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INJURY BY ORGANIC MERCURY SEED TREATMENT IN PEAS

BY

ALAIN FRANCOIS CORCOS
W‘-

A THESIS

Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Botany and Plant Pathology

1952.

THESIS

ACKNOWLEDGMENTS

The writer wishes to thank Dr. D. J. deZeeuw and Dr.
A. L. Andersen for suggesting the problem, and Dr. J. R.
Vaughan, under the guidance of whom his studies were made,
for help in preparing the manuscript. The writer wants to
express his deep appreciation to Dr. deZeeuw for all technical
and helpful advice that he gave him during the course of these
investigations and the making of the manuscript. Valuable crit-
icism of the manuscript was also given by Dr. S. H. Wittwer

and Dr. L. O. Mericle.

t“ 3V3 1‘1 “ :1»; L:

."ur(‘.‘3sr~

TABLE OF CONTENTS

INTRODUCTION ...... . ....................
REVIEW OF LITERATURE . ..................
History of Organic Mercurials ...............

' Injury to Cereals ........................
Injury to Ornamentals .....................
Injury to Miscellaneous Crops . . . ............
Injury to Field Legumes ...................
Injury to Vegetables Other than Peas ..........
Injury to Peas ..... . ....................
MATERIALS AND METHODS ............ . .....
EXPERIMENTAL RESULTS ...................
Field Experiment . ........ . ..............

Effect of Storage on Organic Mercury
Treated Seed . ..... . . . . . . ...............

Histological Studies ....... . ..............

DISC USSION ..............................

10

14

16

20

20

25

27

39

44

46

INTRODUCTION

Seed treatment occupies a prominent place in agriculture
because the first step towards growing a good crop is to sow
healthy seeds. Four kinds of seed treatment have been recog-
nized according to their nature and their purpose: (1) Seed dis—
infection aims to eradicate the fungus or bacterium which has
infected the seed and is established within the seed coat or in
deeper-seated tissue. (2) Seed disinfestation destroys the micro—
organisms on the seed coat. (3) Seed protection, as the name
implies, is intended to protect the seeds from the attacks of
rotting organisms. (4) Seed inoculation with nitrogen-fixing bac-
teria, though not a recent development, has become more and
more important in field legumes.

The most prominent type of treatment is seed protection,
because it is easier to protect than to cure. This involves at
the present time the use of fungicides, either in a dust form
or semiliquid paste, which are applied to the seed coat. Be-
fore the advent of the nonmetallic seed protectants, the poison-

ous and volatile organic mercury dusts were prominent in the

field of seed treatment. Although injury from such dusts has
been known to occur under excessive moisture, poor ventilation
and high temperature, they have been more effective in cereal
disease control than any other type of fungicide. A slurry
method of treating seeds has been devised to prevent the worker
from inhaling the dusts and to apply more efficaciously the fun-
gicide onto the seed coat and the dust containing a wettable
carrier adheres more tightly to it. However, because of the
moisture involved,,chemical injury is more likely to occur.
Serious injury following such a method has been reported on
peanuts (55) and on peas (13). Such injury and inconvenience
partly impairs benefits gained from the slurry method. Little
is known concerning the mechanism by which the slurry method
injures the seeds, and there is some question as to when injury
to the embryo occurs. Extensive histological and cytological
studies have been made on corn seedlings poisoned with ethyl
mercury phosphate (46), but no reports of such studies can be
found in the literature on dicotyledonous plants. For these
reasons, an investigation was begun in the spring of 1951 to
study the mechanism by which the slurry method of seed treat-

ment causes injury to peas. Certain anatomical and histological

deformations resulting from organic mercury treatment were

studied.

REVIEW OF LITERATURE

History of Organic Mercurials

About 1900, European scientists found that certain or-
ganic mercury compounds were highly efficient in destroying
bacteria and their spores (16). Their results led to further in-
vestigations on the use of these materials. According to Gabel
(l7), mention of organic mercurial salts in seed treatment was
first made in 1913. The simplest organic compound used, cya-
nide of mercury, was reported to be completely effective in
preventing stripe disease of barley when used as a steep for
the grain. Mercury chlorophenol, introduced by Remi in Ger-

many for the steeping of cereals attacked by Fusarium, was the

 

chief component of the fungicide "Uspulum." Another organic
salt of mercury, mercury cresol sodium cyanide, was the chief
component of the fungicide "Germisan." These fungicides were
introduced, in the United States about 1925 (20), and soon were
followed by other new organic mercurials. Of these newer
fungicides, the organic mercurials known to have been injurious

under various conditions are listed in Table 1.

Table 1. Organic mercurials occasionally injurious to seeds.

