y., and Byrne, A. R., "Accumulation of Mercury by, and
Occurrence of Methylmercury in, Some Fungi," Chemosphere, 2, No. 2,
pp 57-63 (1973).
Storm, D. R., and Gunsalus, R. P., "Methylmercury is a Potent Inhibitor
of Membrane Adenyl Cvclase." Nature. 250, No. 5469, pp 778-779
(1974).
Sumino, K., "Analysis of Organic Mercury Compounds by Gas Chromatography
Part I. Analytical and Extraction Method of Organic Mercury
Compounds," Kobi J. Med. Sci., 14, p 115 (1968).
Sumino, K., "Analysis of Organic Mercury Compounds by Gas Chromatography.
Part II. Determination of Organic Mercury Compounds in Various
Samples." Kobi J. Med. Sci.. 14, p 131 (1968).
Talmi, Y., "The Rapid Sub-microgram Determination of Volatile Organo-
mercury Compounds by Gas Chromatography with a Microwave Emission
Spectrometric Detector System," Anal. Chim. Acta, 74, No. 1, p 107
(1975).
23
-------
Tatton, J. 0. D., and Wagstaffe, P. J., "Identification and Determina-
tion of Organomercurial fungicide Residues by TLC and GLC," J^_
Chrdmatbg., 44, p 284 Q969).
Tonomura, K., and Kanzaki, F., "Reductive Decomposition of Organic
Mercurials by Cell-free Extract of a Mercury Resistant Pseudomonas,"
Biochem. Biophys. Acta. 184, pp 227-229 (1969).
Tonomura, K., Maeda, K., Futai, F. et al., "Stimulative Vaporization of
Phenylmercuric Acetate by Mercury Resistant Bacteria," Nature, 217,
pp 644-646 (1968).
Toribara, T. Y., Shields, C. P., and Koval, L., "Behavior of Dilute
Solutions of Mercury," Talanta. 17, p 1025 (1970).
Van der Linden, W. E., and Beers, C., "Determination of Composition and
Stability Constants of Mercury II with Amino Acids," Anal. Chim.
Acta. 68, No. 1, pp 143-154 (1974).
Van Faassen, H. G., "Effects of Mercury Compounds on Soil Microbes,"
Plant Soil. 38, pp 485-487 (1973).
Van Loon, J. C., "Agricultural Use of Sewage Treatment Plant Sludge,
a Potential Source of Mercury Contamination," Environ. Lett., 4,
No. 4, pp 259-265 (1974).
Van Loon, J. C., "Mercury Contamination of Vegetation Due to Application
of Sewage Sludge as Fertilizer," Environ. Lett., 6, No. 3, pp 211-218
(1974).
Villermaux, J., "Deformation of Chromatographic Peaks Under the Influence
of Mass Transfer Phenomena," J. Chromatog. Sci., 12, No. 12, p 822
(1974).
Watanabe, N., "Reaction of Alkyl Halides with Mercuric Thiocyanate,"
Bull. Chem. Soc., Japan, 47, No. 11, p 2745 (1974).
Weed, R., Eber, J., and Rothstein, A. J., "Interaction of Mercury with
Human Erythrocytes," J. Gen. Physiol., 45, p 395 (1962).
Yamada, T., "Uptake of Phenylmercuric Acetate Through the Root of Rice
and Distribution of Mercury in the Rice Plant," Nippon Nogei
Kaishi. 42, No. 7, pp 435-439 (1968).
Zepp, R. G., Wolfe, N. L. N., and Gordon, J. A., "Photodecomposition of
Phenylmercury Compounds in Sunlight," Chemosphere, 3, pp 93-99
(1973).
Zimmerman, P. W., and Crocker, W., "The Injurious Effect of Mercury
Vapor from Bichloride of Mercury in Soil of Rose Houses," Boyce
Thompson Instit. Prof. Paper. 1, p 222 (1933).
24
-------
BIBLIOGRAPHY
Aarkrog, A., and Lippert, J., "Direct Contamination of Barley with
5TCr, §9Fe, 58Co, 65Zn, 203Hg, 210Pb." Radiat. Bot., 11. No. 6,
pp 462-472 C1971).
Ahmed, M., and Grant, W. F., "Cytological Effects of the Mercurial
Fungicide Panogen 15 on Tradesoant-La and V-ieia fdba root tips,"
Mutat. Res., 14, pp 391-396 (1972).
Akagi, H., and Takabatake, E., "Photochemical Formation of Methylmercury
Compounds from Mercuric Acetate," Chemosphere, 2, No. 3, pp 131-133
(1973).
