EPA-600/3-77-122
October 1977
Ecological Research Series
          MOVEMENT OF MERCURY-203  IN  PLANTS
                                  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
Information Service, Springfield, Virginia 22161.

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                                                 EPA-600/3-77-122
                                                 October 1977
          MOVEMENT OF MERCURY-203 IN PLANTS
           Don D. Gay and Gene P. Butler
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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                                 FOREWORD
     Protection of the environment requires effective regulatory actions
which are based on sound technical and scientific information.  This infor-
mation 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 involved, 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 multi-
disciplinary, 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

     Two varieties of peas were each planted in soil containing one of
three radioactive forms of mercury.  The total uptake and distribution
of the three forms of mercury in the two pea varieties are not equal,
suggesting at least two separate pathways for mercury uptake and distri-
bution in plants.  The conclusions can be beneficial in designing experi-
ments dealing with mercury compounds and plants and also in the interpre-
tation of data gathered by other investigators.  Users who should find
the report of value include the Office of Air Programs, Office of Toxic
Substances, laboratories within the Office of Research and Development,
other Federal agencies, and university and industrial research staffs.
                                         George B. Morgan
                                             Director
                        Environmental Monitoring and Support Laboratory
                                         Las Vegas, Nevada
                                      iii

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                               ACKNOWLEDGMENT
     The authors gratefully acknowledge the assistance of Professor
Aaron Goldman, Department of Mathematics, University of Nevada, Las Vegas,
in the statistical treatment of the data obtained from this experiment.
                                     iv

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                                   INTRODUCTION
     With  the  findings  that the  toxic methylmercury compound  is being  formed
 in the environment  through microorganisms-mediated transformation  of inorganic
 mercury compounds  (Bisogni and Lawrence, 1973; Jensen and Jernelov, 1969;
 Jernelov,  1970; Landner, 1971; McKinney, 1972; Spangler et_ alU , 1973;  Wood
 et^ al., 1968;  Yamada, 1972; Yamada and Tonomura, 1972), through soil-mediated
 transformation of mercury compounds  (Alberts et^ al., 1974; Kimura  and  Miller,
 1964; Jernelov, 1970; Beckert et^ al^. , 1974) and through plant-mediated trans-
 formations of  inorganic and organic mercury compounds (Fukunaga et_ al., 1972;
 Gay, 1975; Gay, in  press), the investigation of plant uptake  of mercury needs
 to be expanded to include the examination of the uptake and distribution of
 different mercury species in plants.

     The movement and storage of mercury in a variety of plants has been
 determined by  numerous  investigators (Aarkrog and Lippert, 1971; Alvarez,
 1974; Anderson and  Nilsson, 1972; Barker, 1972; Bowden and McArthur, 1972;
 Dae _et al. , 1966; DeGoey e± al., 1971; Fang, 1973; F.indenegg  and Haunold,
 1972; Friedman and  Waiss, 1972; Fukunaga e_t al. , 1972; Gerdes £t al. ,  1974;
 Hauskrecht and Hajduuk, 1966; Imre and Berencsi, 1972; James  e* al., 1971;
 John, 1972; Lee e^  al., 1972; Nardin, 1971; Pickard e£ a^., 1963;  Rao  et^ _al. ,
 1966; Ross and Steward, 1962; Saha e£ al. , 1970; Shacklette,  1965; Smart, 1964;
 Smith, 1972; Stewart and Ross, 1967; Tanner et^ al., 1972; Tkachuk  and  Kuzina,
 1972; Van Loon, 1974; Yamada, 1968; Yeaple, 1972).  Almost all of  this work
 has shown the  total levels of mercury in plants and plant parts via analyses
 by atomic absorption spectrophotometry, by neutron activation or by gamma
 spectroscopy using  radioactive tracers.  These investigations assume that
 the mercury compound used to treat the plant remains the same throughout the
 experiment.