 

 

Trade Name

Composition

Company

 

Semesan

Semesan Bel

Ceresan

Ceresan M

New Improved
Ce re san

30% hydroxymercury
ch10 rophenol

12% hydroxymercury
nitrophenol and hydroxy-
mercury chlorophenol

2% ethyl mercury chlo-

ride

7.7% ethyl mercury
p-toluene sulfonanilide

5% ethyl mercury phos-
phate

E. I. duPont de
Nemours, Wil-
mington, Del.

E. I. duPont de
Nemours, Wil-
mington, Del.

E. I. duPont de
Nemours, Wil-
mington, Del.

E. I. duPont de
Nemours, Wil-
mington, Del.

Bayer Semesan
Div. of E. I.
duPont de Nemours,
Wilmington, Del.

 

 

Several causes of poor emergence of seedlings could be
seed decay by rotting organisms and injury by toxic seed pro-
tectants. Injury by organic mercurials, though not always de—
tected, may often contribute to the delay emergence. Numer-
ous reports of visible toxicity by the organic mercurials to
seeds and seedlings of cereals, ornamentals, field legumes and

vegetables can be found in the literature.
Injury to Cereals

When correctly used, organic-mercury seed treatments
give an excellent control of many seed-borne diseases, but under
some conditions injury to the grains results and considerable
loss has been reported. Weston and Brett (54) recorded some
factors which predispose the grain to injury such as excessive
moisture, high temperature and poor ventilation. Crosier (10)
reported abnormal germination in a sample of Marquis spring
wheat, which was treated with Ceresan and stored before plant-
ing. Records of disappointing field stands have been common.
When treated with dry dusts, the seeds germinated well, but
when treated with Ceresan while moist, they produced roots

and plumules that failed to elongate normally and were abnormally

thickened. Porter (45) reported in 1936 that cereal seeds treated
with an overdose (any amount in excess of that required for pro-
tection) of mercurials had thickened leaf primordia with irregu-
lar crenations and lobes. Cell division was inhibited and the
existing cells had become enlarged and multinucleate, either with
small nuclei or with large polyploid "giant nuclei.” Atkins and
Stamps (2) mentioned in 1948 that seed treatments on oats in-

fected with Helminthosporium victoriae had not given good re-

 

sults. Some of the mercurials drastically reduced stands when
used in excess. In 1951 Craley and French (8) found that in—
creasing rates of Ceresan M from 3/4 to 1-1/2 ounce per
bushel progressively injured rice seedlings. Stephenson (47)
reported that grass seeds were killed if soaked in a saturated
solution of Semesan, Semesan Bel, or Ceresan. Hoppe (23)
reported that seeds mechanically injured along the edges of

the embryo developed the stunted and swollen condition typical

of me rcury poisoning .

Injury to Ornamentals

Person and Chilton (43) found that Ceresan, Semesan,

New Improved Ceresan and Semesan Jr. injured ornamental

seeds when applied in full strength. Gladiolus corms are known
to absorb toxic amounts of mercury from mercuric chloride
(41) and are injured by organic mercurials such as Semesan
(21). There was a direct relationship between external injury
and mercury content in Narcissus bulbs treated with New Im-
proved Ceresan, Ceresan M, and 2 percent Ceresan reported

by McClellan (33). Gould and Miller (20) reported that phenyl
mercury acetate (1 pound to 700 gallons of water) used as a

dip was much safer than the standard 2 percent Ceresan dip,

even though some bulbs were injured.
Injury to Miscellaneous Crops

Thomas (48) treated safflower seeds with Ceresan M at
the rate of 0.5 ounce per bushel in an attempt to control rust

(Puccinia carthami). He found partial control and some reduc-

 

tion in stand at 9 ounces per bushel, the maximum dosage that
the seed would hold. Ciferri (6) reported in 1951 that an ex-
perimental mercurial "zeolite" used as a tobacco seed dressing
caused primary lesions of the phloem. The hypocotyl was stunted,

twisted, and hypertrophied. Xylem cells contiguous to the phloem

were interrupted, and endodermal as well as cortical cells were

collapsed.
Injury to Field Legumes

The literature concerning the effects of chemical treat-
ment on nodulation has been conflicting. Appleman (1) found
satisfactory nodulation on peas and soybeans grown from inocu-
lated seed treated with Semesan. In his tests Ceresan prevented
nodulation on canning peas, but not on soybeans. In the latter
case all nodulations appeared to be on lateral roots and not
the taproot, as typically obtained on plants from nontreated
inoculated seeds. Kadow, Allison, and Anderson (29) found a
decrease of nodulation from an'average of 75 nodules (per plant
nontreated) to 3 or 4 nodules per plant as a result of treating
with an unidentified organic mercury. Some of the earlier work
by Miller and Stapp reported by Appleman (1) demonstrated
that if nodule bacteria are in the soil at the time of planting,
seed treatments do not hinder the development of root nodules.
In trials testing the importance of seed inoculating with a bac-
terial inoculum such as ”Nitragin," Vlitos and Preston (50)

