EPA-600/3-78-037
April 1978
Ecological Research Series
ETHYLMERCURY: FORMATION IN PLANT TISSUES
AND RELATION TO METHYLMERCURY FORMATION
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 nine series. These nine broad categories
were established to facilitate further development and application of environmental
technology. Elimination of traditional grouping was consciously planned to foster
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1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy—Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
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. Investiga-
tions include formations, transport, and pathway studies to determine the fate of
pollutants and their effects. This work provided the technical basis for setting standards
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This document is available to the public through the National Technical Information
Service, Springfield, Virginia 22161
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EPA-600/3-78-037
April 1978
ETHYLMERCURY: FORMATION IN PLANT TISSUES AND
RELATION TO METHYLMERCURY FORMATION
By
L. C. Fortmann, D. D. Gay, and K. 0. Wirtz
Monitoring Systems Research and Development Division
Environmental Monitoring and Support Laboratory
P. 0. Box 15027
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 consti-
tute endorsement or recommendation for use.
ii
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FOREWOKD
Protection of the environment requires effective regulatory actions
which are based on sound technical and scientific information. This
information must include the quantitative description and linking of
pollutant sources, transport mechanisms, interactions, and resulting
effects on man and his environment. Because of the complexities involv-
ed, assessment of specific pollutants in the environment requires a total
systems approach which transcends the media of air, water, and land. The
Environmental Monitoring and Support Laboratory-Las Vegas contributes to
the formation and enhancement of a sound integrated monitoring data base
through multidisciplinary, multimedia programs designed to:
.develop and optimize systems and strategies for moni-
toring pollutants and their impact on the environment
.demonstrate new monitoring systems and technologies by
applying them to fulfill special monitoring needs of
the Agency's operating programs
This report presents data demonstrating the biotransformation of
elemental mercury in plants. The mercury, taken up as a vapor in its
elemental state, is converted by the common garden pea to the toxic
organomercurials, methylmercury and ethylmercury. The significance
of this biotransformation will be considered by EPA and others in making
decisions concerning regulations of activities known to generate mercury
emissions. For additional information, please contact the Pollutant
Pathways Branch, Environmental Monitoring and Support Laboratory-LV,
P. 0. Box 15027. Las Vegas, Nevada 89114.
George B. Morgan
Director
Environmental Monitoring and Support Laboratory
Las Vegas, Nevada
iii
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INTRODUCTION
Increased utilization of coal for energy production and continued
industrial use of mercury may be expected to increase the levels of mercury
pollutants in the environment in the future. Data are needed to define the
effects of mercury pollutants on plants, the uptake and distribution of
mercury within plants, and the species of mercury present and their bio-
transformations within plants. Such data are essential to fully describe
the impact of mercury on plants.
The objective of this study was to examine the fate of one possible
mercury pollutant, elemental mercury, in the tissues of Pisum sativum.
CONCLUSION
Both ethylmercury and methylinercury are formed in the pea following
exposure to elemental mercury vapor. The pattern of change in concentration
suggests both are metabolites of a single pathway. Methylmercury is identi-
fied as an intermediate product of the pathway. The exact role of ethylmer-
cury in this pathway cannot be defined at this time.
MATERIALS AND METHODS
Plant Material; Seeds of Pisum sativum, cv. Little Marvel were grown
hydroponically in a greenhouse for 15 days before exposure.
Exposure Conditions: Seventy seedlings were placed in a Plexiglas ®
chamber of 58,000 cm3 volume, with 10 ml of elemental mercury in a tray
placed on the floor of the chamber. The chambers were placed into a hood
with constant air flow in order to maintain a temperature of 25 ±1 C,
regardless of lighting conditions. Incandescent lighting at 110 uE m 2
sec 1 was used for a 12-hour daily light period. The concentration of
elemental mercury vapor (Hg ) in the chamber was determined by gas chromato-
graphy and calculated to be 1.71 ppb (Long, Scott and Thompson, 1973) with-
in 30 minutes. A saturation level of 7.35 ppb was reached at 3 hours.
Tissue Extraction; Tissue was ground by mortar and pestle with 2.21J HC1
(2 ml/g). The resultant brei was centrifuged at 10,000 rpm for 30 minutes.
The supernatant fluid was then decanted and extracted with an equal volume
of benzene (re-distilled nanograde) by vigorous shaking for 1 minute followed
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by centrifugation at 10,000 rpm for 10 minutes. This benzene fraction was
passed through Whatman Phase Separating paper and then analyzed by gas chro-
matography.