     Recent evidence suggests that this assumption-is not necessarily
 warranted.  Fukunaga et al. (1972) showed a trace amount of methylmercury
 in rice plants treated with various phenylmercury compounds.  Gay  (1975)
 and Gay (in press)  showed that methylmercury is indeed formed in the garden
 pea when it is treated with ionic mercury or phenylmercury compounds through
 in vivo and in vitro studies.  Siegel et al. (1974) have shown that a  volatile
form of mercury is  released from plants harvested near a volcano and allowed
 to stand for periods of time before analysis.  The mercury compound volati-
 lized was not  identified.  Beauford et al. (1975) reported the release of
 heavy metals from plants as being associated with the loss of cuticular wax
 particles.

     It is evident  from these studies that the uptake of mercury by plants
 is not a simple matter.  The biological pathways of the various forms  need
 to be examined for  an understanding of the potential hazard of mercury
 pollution to mankind via the food chain.

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     The present study was designed to assess the uptake, translocation and
storage of three forms of mercury via root uptake from contaminated soil.
Radioactive mercury-203, in the forms of mercuric acetate, methylmercuric
chloride and phenylmercuric acetate, were used.  The garden pea, Pisum
sativum, was the plant of choice and two distinct varieties were used, a
dwarf variety, Little Marvel, and a normal variety, Alaska.
                                 CONCLUSIONS
     Two varieties of the garden pea, Pisurn sativum, were used.  The dwarf
variety, Little Marvel, accumulated and stored about twice as much mercury
through root uptake from contaminated soils as the normal variety, Alaska'.

     Phenylmercury was accumulated and stored in the dwarf and normal
varieties of peas at levels 7 and 10 times greater, respectively, than
methylmercury or ionic mercury.  Phenylmercury was also stored at higher
levels in the pods than in the leaves or stems.

     Two apparent pathways exist in peas for the movement and storage of
the three mercury compounds used.  Ionic and methylmercury utilize one
pathway and phenylmercury utilizes another.
                          MATERIALS AND METHODS
     Soil for the experiment was obtained from the University of Nevada
Agricultural Experiment Station at Logandale, Nevada, and had the follow-
ing characteristics:  54% sand, 11% clay, 35% silt, 1.3% organic carbon
and pH 8.6.

     Three radioactive mercury-203 compounds (mercuric acetate, methyl-
mercuric chloride and phenylmercuric acetate) were obtained from New
England Nuclear at concentrations of one millicurie each.  Each isotope
was handled separately to prevent any cross-contamination.

     Sixty 12.7 centimeter (cm) plastic pots filled with approximately
1.3 kilograms of soil were used for each isotope.  The total amount of soil
needed for each isotope was sieved through 12-mesh screen into plywood boxes
(91 cm in length, 61 cm in width, and 30 cm in height).   The radioactive
mercury compounds were each added to one liter of distilled water and the
original containers were rinsed twice.   Large plastic watering cans were
used to slowly spray the radioactive mercury solutions onto the soil in each
box while the soil was being mixed with a hoe.   After the addition of the
radioactive mercury solutions, it was noted that small wet dirt balls had
formed throughout the soil.  The soil mixture was dumped out of the boxes
onto large heavy pieces of plastic and sieved again a shovelful at a time.
In the sieving of each shovelful of soil, the small wet dirt balls were broken
up by scraping a trowel across the screen.  When all of the soil was sieved
again, the box of soil was mixed again with a hoe and the 12.7 cm plastic

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 pots were  filled.  Four pea seeds were planted  in each pot; 30 pots received
 the seeds  from  the dwarf variety and 30 pots received seeds of the normal
 variety.