excluded Ceresan M because in preliminary work this fungicide

10
reduced considerably the stands of Austrian Winter pea, mung-
bean chinese red cowpea, yellow hop clover and hairy wetch.
Recently Gederman (19) studied the effects of Arasan, Phygon,
and Ceresan M on red clover, Alfafa, and sweet clover in wet
and dry soil. In general the crops were benefited by most
treatments. Phygon and Arasan, nonmercury compounds which
appeared harmless in wet soil, reduced the emergence or in-
jured the seedlings when the seed was planted in dry soil. In
greenhouse tests with 1 percent Ceresan M, red clover failed
to form roots and there was a swelling of the hypocotyl. Seed-
lings of alfalfa and sweet clover were only slightly affected by

the same treatment.
Injury to Vegetables Other than Peas

Bald (3) reported that mercury treatments were toxic to
the cells of potato tubers and probably also to the rotting or-
ganisms. For this reason, when used to treat tubers, organic
mercury dips gave some protection to the sets cut from them,
without noticeably affecting suberization. Its effects on the cut
surfaces, were however to destroy many layers of cells, and

absorption of mercury seriously affected the emergence and

11
subsequent growth of the potato shoots. Muller (34), as reported
by Clayton (7), found "Germisan" and "Uspulum" toxic to to-
mato and celery seed in the concentration usually employed.
Horsfall (24), in 1930, found that Semesan was highly injurious
to tomatoes under certain conditions "not well understood as
yet." Clayton (7) treated tomato seeds with a liquid organic
mercurial and found germination temporarily inhibited. He
concluded that it was safer in this case to use organic mercury
dusts. Vaughan (49) reported that concentrations of ethyl mer-
cury phosphate greater than 1/Z0,000 used on tomato seed caused
a reduction in the percentage of germination as well as a slow-
ing up of the rate of germination. Davis and Haenseler (12)
however, found no visible injury with New Improved Ceresan
to tomato in the soil greenhouse tests, but a slight injury with
New Improved Ceresan in sand tests. Leach (30), in tests
combining fungicidal treatments with pelleting materials, re—
marked that the delay in emergence of pelleted tomato seeds,
previously treated with mercuric chloride or New Improved
Ceresan dip, was greater than the additive delays from coating
and treating separately. The inclusion of organic mercury

compounds in coating resulted in reduced and retarded emergence.

12
Miller and Grogan (35) treated tomato seed with mercuric chlo-
ride (1/3,000) and with New Improved Ceresan (1/1,200). They
concluded that when the ratio of seed weight to the volume of
treating solution was increased above 1:8, germination was im-
paired. Dickey and Ark (14, 15) investigated injury to tomato
seeds treated with mercurials. They determined the location
and the concentration of the mercury within the seeds by using
the dithizone method (35). Seeds treated with mercuric chlo-
ride (1:1000) for 10 minutes and washed in water for 15 min-
utes were germinated in petri dishes and in pots in the green-
house. No trace of mercury was detected at any time in the
embryo after this treatment. On the sixth day after planting,
the mercury in the remaining parts of the seed showed a uni-
form lowering of concentration to 30 to 35 percent of the amount
present immediately after treatment. The endosperm, with the
inner cellular layer attached, contained 14 to 18 percent as
much mercury as the seed coat. The critical amount of mer-
cury in the endosperm of nongerminated seeds was shown to
be 0.6 to 0.7 p.p.m. Treated seeds were germinated in pots
in the greenhouse receiving definite amounts of water each day.

In both loam and sandy soil a relationship was found between

13
the amount of mercury removed, the amount of water supplied
and germination of seeds. These authors studied the penetration
of mercury into tomato seeds and found it to be proportional
to the logarithm of the treatment time. Penetration was more
rapid in the case of mercuric chloride and proceeded at an
even rate from the epidermis to the embryo.

Dried shell and snap beans are known to be only slightly
susceptible to mercury injury in common practice even though
often overdosed. However, snap beans (43) germinated poorly
when soaked in a solution of 1:1500 mercuric chloride in 70
percent alcohol plus 2 percent acetic acid to control bacterial
blight. No report has been found on injury by organic mercury
compounds.