Alvarez, R., "Sub-microgram per Gram Concentrations of Mercury in
Orchard Leaves Determined by Isotope Dilution and Spark-Source
Mass Spectrometry," Anal. Chim. Acta, 73, pp 33-38 (1974).
Anelli, G., Pelosi, P., and Galoppini, C., "Influence of Mercury on the
Amino Acid Composition of Tobaccq Leaves," Agri. Biol. Chem., 37,
No. 7, pp 1579-1582 (1973).
Bache, C. A., Gutenmann, W. H., St. John, L. E., et al., "Mercury and
Methylmercury Content of Agricultural Crops Grown on Soils Treated
with Various Mercury Compounds," J. Agr. Food Chem., 21, No. 4,
pp 606-613 (1973).
Balicka, N., Kosinkiewicz, B., Musial, M., and Stankiewicz, M., "Deacti-
vation of the Hg Seed Dressing by Avthvobaotev spp.," Acta Microbiol.
Pol. Ser. B. Microbiol. Appl.. 5, No. 1, pp 3-8 (1973).
Balicka, N., and Musial, M., "The Effect of Microorganisms on Phyto-
toxicity of Herbicides. Part I. Complex Activity of Some Pesticides
on Plants," Acta Microbiol. Polon., 4, No. 21, p 183 (1972).
Bisogni, J. J., and Lawrence, A. W., "Kinetics of Microbially Mediated
Methylation of Mercury in Aerobic and Anaerobic Aquatic Conditions,"
U.S. Nat. Tech. Inform. Serv., Pb. Rep.. No. 222025/9, p 195 (1973).
Bock, K. R., Robinson, J. B. D., and Chamberlain, G. T., "Zinc
Deficiency Induced by Mercury in Coffea grab-Lea," Nature, 182,
pp 1607-1608 (1958).
Bradeen, D. A., Winget, G. D., and Gould, J. M., "Site Specific Inhibition
of Photophosphorylation in Isolated Spinach Chloroplasts by Mercuric
Chloride," Plant Physiol., 52, No. 6, pp 680-682 (1973).
25
-------
Clendenning, K. A., and North, W. J., "Effects of Wastes on the Giant
Kelp, Maaxoay&tis pyrifeva," pp 82-91, In E. D, Pearson (Ed.),
Prbc. Int. Cbrif. Waste Disposal Marine Environ., Berkeley,
California, Pergamon Press, New York (1958).
Dobrolyubskii, 0. K., "Mercury Trace Element as the Influencing Factor
on Plant Organisms," Doklady Akad. Nauk SSSR (Bot. Sci. Sec.
Transl.), 128, No. (1/6), pp 253-255 (1959). Translated? (from)
Doklady Adad. Nauk SSSR. 125, No. 5, (I960).
Fang, S. C., "Uptake and Biotransformation of Phenylmercuric Acetate
by Aquatic Organisms," Arcji. Enyiron. Coritam.^ Toxicol., 1, No. 1
pp 18-26 (1973).
Fujita, M., and Hashizume, K., "Accumulation of Mercury by Fresh Water
Planktonic Diatom," Chemosphere, 1, No. 5, pp 203-207 (1972).
Fukunaga, K., Tsukano, Y., and Kanazawa, J., "Residue Analysis of
Organomercury Fungicides Sprayed on Rice Plants," Environ. Toxicol.
of Pesticides. Academic Press, New York, pp 177-191 (1972).
Furukawa, K., and Tonomura, K., "Cytochrome c Involved in Reductive
Decomposition of Organic Mercury," Biochim. Biophys. Acta, 325,
No. 3, pp 413-423 (1973).
Gerdes, R. A., Hardcastle, J. E., and Stabenow, K. T., "Mercury Content
of Fresh Fruits and Vegetables," Chemosphere, No. 1, pp 13-18
(1974).
Gilmour, J. T., and Miller, M. S., "Fate of a Mercuric-mercurous
Fungicide Added to Turfgrass," J. Environ. Qual., 2, No. 1,
pp 145-148 (1973).
Haney, A., and Lipsey, R. L., "Accumulation and Effects of Methyl-
mercury Hydroxide in a Terrestrial Food Chain Under Laboratory
Conditions," Environ. Pollution, 5, pp 305-316 (1973).
Harriss, R. C., White, D. B., and Macfarlane, R. B., "Mercury Compounds
Reduce Photosynthesis by Plankton," Science, 170, p 736 (1970).
Imura, N., Pan, S. K., and Ukita, T., "Methylation of Inorganic Mercury
with Liver Homogenate of Tuna," Chemosphere, 1, No. 5, pp 197-201
(1972).