     To prevent  cross-contamination three special exposure chambers were
 designed and built for each of the three radioactive mercury compounds.
 The chambers were made of redwood and cedar frames covered with clear ace-
 tate.  All joints were sealed with 5 cm Permaseal plastic tape.  The dimen-
 sions of each chamber were 137 cm in height, 152 cm in length, and 91 cm in
 width.  A  constant air flow of 1.7 m3 min~l was maintained through each
 chamber.  The air flow was through holes in the lower edge of the long sides
 of each chamber, through a 12.7 cm diameter acetate plenum mounted length-
 wise across the  top of each chamber, through 12.7 cm stove pipe mounted down
 one end of each  chamber with a damper control,  through a prefilter and two
 activated charcoal filters mounted inside a fiberglass-lined plywood box and
 through an exhaust fan, rated at 0.235 m^s"1 air flow,^ to the outside.  Air
 from all three chambers converged into the box  containing the filters.  A
 platform made of redwood slats was placed 15.24 cm above the bottom of each
 chamber on which the pots were placed.  Each pot was watered twice a day at
 8 a.m. and 4 p.m.  A door 61 cm by 61 cm was made in the middle of one long
 side of each chamber and a rubber gasket surrounded each opening.  A 0.64 cm
 thick piece of plexiglass was used for each door and each was secured by
 eight bolts.

     Two varieties of Pisum sativum in terms of overall growth were selected
 for the experiment.  A dwarf variety, Little Marvel, was selected because
 it is a short plant displaying thick stems and  large leaves.  A normal
 variety, Alaska, was selected because it attains a height 2 to 3 times
 that of the dwarf and has smaller leaves and much less thickened stems
 than displayed by the dwarf variety.

     The peas were planted December 23 and the  first harvest was on February
 21 with subsequent harvests on February 28 and March 7.   The plants were
 divided into stems, leaves and pods to assess the amount of translocation
 and storage occurring with each form of mercury.  The plants from five pots,
 randomly selected for each variety of pea, were harvested and pooled to
make up the sample from each mercury-amended soil.  Each fraction was
weighed and sealed in 5 cm aluminum cans.   The samples were analyzed at
 the Las Vegas Laboratory and the results were corrected for background and
 decay.

     To assess significant differences among all the variables, a least
 squares three-way analysis of covariance statistical procedure was used.
With the collection of plant samples weekly for 3 weeks, a test was needed
which would determine if there were any differences in sampling over time.
 In the analysis of covariance test the covariant is the weekly sampling of
plant parts and is represented by the regression factor.

     The interactions having significant differences at  the 95% confidence
 interval were further broken down by way of a Studentized Range Test to
determine where the differences occurred.

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                              RESULTS AND DISCUSSION
      The  soil-mixing  technique was  evaluated  for  thoroughness  of distri-
 bution  of the  radioactive mercury isotopes by taking  12 random samples
 from each of the  contaminated soils.  The amount  of soil used  for  each
 sample  was 100 grams  and the soil was sealed  in 5 cm  aluminum  cans and
 analyzed  by Nal (Tl)  gamma  spectroscopy.  The 95% confidence intervals
 for  the different  contaminated soils was the  mean value ±1.3%  for  ionic
 mercury,  ±1.2% for phenylmercury and ±1.1% for methylmercury.  The mixing
 technique,  though  simple, is extremely thorough in distributing the radio-
 active  mercury throughout the soil.

      The  data  realized from the experiment (see Table 1) is expressed in
 picocuries  per gram fresh weight of tissue.   The  data show the two pea
 varieties  used did not take up and store in their respective tissues, pods,
 leaves, and stems,  equivalent amounts of the  three forms of mercury added
 to the  soil.

      To determine  where significant differences in mercury levels occurred
 a least squares three-way analysis of covariance  statistical procedure was
 applied to  the data (see Table 2).  This analysis showed that significant
 differences exist  (1) among the three forms of mercury used, (2) between
 the dwarf  and  normal varieties of peas, and (3) among selected pea tissues
 analyzed.

      A  Studentized Range Test (SRT) at the 95% probability level showing
 significant differences was used to determine where the significant dif-
 ferences occurred.

      When  the  measured levels of the three forms  of mercury used in this
 experiment are treated with the SRT, we find  that the methylmercury level
 does  not significantly differ overall from ionic mercury level.  However,
 both  methylmercury and ionic mercury levels differ significantly from the
 phenylmercury  level.