Lima beans are known to be very sensitive to mercury
injury. Clayton (7) observed in 1931 that after two weeks of
growth, plants from nontreated seeds were bigger than the plants
from Semesan treated seeds. His photographs showed the stunt-
ing effect of the plants as well as the poor development of the
root system. New Improved Ceresan also injured lima beans

(11, 37).

l4

Peanut seed has been reported to be affected by Ceresan
M (55). As the dosage of Ceresan M was increased, the emer-
gence also increased reaching a peak at 1-1/2 to 3 ounces per
100 pounds of seed. At 4-1/2 ounces per 100 pounds, the emer-
gence was less than that for the untreated seeds. Occasionally
Ceresan M was found to be toxic to peanut seed even at 3 ounces
per 100 pounds of seed. In this case the primary root of the
seedling typically grew for an inch or slightly more, but the
epicotyl did not develop. The hypocotyl continued to increase

in diameter and resulted in a deformed seedling.
Injury to Peas

Jones (27) was the first to report application of organic
mercurials on peas. Haenseler (22) found a decrease in germ-
ination of pea seed after liquid Semesan treatment. He attrib-
uted however the decrease in germination to a mechanical in-
jury resulting from handling the swollen pea rather than injury
to the germs by chemicals. Crosier and Patrick (10) mentioned
injury in peas caused by New Improved Ceresan. McNew (38,
39, 40) showed that ethyl mercury phosphate retarded plant

growth of Green Admiral, Wisconsin, and Surprise peas and

15
in addition reduced the yield of Surprise variety. He concluded
that fungicides other than organic mercurials should be used on
peas. Walker e_t a_1_. (53) found in 1940 that Ceresan was not
as effective as were the common copper oxide seed treatments.
Walker suggested that low emergences from these treatments
might have been due either to low protective values or to in-
jurious effects of the fungicide. In 1941 New Improved Ceresan
was found to be injurious to peas in New York State (42). Hull
(25) noticed that the tap roots of Alaska pea seedlings were
noticeably more stunted by heavy dosage of a dry organic mer-
cury seed dressing than were those of Gradus. It was found by
deZeeuw and Andersen (13) that response of pea varieties to
Ceresan M varied with the method of applying fungicide to the
seed. Dry applications of 4 ounces per 100 pounds of seed re-
sulted in significant stand increases, whereas stands of many

varieties were reduced significantly with the same rate of ap-

plication of the fungicide in water slurry.

MATERIALS AND METHODS

The effects of mercury poisoning on peas by organic
mercury slurries were studied first in the field, then in the
laboratory. Anatomical and histological studies were made on
three of the pea varieties, Alaska wilt resistant, Dwarf Gray
Sugar and Wisconsin Perfection; which deZeeuw and Andersen
(13) found to respond differently when treated with Ceresan M.
Alaska was injured severely, Dwarf Gray Sugar slightly, and
Wisconsin Perfection was ordinarily not injured at the rate
they used.

In addition to Ceresan M, another organic mercurial,
Agrox, whose active ingredient is phenyl mercury urea, and
mercuric chloride were included in the field tests to see whether
or not they would cause a similar injury. Phygon X1, a non-
metallic and nontoxic fungicide, which had been shown to be
effective in pea protection, was used as a control in order to
determine whether poor emergence, if any, was due to the ef-
fects of rotting organisms or to the toxicity of the materials.

All these seed protectants were applied to the seeds by

the slurry method. As has been mentioned above, the slurry

17
method of treating seeds was devised to eliminate dust in the
atmosphere and to provide a more uniform and accurate appli-
cation of the fungicide on the seed coat. Because of the small
amount of seeds used in these experiments the slurry method
was modified as follows:

Six hundred seeds were accurately weighed in order to
determine the required amount of fungicide (4 ounces per 100
pounds or 0.0025 of the seed weight). This amount of fungi-
cide was mixed with 5 drops of water or less in a beaker.

The percentage of moisture of this semipaste is very impor-
tant, for too much or too little of water will prevent the even
coating of the seeds. Only by trial and error the quantity of
water can be determined. However, the best method was found
to be a rotating of the beaker containing the seeds and the fun-
gicide in semipaste form and to add if necessary one drop of
water at a. time until no residual dust remained on the side of
the vessel. After treatment the peas were dried thoroughly
before being germinated in the field or in the laboratory.

In the field experiment, pea seeds of the three varieties
were treated with Ceresan M and Agrox at the rate of 2 ounces

and 4 ounces per 100 pounds, with Phygon X1 at the rate of 4

18
ounces per 100 pounds and with mercuric chloride (1:1000 solu-
tion) at the rate of 5 cubic centimeters per 100 seeds. The
field layout was a split plot design. Because one of the main
objects was to determine varietal differences in peas to the
seed protectants, the smallest split in the plot design was the
varieties within each treatment. Each row was 25 feet long,
spaced 28 inches apart and 100 seeds were planted in a row
with a V-belt planter. Stand counts, height measurements and
observations of any visible injury were recorded.