Jensen, S., and Jernelov, A., "Biological Methylation of Mercury in
Aquatic Organisms," Nature, 223, p 753 (1969).
John, M. K., "Mercury Uptake from Soil by Various Plant Species," Bull.
Environ. Contam. Toxicol., 8, No. 2, pp 77-80 (1972).
Kasahara, M., and Anraku,.Y», "Inhibition of Respiratory Chain of E. ooH
by Zn and Hg Ions," J. Biochem., 72, No. 3, pp 777-781 (1972).
26
-------
Kimura, Y., and Miller, V. L., "The Degradation of Organomercury
Fungicides in Soil," Agr. Food Chem., 12, No. 3, pp 253-257
(1964).
Kimura, Y., and Miller, V. L., "Vapor Phase Separation of Methyl or
Ethyl Mercury Compounds and Metallic Mercury," Anal. Chem., 32,
p 420 (1960).
Kressling, H., "Some Pactors Concerning the Detoxification of Organic
Mercurial Fungicides,"Syensk. Papperstidning. 64, p 689 (1961).
Landner, L., "Biochemical Model for the Biological Methylation of
Mercury Suggested from Methylation Studies in vivo with Neurospora
crosso." Nature, 230, pp 452-453 (1971).
Law, S. L., "Methyl Mercury and Inorganic Mercury Collection by a
Selective Chelating Resin," Science, 174, No. 4006, pp 285-287
(1971).
Lee, D. F., Thomas, B., Roughan, J. A., and Watters, E. D., "Mercury
Content of Some Foodstuffs of Vegetable Origin," Pestic. Sci., 3,
pp 13-17 (1972).
LeFevre, P. A., and Daniel, J. W., "Properties of Organomercury-degrading
System in Mammalian Liver," Febs (Fed. Eur. Biochem. Soc.) Lett.,
35, No. 1, pp 121-123 (1973).
Magos, L., Tuffery, A. A., and Clarkson, T. W., "Volatilization of
Mercury by Bacteria," Brit. J. Industr. Med., 21, pp 294-298 (1964).
Matsumura, F., Gotch, Y., and Bousch, G. M., "Factors Influencing
Translocation and Transformation of Mercury in River Sediment,"
Bull. Environ. Contain. Toxicol., 8, No. 5, pp 267-272 (1972).
McKinney, M., "Factors in the Biochemical Conversion of Mercury
Pollutants to Toxic Methylmercury Effected by Micro-organisms in
Marine Sediment." U.S. Nat. Tech. Infor. Serv. Ad Rep., No. 749674
(1972).
Nelson, J. D., Blair, W., Bunkman, F. E., et al., "Biodegradation of
Phenylmercuric Acetate by Mercury Resistant Bacteria," Appl.
Microbiol., 26, No. 3, pp 321-326 (1973).
Newsome, W. H., "Determination of Methylmercury in Fish and Cereal Grain
Products," J. Agr. Food Chem., 19, p 567 (1971).
Norseth, T., and Clarkson, T. W., "Studies on the Biotransformation of
203Hg-labeled Methyl Mercury Chloride in Rats," Afchlv. Environ.
Health, 21, p 717 (1970).
Ostlund, K., "Studies on Metabolism of Methylmercury and Dimethylmercury
in Mice," Acta Pharmacol. Toxicol.. 27, (Suppl 1), pp 13-132 (1969).
27
-------
Puerner, N. J., and Stegel, S. M., "Effects of Mercury Compounds on
Growth, and Orientation of Cucumber Seedlings," Pnystiol. Plant.,
26, No. 3, p 310 (1972).
Radmer, R., and Kok, B., "Kinetic Observation of the System II Electron
Acceptor Pool Isolated By Mercuric Ion," Bibchem. Biophys. Acta,
357, pp 177-180 (1974).
Rao, A. V., Fallin, E., and Fang, S. C., "Comparative Study of Uptake
and Cellular Distribution of 203Hg-laBeled Phenyl-mercuric Acetate
and Mercuric Acetate By Pea Roots," Plant Physiol., 41, p 443 (1966).
Rose, A. H., Chemical Microbiology, Butterworths, London (1968).
Ross, R. G., and Stewart, D. K. R., "Movement and Accumulation of Hg
in Apple Trees and Soil," Can. J. Plant Sci.. 42, pp 280-285
(1962).
Siegel, S. M., Puerner, N. J,, and Speitel, T. W., "Release of Volatile
Mercury from Vascular Plants," Physiol. Plant., 32, No. 2, p 174
(1974).