      When the  two pea varieties are treated with  the SRT,  we again find
 overall significant differences in mercury levels between stems and leaves
 and stems and  pods but no significant difference between leaves and pods.

      Applying  the SRT to the form of mercury versus the pea variety factor,
we find that ionic mercury and methylmercury levels in Little Marvel or
Alaska peas differ significantly from phenylmercury levels in either Little
Marvel or Alaska peas and that the phenylmercury level in the Alaska peas
 differs significantly from the phenylmercury level in the Little Marvel
peas.  No significant difference was found between methylmercury and ionic
mercury levels in either Little Marvel or Alaska peas.  Figure 1 depicts
 the mean values for each form of mercury found in Alaska or Little Marvel
varieties of peas.

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TABLE 1.  THE WET WEIGHTS AND AMOUNTS OF MERCURY-203 IN pCl/g
          OF SELECTED PLANT PARTS HARVESTED WEEKLY FOR THREE
          WEEKS FROM LITTLE MARVEL AND ALASKA PEAS

February 21
Hg Source
Methyl


Methyl

Ionic


Ionic

Phenyl


Phenyl

Peas
Alaska


Little
Marvel

Alaska


Little
Marvel

Alaska


Little
Marvel

Parts
Pods
Leaves
Stems
Pods
Leaves
Stems
Pods
Leaves
Stems
Pods
Leaves
Stems
Pods
Leaves
Stems
Pods
Leaves
Stems
Weight
0.69
3.74
3.33
7.20
4.62
2.00
1.46
3.01
2.78
6.27
3.67
1.57
1.79
5.47
5.48
4.40
3.20
1.17
pCi/R
39
62
38
31
140
57
18
79
23
110
220
140
780
540
480
980
880
630
February 28
Weight
(R)
1.97
6.14
6.60
7.88
4.30
1.89
3.00
5.37
4.98
5.07
2.59
1.22
3.49
4.89
5.68
3.48
3.11
1.20
pCi/l
44
51
28
140
180
130
39
65
24
65
150
74
710
470
370
1200
690
670
March 7
Weight
* (R)
1.24
4.25
5.51
8.23
4.07
1.38
4.77
3.17
3.55
3.37
1.40
0.87
2.72
7.30
8.02
2.73
0.98
0.99
pCi/s
42
43
14
70
110
57
53
110
27
150
380
270
490
310
230
1300
1100
540

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        TABLE 2.  LEAST SQUARES THREE-WAY ANALYSIS OF COVARIANCE
    Source
DF
   SS
MS
Form of Mercury

Pea Variety

Plant Parts

Form of Mercury
     vs.
Pea Variety

Form of Mercury
     vs.
Plant Parts

Pea Variety
    vs.
Plant Parts
2

1

2
4,177,030   2,088,520     230.45*

  494,256     494,256      54.54*

  179,106      89,553       9.88*
           285,798
           399,450
            17,562
              142,899
               99,863
                8,781
*Denotes significance at the 95% confidence level,
DF = degrees of freedom
SS = sums of squares
MS = mean square
 F = Fisher variance ratio
            15.77*
            11.02*
             0.97
Form of Mercury
vs.
Pea Variety 4
vs.
Plant Parts
Regression 1
Error 35


42,547 10,637 1.17


67 67 0.007
317,203 9,063

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 1000

  900

  800
METHYLMERCURY
             ALASKA
                         LITTLE MARVEL
           PEA VARIETY

Figure  1.  Mean values of the total mercury found in the Alaska and
          Little Marvel varieties of peas.

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 1000
                                            IONIC MERCURY

                                            PHENYLMERCURY
             POOS
     LEAVES
PLANT PARTS
STEMS
Figure 2.  Mean values of  the amount of mercury in various parts  of
          the pea plant.

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      Looking at the interaction between the three forms of mercury and
 the three plant parts by way of the SRT, we find again the major differ-
 ence between phenylmercury and each of the other two forms.  Methylmercury
 in the stems, leaves or pods and ionic mercury in the stems, leaves or
 pods both differ significantly from phenylmercury in the stems, leaves or
 pods.  In addition it was noted that phenylmercury in the stems and leaves
 differs significantly from phenylmercury in the pods.

