EFFECTS OF AERBICIDAL SPRAYS ON NITRATE
ACCtJMULAT10N IN CERTAIN WEED SPECIES

By
Peter Andrew Frank

AN ABSTRACT
Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
In partial fulfillment of the reqiiirements
for the degree of
DOCTOR OF PHILOSOPHY
Department of Botany and Plant Pathology
Year

Approved

(3 N.
■

1955

ProQuest Number: 10008305

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1

Peter Andrew Frank
Nitrate poisoning of livestock feeding on plants containing
high concentrations of nitrate has been recognised since 1888.
In recent years there have been a number of reports of live­
stock becoming poisoned after grazing on herbicidally treated
vegetation.

This poisoning was believed to have been caused

by the ingestion of toxic quantities of nitrate which had been
accumulated by the plants as a result of chemical treatment.
A study of the problem of nitrate accumulation was ini­
tiated during the summer of 1953 and continued through the
summer of 195*+•

For this work, a group of weeds commonly found

in cropped and pasture land in Michigan was selected.

The

following species were used;
1.

Amaranthus retroflexus L.
Ambrosia elatior (L.) Descourtils
3 • Cheno oodium album L .
*+• Cirsium arvense (L.) Scop.
5* Eupatorium maculaturn L.
6. Imnatiens biflora Walt.
7. Poa nratensis L.
8. Polygonum Convolvulus L*
9 • Polygonum Persicaria L.
10. Prunus virginiana L,
11. Sambucus canadensis L.
12. Solanum Dulcamara L
13- Spiraea alba Du Roi
Thalictrum dioicum L.
w*

i

ii iii

i■

—

71«« n

» i■* >
jm

These weeds were treated with the following herbicides:
1*.
2.
3.
*+.
5.
6.

Isonropvl ester of 2 ^-dichlorophenoxyacetic acid
(2,4~D)
butyl ether ester of 2 ,*+,5-trichloroohenoxvacetic acid
(2,1+,5-T)
Sodium salt of 2-me thy1-*+-chlorophenoxyacetic acid
(MCP )
Alkanolamine salt of dinitro-o-sec-butyl-phenol (DNBP)
Isopropyl-N-(3-chlorophenyl) carbamate (CIPC)
Diethanolamine salt of maleic hydrazide (MH).

2

Peter Andrew Prank
The weeds were sprayed with sub-lethal dosages of these
chemicals.

This was done to prevent excessive deterioration

or death of the plants during the period in which analyses
were to be made.

The herbicidal applications were timed so

that the period of analysis coincided as closely as possible
to the stage preceding reproduction.
Samples for analysis were obtained at 2b hour intervals
for four days following.treatment and an additional sampling
was made two weeks after treatment.

Samples were obtained in

the early morning and immediately taken to the laboratory where
extracts were prepared from the fresh materials.

The nitrate

content of the samples was determined by a colorimetric com­
parison with standard solutions of potassium nitrate, using
a method involving the nitration of 3,*i-xylenol.
Herbicidal treatments used in this study did not affect
the nitrate content of the following, species: Chenopodium album.
Cirsium arvense* Prunus virminiana and Thalictrum dioicum.
The treatments resulted in significant increases in the
nitrate content of five of the weeds.

These weeds and treat­

ments were:
1.
2.

The DI'JBP treatment of Anaranthus retroflexus
The 2,*f-D, DHBP, CIPC and KH treatments of Eunatorium
maculaturn
3.. All treatments of Impatiens biflora
k. The 2,*+-D, 2 ,j+,5“T and MCP treatments of Polygonum
Convolvulus
5. The DNBP treatment of Polygonum Persicarla.
Ten of the fourteen weeds studied contained nitrate in
sufficient quantities to cause nitrate poisoning of livestock
even though no sprays had been applied.

3
Peter Andrew Frank
In only two weeds * Bupatorium macula turn and Imoatiens
biflora. could the accumulation of toxic concentrations of
nitrate be attributed solely to the effect of herbicidal
treatment.

None of the herbicides used appeared to- have the

same effect on nitrate accumulation in all of the weeds tested.
Chenopodium album and Amaranthus retroflexus were pre­
viously reported to accumulate nitrate following treatment
with 2,k-D.

In this study it was found that nitrate in Cheno-

podium was not affected by any of the treatments and Amaran­
thus accumulated nitrate following the DNBP treatment only*

EFFECTS OF HERBICIDAL SPRAYS ON NITRATE
ACCUMULATION IN CERTAIN WEED SPECIES

by
Peter Andrew Frank

A THESIS
Submitted to the School of Graduate Studies of MI chi
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Department of Botany and Plant Pathology
School of Science and Arts
1955

it#

TABLE OF CONTENTS
Page
INTRODUCTION

........................

REVIEW OF LITERATURE

................ 1+

MATERIALS AND PROCEDURES .

....................18

EXPERIMENTAL RESULTS ..........................
DISCUSSION . . . . . .

1

21

......................... h2

SUMMARY AND CONCLUSIONS.......................... *+?
LITERATURE CITED

..........