The efficiency of the benzene extraction, as determined in our laboratory,
agreed with the average of 76% reported by Talmi (1975). The efficiency of
the extraction procedure for plant material was determined by extraction of 5-g
plant samples to which known quantities of methylmercury (CH3HgCl) and ethyl-
mercury (CaHsHgCl) had been added. Efficiency of the extraction was deter-
mined to be 74% ± 2% for CH3HgCl and 69% ± 2% for C2H5HgCl. When a second
benzene extraction of the aqueous phase was performed, an additional 21% of
the original CH3HgCl and 20% of the added C2H5HgCl was obtained.
For convenience and reduced handling of large numbers of samples during
time-course experiments, the second benzene extraction was dropped. Having
determined the benzene extracted to be 76% efficient, standards were pre-
pared by a method similar to the plant extractions, i.e., known amounts of
CH3HgCl and C2H5HgCl were added to 2.21J HC1, which was extracted with an
equal volume of benzene. When this is used as the standard, reported values
can be considered to be 95% ± 3% of actual concentration, without the neces-
sity of using a correction factor.
Sample Analysis: A Gas Chromatograph-Microwave Emission Spectrometer
(GC-MES) system (Talmi, 1975) with a Gay-Frank optical system modification
(Gay and Frank, 1977) was used for analysis of both air and benzene samples
with the following operating conditions: Column: 3-foot by V-inch glass,
packed with 4% FFAP (80/100 mesh) on Supelcoport® (Supelco Inc., Beliefonte,
PA); Column temperature: 150 C for benzene samples, 30 C for air samples;
Carrier gas: ultrapure argon at a flow rate of 11 ml/min; Temperature of
injection port and detector; 225° C; Microwave generator output: 50 W
incident, 3 W reflected power; Photomultiplier voltage: 500 V; Monochroma-
tor slit width: 70 ym; and Spectral wavelength: 253.7 nm. The concentra-
tion of mercury compounds, expressed as nanograms per gram (ng/g) in this
report, represents nanograms Hg+ per gram fresh weight of identical tissues
of control plants. This convention was necessary since the effect of Hg°
toxicity is dessication of the leaf.
RESULTS
Initially, plants were exposed to elemental mercury for 24 hours to
determine whether any organomercury compounds would be formed. As indicated
in Table 1, C2H5Hg+ and CH3Hg+ were detected in all parts of the plant. There
was no definite pattern of distribution of either metabolite within the plant
with the exception of consistently high levels of both mercury species in the
oldest lateral branch (stem and leaf). This lateral was the first to show
visible signs of mercury toxicity. The pea, having a single growing point,
was an ideal plant for study of a gas known to effect plant senescence
(Speitel and Siegel, 1975). Damage to the plant occurred in a gradient from
the first lateral to the top of the plant. The effects of mercury vapor
®
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toxicity consisted of wilting, followed by severe dessication of the leaf.
An exposure duration was lengthened, each successive lateral up the stem
wilted, then became severely dessicated, although no abscission occurred
during the 48-hour period.
TABLE 1. THE FORMATION OF ETHYLMERCURY AND METHYLMERCURY IN THE
PEA AFTER EXPOSURE TO ELEMENTAL MERCURY VAPOR FOR 24
HOURS (Values reported are the average of triplicate
samples and represent ng Hg+/gram fresh weight of
identical tissue of control plants.)
Plant Part
1st Lateral*
2nd Lateral*
3rd Lateral*
4th Lateral*
5th Lateral*
Apex
Stem
Root
Control
C2H5Hg+ (ng/g)
12.82 ± 0.60
9.93 ± 0.35
5.40 ± 0.05
8.05 ± 1.81
6.99 ± 3.59
11.00 ± 1.67
5.22 ± 3.33
4.85 ± 1.34
N.D.**
CH3Hg+ (ng/g)
19.21 ±
8.07 ±
4.21 ±
9.95 ±
6.49 ±
17.26 ±
10.77 ±
11.55 ±
N.D.**
12.82
1.77
0.79
7.05
0.47
5.66
7.59
6.35
*Includes both leaf and stem, the 1st lateral branch being the lowest and
oldest branch off the central axis.
**None detectable in any plant part.
In an attempt to define the relationship between the two mercury com-
pounds, a time-course of exposure was performed. Concentrations of both
CHsHg"1" and C2HsHg+ varied considerably over the 48 hours of exposure (Figure 1)
In the aerial portion of the plant CaHsHg"*" concentration rose during the 48-
hour period from 2.20, 4.05, and 4.25 ng/g to 25.92, 19.48, and 24.07 ng/g
for the stem, apex, and laterals, respectively. A similar rise in concentra-
tion was not observed for CH3Hg . Concentrations ranged from 1.79 ng/g to
17.28 ng/g in the aerial portion of the plant, with an average concentration
of 4.66 ng/g. It is significant that in all aerial parts the CH3Hg+ concen-
tration after 48 hours of exposure was nearly the same as that recorded at
the first extraction (4 hours).