In the laboratory seeds were treated either with Ceresan
M at the rate of 4 ounces per 100 pounds of seed or with an
overdose of wet Ceresan M. Such treated seeds and nontreated
(control) seeds were germinated in wet rolled paper towels put
sidewise in covered jars so that the lower ends of the towels
were in an inch of water at the bottom. Roots from treated
and untreated peas were killed in a standard strong Formalin
Aceto-Alcohol solution, dehydrated in a standard tertiary butyl
alcohol series and embedded in paraffin (26). Sections were
made at 10 /u and 30 /u and stained by the Foster tannic acid
method (16). Measurements of cell and nucleus size were made

with a calibrated ocular micrometer. The smear teclmique was

19
used to study polyploidy in root cells. Stains used to color the

nuclei included Fuelgen, aceto-carmine and propionic acid.

EXPERIMENTAL RESULTS

Field Expe riment

The seeds were treated on August 20, 1951, planted three
days later, and seedling counts made on September 4. Table 2
shows the data from these counts together with the average stands
for the various treatments.

Analysis of the stand data showed that Ceresan M used
at the rate of four ounces per 100 pounds of seed in slurry
form was injurious to all three varieties (average stands of
23.5, 39.5, and 50, as compared to Phygon X1 with 89, 61.7,
and 87.51). No varietal difference could be found in this re-
spect. Agrox at both rates was a good seed fungicide and mer-
curic chloride was of no value in controlling damping off. In-
spection in the field of the growing plants in the 4 ounces
Ceresan M rows disclosed that they were stunted. Some varietal
difference could be found. Alaska peas were the most injured,
Dwarf Gray sugar less, and Wisconsin Perfection the least. The
average height of the plants from the various treatments taken

from one representative replication is shown in Table 3. Data

21

Table 2. Stand counts of three varieties of peas treated with
four chemicals.

 

 

 

 

 

 

 

Replications Avg.
Treatments Total No.
and Rate I II III IV Plants P12: ts
Alaska Wilt Resistant
Ceresan M - 2 oz. a/72 64 78 91 305 76.5
Ceresan M - 4 oz. 13 39 12 29 93 23.5
Phygon X1 - 4 oz. 86 89 91 90 356 89
Agrox - 2 oz. 84 . 91 86 80 341 85
Agrox - 4 oz. 85 74 66 88 313 78
l/l,000 HgClz — 5 CC. 58 52 74 63 247 61.7
Check (no treatment) 37 46 47 59 189 47.5
Wisconsin Perfection
Ceresan M - 2 oz. 65 71 56 59 251 62.7
Ceresan M - 4 oz. 25 56 3O 37 158 39.5
Phygon X1 - 4 oz. 58 70 54 65 247 61.7
Agrox - 2 oz. 46 53 46 37 182 45.5
Agrox - 4 oz. 55 65 57 73 244 61
1/l,000 HgCl2 - 5 cc. 4 7 5 3 19 4.75

Check (no treatment) 5 4 1 7 17 4.25

 

22

Table 2 (Continued)

 

 

 

 

 

 

 

Re lications Avg.
Treatments P Total No.
and Rate I II III IV Plants of
Plants
Dwarf Gray Sigar
Ceresan M - 2 oz. 94 92 93 99 378 94.5
Ceresan M - 4 oz. 50 59 50 42 201 50
Phygon X1 - 4 oz. 72 87 93 99 351 87.75
Agrox - 2 oz. _ 100 87 96 99 382 95.5
Agrox - 4 oz. 88 89 95 95 357 91.5
l/1,000 HgCl2 - 5 cc. 97 91 79 99 362 90.5
Check (no treatment) 93 99 94 93 379 94.7
a. 100 seeds planted per row.
Inte rp retation:
L.S.D. for treatment within each variety at 5 percent
level — 10.8.

L.S.D. for treatment within each variety at 1 percent
level - 13.8.

23

Table 3. Average height of three pea varieties grown from
seeds treated with various seed treatment fungicides.*

 

 

 

 

Slurry Treatment Dwarf Wisconsin Alaska
Gray , Wilt
and Rate Perfection ,
Sugar Re51stant
Ceresan M - 2 oz. 9.1 cm. 7.1 cm. 12.3 cm.
Ceresan M - 4 oz. 6.4 4.8 3.6
Phygon X1 - 4 oz. 9.1 9.6 16.0
Agrox - 2 oz. 9.4 8.3 13.9
Agrox - 4 oz. 9.3 6.8 13.3
1/1,000 HgClz - 5 cc.
for 150 seeds 9.3 5.05 13.4
Check 9.3 5 16
Average 9.3 6.66 12.63

 

 

* Plants from one representative replication.