Simpson, P. G., Hopkins, T. E., and Haque, R. J., "Binding of Methyl-
mercury Chloride to the Model Peptide, N-acetyl-L-crysteine,"
J. Phys. Chem., 77, p 2282 (1973).
Smart, N. A., "Mercury Residues in Potatoes Following Application of
Foliar Spray Containing Phenylmercuric Chloride," J. Sci. Food Agr.,
15, pp 102-108 (1964).
Tkachuk, R., and Kuzina, F. D., "Mercury Levels in Wheat and Other Cereals,
Oilseed and Biological Samples," J. Sci. Food Agr., 23, No. 10, pp
1183-1185 (1972).
Tonomura, K. et al., "Microbial Conversion of Mercury Compounds," Environ.
Toxicol. Pestic., Proc. U.S.-Japan, pp 115-133 (1971).
Watling-Payne, A. S., and Selwyn, M. J., "Inhibition and Uncoupling of
Photophosphorylation in Isolated Chloroplasts by Organotin, Organo-
mercury and Diphenyleneiodonium Compounds," Biochem. J., 142, pp
65-74 (1974).
Wedding, R. T., and Kendrich, J. B., "Toxicity of N-methyl Dithiocarbamate
to Rhizoctonia solani," Phytopathology, 49, p 557 (1959).
Westoo, G., "Determination of-^ethylmercury Compounds in Foodstuffs.
I. Methylmercury Compounds in Fish, Identification and Determination,"
Acta Chem. Scand., 20, p 2131 (1966).
Westoo, G., "Determination of Methy linercury Compounds in Foodstuffs.
II. Determination of Methylmercury Compounds in Fish, Egg, Meat,
and Liver," Acta Chem. Scarid., 21, p 1790 (1967).
28
-------
Westoo, G., "Determination of Methylmereury Salts in Various Kinds of
Biological Material." Acta Chem. Scand., 22, p 2277 (1968).
Westoo, G., "Methylmercury as Percentage of Total Mercury in Flesh, and
Viscera of Salmon and Sea Trout of Various Ages," Science, 181,
No. 4099, pp 567-568 (1973).
Wong, Y. S., Chieh, P. C., and Carty, A. J., "Binding of Methylmercury
by Amino Acids," J. Ch.em. Soc., Ch,em. Cbmmun., No. 19, pp 741-742
(1973a).
Wong, Y. S., Chieh, P. C., and Carty, A. J., "Interaction of Organo-
mercury Pollutants with Biologically Important Sites. X-ray Study
of 2:1 Complex Between Methylmercury and Penicillamine," Can. J.
Chem., 51, No. 15, pp 2597-2599 (1973b).
Wood, J. M., Kennedy, F. S., and Rosen, C. G., "Synthesis of Methyl-
mercury Compounds by Extracts of a Methanogenic Bacterium,"
Nature. 220, pp 173-174 (1968).
Yamada, M., "Microbial Methylation in an H2S Environment," Hakko Kogalu
Zasshi, 50, No. 12, pp 901-999 (1972).
Yamada, M., and Tonomura, K., "Formation of Methylmercury from Inorganic
Mercury by ClostiridLwn," Hakko Kogalu Zasshi, 50, No. 12, pp 893-899
(1972).
/
Zimmerman, P. W., and Crocker, W., "Plant Injury Caused by Vapors of
Mercury and Compounds of Mercury," Contrib. Boyce Thompson Instit.,
6, p 167 (1934).
29
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
i. REPORT NO.
EPA-600/3-76-049
2,
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
METHYLMERCURY: FORMATION IN PLANT TISSUES
5. REPORT DATE
May 1976
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
Don D. Gay
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Environmental Monitoring & Support Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Las Vegas, NV 89114
10. PROGRAM ELEMENT NO.
1AA602
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
Same as Above
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY COPP
EPA-ORD, Office of Health
and Ecological Effects
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Methylmercury was found in the tissue of the pea plant (Pisim sativum)
after spraying mercuric nitrate onto the leaves, after planting in mercuric
nitrate or phenylmercuric acetate contaminated soil and after infiltration
and incubation of stems, leaves, and apices in mercuric nitrate or phenyl-
mercuric acetate solutions. The concentration of mercury added in each
experiment was 10 micrograms/gram. Younger pea tissue formed more methyl-
mercury than older tissue. Methylmercury was also found in three different
species of plant growing near an abandoned mercury mine.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Plant physiology
Plant chemistry
Mercury
Trace elements
Legumes
Absorption
Methylmercury
Mercurial transformation
Mercury uptake
Biotransformation
Phenylmercury
Pisum sativum
17C
06C
06F
07B
07C
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
36
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
•&GPO 691- 218-1976
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