Comparison of graphs a, b, and c of Figure 1 indicates a distinct pat-
tern of concentration change in the aerial portion of the plant. Periods of
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8 12 16 20 24 28 32 36 40 44 48
HOURS OF EXPOSURE
8 I? 16 20 24 ?8 32 36 40 44 4B
HOURS OF EXPOSURE
38-
32-
28-
24-
.20-
:16-
12-
|B) APEX
8 12
16 20 24 28 32
HOURS OF EXPOSURE
i-'"r
CONTROL
f""i
36 40 44 48
12 16 20 24 28 32
HOURS OF EXPOSURE
36 40 44
Figure 1. Changes in the concentration of ethylmercury and methylmercury
in various plant parts of peas exposed to elemental mercury
vapor for 48 hours.
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high CgHsHg"^ concentration are periods of low CH3Hg+ concentration, and vice
versa, so that the two curves are similar but are out of phase.
To elaborate this relationship more fully, peas were exposed to elemental
mercury vapor for a period of either 24 hours light or 24 hours continual
darkness. The results indicate nearly twice as much CaHsHg"*" formed in
the laterals and apex during the light period than in the dark (Figure 2). No
C2HsHg+ could be detected in the stem or root under conditions of continuous
darkness, despite concentrations of CHaHg comparable to those recorded for
the plants exposed in the light.
levels of CHsHg"1" in the plant.
Light had no significant effect on the
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20-
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^ETHYLMERCURY
[^METHYLMERCURY
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-X- NONE DETECTABLE
1. S.E. OF THE MEAN
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DARK LIGHT DARK LIGHT DARK LIGHT DARK
LATERALS STEM
APEX ROOT
Figure 2.
The effect of light on ethylnxercury and methylniercury concen-
tration in Pisuax sativua after a 24-hour escposure to mercury
vapor.
Both C2H5Hg+ and CH3Hg+ were detected in the root, although appreciable
concentrations of CaHsHg+ were not observed until after 12 hours of exposure
to Hg . The presence of these compounds can be explained by two possible
mechanisms. Either they were formed in the root from mercury taken up from
the water in which plants were grown, or they were transported from the aerial
portion of the plant to the root. A less likely possibility is that all
mercury detected in the aerial portion of the plant originated in the root.
To determine the source of the organic mercury, the roots were "isolated"
from the mercury vapor by placing the roots of seedlings grown in 5-ml micro-
beakers into a 50-ml test tube, care being taken to form a tight seal between
the beaker and test tube. A layer of activated charcoal was placed across the
bottom of the microbeaker and was covered by a layer of vermiculite. As is
indicated in Table 2, no C2H5Hg+ or CH3Hg+ was detected in the "isolated"
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roots after a 24-hour exposure, despite formation of appreciable concentra-
tions of both organomercury compounds in the aerial portions of the plant.
TABLE 2. THE EFFECT OF ISOLATING THE ROOT MEDIUM FROM ELEMENTAL
MERCURY VAPOR. (Exposure time, was 24 hours.)
Root Medium Isolated Root Medium Exposed
C2H5Hg+ (ng/g) CH3Hg+ (ng/g) C2H5Hg+ (ng/g) CH3Hg+ (ng/g)
Apex
Lat
Stem
Root
15.99
15.6
15.88
N.D.*
15.45
14.35
N.D.*
N.D.*
12.67
6.73
8.04
4.25
11.62
4.99
3.18
6.43
*None Detectable.
DISCUSSION
The formation of both C2H5Hg+ and CH3Hg+ in peas exposed to Hg offers
some fresh insight into the nature of the mercury pathway of the pea. Methyl-
mercury formation in the pea has been reported previously by Gay (1975). +In
both in vivo and in vitro experiments with inorganic mercury salts, CH3Hg
was identified as an intermediate product. The data reported hereQsuggest
that it may also be an intermediate in the biotransformation of Hg . This
is supported by the following observations.
(a) Although the level of C2H5Hg+ increased during the 48-hour exposure
period, no corresponding rise in CH3Hg+ was observed. A baseline average
of 4.66 nanograms CH3Hg+ per gram was maintained in the aerial portion of
the plant with concentrations at 48 hours being nearly the same as those
recorded at 4 hours.