24
observations show that the height of the three pea varieties was
severely reduced by Ceresan M at the rate of four ounces per
100 pounds. The height of the Dwarf Gray Sugar peas, a vari—
ety resistant to damping off, seemed to be unaffected by seed
treatment with Agrox, mercuric chloride, or Phygon X1. On
the other hand better results were obtained with the Alaska
and Wisconsin Perfection varieties when their seeds were treated
with Phygon X1 and the plants were more vigorous.

Representative plants from rows treated with Ceresan
M at four ounces were carefully dug and the roots examined
for signs of mercury injury. The photographs (Plates 1, II, and
III) taken three weeks after planting illustrate the typical symp-
toms in each case. The roots of affected plants were shortened
and bronzed at the tip; the hypocotyl was greatly enlarged and
the stem was apparently normal, although it was short in pro-
portion to the root system. However, as the photographs show,
by the end of the season the plants had grown adventitious roots
above the cotyledons. These started to elongate and allow par-
tial recovery but the cold weather stopped any additional growth

that might have occurred.

25
Effect of Storage on Organic Mercury
Treated Seed

Chemically treated seeds may be injured under various
environmental conditions during storage, such as high tempera-
ture, high humidity, and lack of aeration. According to Leach
9i a_l_. (29), addition of 10 percent moisture to sugar beet seeds
treated with Ceresan and subsequently stored for 10 days before
planting, retarded germination and produced stunted seedlings.
Addition of 6 percent moisture and confinement in a dry space
for 30 days resulted in injury to part of the seedlings, whereas
2 percent moisture was noninjurious. Baylis (4) treated sam-
ples of wheat seed with Agrosan G. and Ceresan U. T. 1875 A.
at dosages of 2 and 4 ounces per bushel, and stored them for
12 months. The moisture content of the seed samples ranged
from 12.8 to 16.7 percent. In general the lower the moisture
content of the seed, the higher was the field germination of both
treated and untreated seeds. Brett and Weston (5) dusted seeds
of wheat, oats, and barley with organic mercury protectants and
found that seeds of good quality and proper moisture content

were not injured after one year storage.

26

Because of the high moisture content of the seed follow-
ing the slurry method of seed treatment used in these trials,
the question arises as to when the mercurial injury occurs.

Is it immediately after treatment and during storage, or is it
during germination? It has been proved by Wain (49) that in
the case of copper seed treatments in peas, the injury occurred
during germination. The theory was that pea seeds, when they
germinate, render soluble the insoluble copper complex ion, which
in turn injures the embryo after passage through the micropyle
and the seed coat. It was thought that mercury might injure

in a similar manner during germination. To test this hypoth-
esis, seeds which had been treated with an excess of wet Cere-
san M and stored for a month were divided into two lots. The
first lot had their seed coats removed immediately after a 25
minute soaking in water. The second lot was soaked for 25
minutes, but their seed coats were not removed. Both lots
were germinated and the seedlings obtained were compared to
seedlings from nontreated seeds. Results showed that seedlings
obtained from treated seeds with seed coats removed germinated
as well as those from nontreated seeds, though the roots were

slightly injured and curved. The lot of treated seeds with seed

27
coats left on was completely inhibited. This indicated that in-
jury to peas by mercury is more likely to occur during germ-

ination.
Hi stologic a1 Studie s

As stated above, the germination of peas treated with
an overdose of wet Ceresan M was completely inhibited. Such
seeds had necrotic spots visible on their seed coats (Plate IV,
Fig. D.). When they were split for examination the tip of the
radicle and the testa were particularly darkened.

Nontreated seeds produced seedlings with straight thin
roots (Plate IV, Fig. A). Such roots seen in longitudinal sec-
tion had regular polyhedral cells. These cells from the tip
of the meristematic tissue to the beginning of the differentia-
tion layer, were nearly square: 25 to 30 /u in diameter, and
their nuclei were always present and occupied nearly half the
cell with a diameter of 10.8 /u to 13.5 In, giving a ratio,
nucleus to cell, of 1/2. Cross sections made in the beginning
of the differentiation layer showed that these cells were usually

octogonal (Plate IV, Fig. F).