      Figure 2 depicts the mean values of the amounts of mercury found
 in the various plant parts.  The graph shows the close relationship
 between methylmercury and ionic mercury and the disparity in relation
 of each of these to phenylmercury.

      Because no significant difference was noted for pea variety versus
 plant parts in the analysis of covariance, the Studentized Range Test was
 not applied.

      The results from this experiment consistently show that phenylmercury
 does not have the same pathway in either variety of peas as ionic or methyl-
 mercury,  which utilize the same pathway.

      In a previous paper (Gay,  1975),  it was  shown that phenylmercury and
 ionic mercury had different sites  for their transformation to methylmercury.
 Although both mercurials give  rise  to methylmercury when incubated with
 plant tissues,  maximum formation of methylmercury from phenylmercury
 occurred in the apical regions  while maximum  formation of  methylmercury
 from ionic mercury occurred in  subtending internodal regions.   That
 methylmercury is formed from either mercury compound is unusual,  but  to
 have  two  apparent  pathways  for  these transformations is totally an unexplor-
 ed region.

     This  experiment supports the theory of separate but distinct  pathways
 for  the metabolism and  storage  of different mercury  compounds  in  plants.

     Ubiquitous  mercury  levels  in similar  plants  cannot be  assumed  because
 this  experiment, utilizing  peas  from the same genus  and species but different
 varieties,  shows that highly significant differences  in mercury levels  exist
 between two  pea  varieties.


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                                      11

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                                       12

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Yeaple,  D.  S.   "Mercury in  Broyophytes."  Nature 235:5335,  pp.  229-230
1972
                                         13
                                                  *U. S. GOVERNMENT PRINTING OFFICE: 1977-785-182

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                                    TECHNICAL REPORT DATA
                            (Please read Instructions on the reverse before completing}
 1. REPORT NO.
    EPA-600/3-77-122
                                                            3. RECIPIENT'S ACCESSION-NO.
 4. TITLE ANDSUBTITLE

    MOVEMENT OF MERCURY-203 IN PLANTS
              5. REPORT DATE
               October 1977
                                                            6. PERFORMING ORGANIZATION CODE
 7. AUTHOR(S)
    Don D.  Gay and Gene P.  Butler
                                                            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. NV  89114	
              10. PROGRAM ELEMENT NO.

                1AA602
              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

    Seeds of Pisum sativum, varieties Little Marvel  and Alaska, were planted  in soils
    contaminated with radioactive ionic mercury, methylmercury or phenylmercury
    compounds.   After maturation, stems, leaves, and pods were harvested and  analyzed
    by  gamma spectroscopy.  Utilizing a least squares three-way analysis of covariance
    coupled  with a Studentized  Range Test, significant differences were noted among
    the levels  of the three mercury compounds in the plants,  between mercury  levels
    in  the two  pea varieties and  among mercury levels in the  different pea tissues
    examined.

    Phenylmercury levels differed consistently from  levels of ionic mercury and
    methylmercury suggesting a  separate pathway for  it in peas.
 7.
                                KEY WORDS AND DOCUMENT ANALYSIS
                  DESCRIPTORS
b.lDENTIFIEBS/OPEN ENDED TERMS  C. COSATI Field/Group
   Plant  physiology
   Plant  chemistry
   Mercury
   Trace  elements
   Leguminous plants
   Absorption
    Methylmercury
    Phenylmercury
    Mercurial  transforma-
      tion
    Mercury uptake
    Pisum sativum
     06A
     06C
     06F
     07B
     07C
     17C
 8. DISTRIBUTION STATEMENT

   RELEASE TO PUBLIC
19. SECURITY CLASS (THIS Report)
    UNCLASSIFIED
21. NO. OF PAGES
      20
20. SECURITY CLASS (Thispage)
    UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)

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