(b) Fluctuations in CH3Hg concentrations between replicate samples
(Table 1 and Figure 2) suggest an intermediary role. The release of volatile
mercury products from plants has been reported by Siegel (1974). His work
resulted from the observation of analytical inconsistencies in data from re-
plicate leaf samples. In this study, we observed similar inconsistencies in
the CH3Hg+ data. However, standard error for C2H5Hg+ data was small. The
efficiency of the extraction and analytical procedures being similar for
both CH3Hg+ and C2H5Hg+, inconsistencies should have been observed for both,
but were not. The apparent inconsistencies appear to be artifacts of the
time intervals between extractions and the inability to detect other inter-
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mediates or final mercury products, possibly because of their volatile nature.
(c) Further verification of this role as intermediate is indicated by
the failure of light to have any significant effect on CH3Hg"1" concentration
despite a two-fold increase of CaHsHg"1" in the same tissue (Figure 2).
Following the report that Hg vapor induces ethylene formation and
abscission in Coleus and Citrus explants (Goren and Siegel, 1976),
was considered a likely metabolite in the mercury pathway. The accumulation
of CaHsHg"1" observed in this investigation verifies this initial assumption,
but does not enable definition of its role as initial product, intermediary,
or final product in this biochemical pathway.
The pattern of concentration changes observed (Figure 1) does indicate
that CH3Hg+ and C2H5Hg+ are both metabolites of a single pathway of mercury
in the pea. In the aerial portion of the plant, a high concentration of
either metabolite is followed by a high concentration of the other metabolite,
with a simultaneous decrease in the concentration (or rate of increase) of
the former. The net result of this would be to prevent accumulation of high
concentrations of any single mercury metabolite, presumably by conversion to
other, presently unidentified, mercury products.
REFERENCES
Gay, D. D. 1976. Methylmercury: Formation in plant tissues. Ecological
Research Series. U.S. Environmental Protection Agency report. EPA-600/3-
76-049, Las Vegas, Nevada, May 1976.
Gay, D. D. 1976. Biotransformation and chemical form of mercury in plants.
Ecological Research Series. U.S. Environmental Protection Agency report.
EPA-600/3-76-082, Las Vegas, Nevada, July 1976.
Gay, D. D. and C. W. Frank. Personal Communication, July 1976.
Goren, R. and S. M. Siegel. 1976. Mercury induced ethylene formation and
abscission in Citrus and Coleus explants. Plant Physiology 57, 628-631.
Long, S. J. , D. R. Scott, and R. J. Thompson. 1973. Atomic absorption of
elemental mercury collected from ambient air on silver wool. Analytical
Chemistry 45_: 22-27.
Siegel, S. M., N. J. Puerner, and T. W. Speitel. 1974. Release of volatile
mercury from vascular plants. Physiologia Plantarum 32:174-176.
Speitel, T. W. and S. M. Siegel. 1975. Auxin and carbon dioxide sensitive
effects of mercury and iodine vapors in plant senescence. Plant and Cell
Physiology 16_: 383-386.
Talmi, Yair. 1975. The rapid sub-picogram determination of volatile
organo-mercury compounds by gas chromatography with a microwave emission
spectrometric detector system. Analytica Chimica Acta 74:107-117.
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/3-78-037
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
ETHYLMERCURY: FORMATION IN PLANT TISSUES AND
RELATION TO METHYLMERCURY FORMATION
5. REPORT DATE
April 1978
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
L. C. Fortmann. D. D. Gay, and K. 0. Wirtz
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Environmental Monitoring and Support Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Las Vegas, Nevada 39114
10. PROGRAM ELEMENT NO.
1AA601
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
U.S. Environmental Protection Agency-Las Vegas, NV
Office of Research and Development
Environmental Monitoring and Support Laboratory
Las Vegas. Nevada 89114
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
EPA/600/07
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Seedlings of the common dwarf garden pea, Pisum sativum, cv. Little Marvel,
exposed to elemental mercury vapor formed both methylmercury and ethylmercury
in all parts of the plant. Concentrations of both organomercury compounds
fluctuated considerably over a 48-hour exposure period, but the total of
detectable forms of mercury continued to rise due to increased ethylmercury
formation. Ethylmercury formation was greater in the light than in the dark,
but methylmercury concentration did not differ significantly. The pattern
of change in the concentrations of methylmercury and ethylmercury suggests
both are metabolites of a single mercury pathway in peas.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Plant physiology
Plant chemistry
Mercury
Trace elements
Leguminous plants
06A,CrF
07B,C
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
12
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
«U. S. GOVERNMENT PRINTING OFFICE: 1978-785-627
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