28

Seeds treated with Ceresan M at the rate of 4 ounces
per 100 pounds produced seedlings with slightly to seriously
injured enlarged roots. The tip of these roots was generally
necrotic and seen in longitudinal section had distorted cells
(Plate IV, Fig. B and C). The cell wall instead of being a
straight line, curved in and out and often seemed to be col-
lapsed. Except for the epidermal cells there was no uniform-
ity in cell size. These distorted cells were greatly enlarged
in comparison to normal cells, being at least 50 /u in diam-
eter. When the roots were cut at 10 /u the cells contained
few nuclei. Table 4 gives an average of 20 measurements of
abnormal root cell diameter and nucleus diameter. The ratio
of nucleus diameter to cell diameter was 0.16, and showed to
be inferior to the one in normal cells 1/2. This gave an indi-
cation that the cell enlarged more than the nucleus. When seen
in cross-section,~the cells seemed less distorted (Plate IV, Fig.
H), but they were twice as large as normal cells and less dis-
tinctly octogonal.

The average number of cells across the width of an
abnormal root was found to be 17, as compared to 21 from

the same region of a normal root. This indicated that the

29

Table 4. Twenty measurements of root cell and nucleus diam-
eter from peas treated with Ceresan M at the rate
of four ounces per one hundred pounds and from non-
treated peas.

 

 

 

 

 

 

Abnormal No rmal

Cell Nucleus Cell Nucleus

Diameter Diameter Diameter Diameter
81 /u 18.9 27 13
121.5 24.3 27 13
67.5 10.8 27 13
135 27 27 13
108 18.9 27 13
76.5 8.1 27 13
67.5 13.5 27 13
108 16.2 27 13
135 27 27 13
94.5 16.9 27 13
81 13.5 27 13
108 16.2 27 13
140 40 27 13
135 27 27 13
140 26 27 13
80 8.1 27 13
108 15.9 27 13
63.5 16.2 27 13
110 16.2 27 13

Total 2042 327.4

Average 10.2 16.2 27 13

Ratio:
Nucleus/Cell 0.16 0.49

 

 

30
obvious swelling of the abnormal roots was due to the enlarge—
ment of the individual cells.

Shoots from peas that had been treated with Ceresan M
in excess were cross-sectioned. No visible injury could be

found in the shoot cells which appeared normal.

31

PLATE I

Injury of Alaska Wilt Resistant pea caused by an organic
mercury seed treatment material, Ceresan M.

Left: Control, nontreated.

Right: Treated with Ceresan M in aqueous slurry at the

rate of 4 ounces per 100 pounds of seed.

32

 

33

PLATE II

Injury of Wisconsin Perfection caused by an organic mer-
cury seed treatment material, Ceresan M.

Left: Control, nontreated.

Right: Treated with Ceresan M in aqueous slurry at the

rate of 4 ounces per 100 pounds of seed.

34

 

35

PLATE III

Injury of Dwarf Gray Sugar by an organic mercury seed
treatment material, Ceresan M.

Left: Control, nontreated.

Right: Treated with Ceresan M at the rate of 4 ounces

per 100 pounds of seed.

36

 

37

PLATE IV

Anatomical aspects of uninjured pea seedlings and those
injured by mercury containing seed treatments.

A. Normal seedling.

B, C, D. Slight to serious injury.

E. Complete inhibition.

F, G. Longitudinal sections of normal and abnormal pea
roots.

H, 1. Cross sections of normal and abnormal pea roots

at the beginning of the differentiation layer.

fie1cm

 

         

   
 

 

I ' D) ‘ '
' '0... . ‘ ‘
"‘ {fit-gm: .

 

DISCUSSION

The results obtained in this investigation are similar to
those obtained by others (10, 13, 45, 50, 55) on cereals, clover,
peanuts, and peas. Mercurial toxicity resulted in an inhibition
in length of the developing primary root and in a thickening of
the root due to an enlargement of the cells. In peas, the stem
was not directly affected by mercurial poisoning as shown by
the presence of adventitious roots from the epicotyl. This field
observation was confirmed in the laboratory by cross-sectioning
shoot tips of pea seedlings. No gross anatomical differences
could be found between abnormal and normal seedlings. This
is of importance because according to Sass (46), the plumules
of corn as well as the roots were injured by organic mercurials.
No reason can be given on the strength of this work as to why
pea stems were noninjured by Ceresan M and further work should
be done along this line.

The field results were conclusive and confirmed that
Ceresan M was injurious to the three pea varieties. However,

some varietal difference could be found in response to this

40
fungicide. Differences in seed coat structure or in the physi-
ology of these varieties may be the answer. Besides direct
observations of mercurial injury to peas, it should be noted
that Phygon X1 was found to be a very good seed protectant
for Alaska and Wisconsin Perfection. Dwarf Gray Sugar was
in these tests a variety resistant to damping off as shown by
the stand data of the control plants.

In all histological preparations made, the cells had lost
their meristematic aspect. No mitosis was seen. Figure G,
Plate IV, showed no differentiation of plerone and periblem
layers. The question arose is this a median longitudinal sec-
tion. For if it is not a median longitudinal section, this dif-
ferentiation would not be seen. Because of the bending effect
that the root takes after treatment, straight sections through
the differentiation layers are hard to obtain. From observations
of histological preparations the root appeared to have started
differentiation but had stopped. More histological preparations
should be made to understand how the plerone and periblem
layers are affected by mercury.

Porter (45) reported that the cells of abnormal roots

injured by an overdosage of New Improved Ceresan became

41
multinucleate. This was confirmed by Sass (44) on the basis
of extensive histological and cytological studies on the malfor-
mation of corn seedlings treated with a 1 to 1:1500 solution of
New Improved Ceresan. The seedlings exhibited various de—
grees of distortion of cells, tissues, and organs in proportion
to the severity of the gross external symptoms. "Isolated areas
of enlarged cells were found to occur in the older leaf primordia
of the plumule. Hypertrophy progressed from the end of the
primordium and spread to the apical meristem. These cells
were binucleate." Because of these findings the writer looked
for multinucleate or giant nucleate cells for confirmation. He
was unable to find multinucleate cells by any of the common
techniques. They did not appear in any of the preparations of
root tip smears or in the smears of more mature tissues. Nor
was the writer successful in finding them in paraffin sections.
This does not mean that Ceresan M under certain conditions
cannot cause polyploidy or polyteny (or both) by failure of mi-
tosis thus resulting in giant nucleate cells or multinucleate
cells. In fact, the organic mercury dusts which are known to
cause these abnormalities have to be used in overdose (45, 18).

One of the difficulties encountered was that pea seeds treated

42
with an overdose of wet Ceresan M were totally inhibited in
their germination and roots from such seedlings are naturally
unavailable. All the work then had to be done with peas in
such a way that injury would be manifested without producing
inhibition. Sass (44), in his cytological studies, used corn roots
from seeds already germinated and subsequently treated with a
solution of New Improved Ceresan. If such a method were used
with pea roots, it should be possible to ascertain whether or not
Ceresan M causes polyploidy. Gassner (17) in 1950 reported
that overdosages of nonsubstituted aliphatic mercury compounds
caused polyploidy. Since Ceresan M is such a compound, it
would be worthwhile to do further work. It was found that
Agrox did not cause any injury to the three pea varieties which
confirms Gassner's statement that nonsubstituted aromatic com-
pounds (of the type of Agrox) are nontoxic.

As stated above, there was a slight injury ’on seeds
treated with an overdose of wet Ceresan, when the seed coat
had been removed. These results were only an indication that
injury is likely to occur during germination. Because there
was a slight injury, mercury in some form could have pene-

trated into the seeds during storage. McCoach (34) described

43
an analytical method for determining minute quantities of or-
ganic mercury materials in plant tissues. The organic mercury
was converted to mercury dithizonate and was extracted with
carbon tetrachloride. Determination of the mercury content
was made by measuring the density of the extract against a
reference solution with a spectrophotometer. Such a method
by which mercury could be determined in or under the seed
coat before and after storage would be very useful in deter-
mining the time when injury occurs. In storage tests it should
be possible to find whether or not toxic quantities of mercury
move to the embryo when the seed is dry or whether injury

begins at germination.

SUMMARY

Organic mercury protectants were applied to three vari-
eties of peas in field and laboratory tests to study the anatom-
ical and histological deformations resulting from mercury in-
jury.

A field experiment lshowed that Ceresan M slurry at the
rate of 4 ounces per 100 pounds of seed affected in varying de-
grees the three varieties of peas tested. Alaska was visibly
more injured than Dwarf Gray Sugar and Wisconsin Perfection.
Agrox did not affect the same varieties under the same condi-
tions.

Symptoms of mercurial poisoning on 'the pea are stunt-
ing of the upper part of the plants due to an inhibition of the
primary root, enlargement of the hypocotyl and secondary for-
mation of adventitious roots from the epicotyl. The adventi-
tious roots may allow the plant to recover (partially from pre-

vious root injury but the plants never attained the vigor of

I

normal ones.

Histological studies showed that the enlargement of the

hypocotyl as well of the primary root was due to an enlargement

45
of the existing cells; These cells were distorted and apparently
had stopped mitosis. There appeared to be a correlation be-
tween the size of cell and size of the nucleus. However, this

trend towards polyploidy or polyteny was not confirmed by any

of the common cytological methods.

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51

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