A P P E N D I X ................

b-7
52

ACKNOWLEDGMENTS
The author wishes to express his sincere thanks and
appreciation to Dr., B. II. Grigsby for his aid and super­
vision throughout the course of this work.
The author also expresses his appreciation to Dr.. G.
P. Steinbauer for his assistance and to Professor C. D.
Ball for his help and advice in preparing the manuscript.
He particularly wishes to thank his wife, Carol, whose
aid and encouragement made this work possible.

INTRODUCTION

Since the advent of the new-and generally more efficient
herbicides, a great deal of work has been done and a voluminous
literature presented concerning their uses and possible mecha­
nisms of action.

While the more obvious effects of the chem­

icals have been described at length, the basic physiological
mechanisms remain undefined..
The rapid incorporation of new herbicides into existing
weed control practices, while a boon to agriculturalists, util­
ity corporations, home owners and others, has not been without
some criticism.

This largely has been due to the failure of

research agencies to conduct extensive experiments concerning
the possible adverse effects in humans and in animals, from
extended exposure to the chemicals and the products of the
treated plants.

There is, In addition to the problem of chron­

ic toxicity, the possibility that application of herbicides
may affect certain plants in such a way as to bring about the
elaboration of a toxic substance by these plants or the accu­
mulation of a toxic substance ordinarily present in non-toxic
concentrations•
In recent years there have been numerous reiDorts of death
of livestock foraging on 2,k-D and 2,k,5-T treated members of
the Prunus and Sorghum genera.

Members of these two genera

2

are known to accumulate hydrocyanic acid or glucosides which,
upon hydrolysis, may produce this acid, sometimes in concen­
trations lethal to livestock.

It has been postulated that the

treated plants accumulated abnormal quantities of hydrocyanic
acid which caused the livestock losses (12).

Data fro i some

experiments have Indicated that treated plants do not accumu­
late 3.arg@ amounts of hydrocyanic acid (27,17).
In addition to the reports of poisoning by hydrocyanic
acid, there have been a number of cases reported where live­
stock losses were attributed to nitrate poisoning (11).

Live­

stock grazing on herbicide treated vegetation were believed
to have ingested toxic quantities of nitrate which had been
accumulated by the plants as a result of chemical treatment.
That nitrate poisoning is not uncommon may be seen from the
numerous cases reported over the past sixty years.

However,

the assumption that crop and weed plants may accumulate ni­
trate as a result of herbicidal treatment is a new develop­
ment which requires careful experimental study.
^Willard (55)? in discussing the indirect effects of herb­
icides, cites numerous reports of ordinarily unpalatable weeds
becoming palatable to livestock following herbicidal treatment.
Many of the weeds tested were found to contain toxic quantities
of nitrate in both the treated and untreated state.

The effect

of herbicides on the palatability of these weeds cou^d be a
factor contributing to the nitrate poisoning of livestock.

3

A study of the problem of nitrate accumulation was initi­
ated during the summer of 1953 and. continued through the sum­
mer of 1951*.

For this work, a group of weeds commonly found

in cropped and pasture land in Michigan was selected.

This

group included certain weeds known to accumulate high concen­
trations of nitrate as well as others suspected of having con­
tributed in some manner to livestock losses.

These weeds were

treated with various herbicides and nitrate analyses were made
at regular intervals following treatment in an attempt to de­
termine the effects of the treatments wipon the nitrate content.

REVIEW OF LITERATURE

High levels of nitrate in green weeds and crops as well
as cured feeds may often have disastrous consequences.

The

first recognized case of nitrate poisoning of livestock was
in 1888 and was reported, along with a number of other cases,
in 1895 (^9)#

The losses were traced to the feeding of both

green and cured corn fodder and hay.

The nitrate content, de­

termined as potassium nitrate, amounted to as much as 25 per­
cent of the dry weight and the salt crystals could be observed
readily with the naked eye.

In more recent years, nitrate poi­

soning from oat hay (h,5^* 31)? hay containing pigweed (36),
sorghum (36,39) and sugar beet tops (*+()) has been reported.
Poisoning from ingestion of high nitrate plants is due to
the reduction of nitrate to nitrite by microorganisms in the
intestinal tracts of animals.

The nitrite is taken into the

blood stream where it combines with hemoglobin to form methemoglobin.

The latter is incapable of giving up its oxygen to

the tissues and thus causes asphyxia (*f).

Bradley et al. (*+),

have determined the minimum lethal dose to be 25 grams of po­
tassium nitrate per one hundred pounds of animal weight.

At

this level, a five-hundred-pound animal would have to eat but
five and one-half pounds of oat hay containing 5 percent ni­
trate to become fatally poisoned.

The absorption and. elaboration of nitrogenous nutrients
in plants is dependent upon such external factors as the pH
and nitrate concentration of the growth medium, and the rela­
tive availability of various mineral solutes.

Such factors as

light, temperature, moisture and oxygen supply are also impor­
tant.

Internal factors of equal importance are the availabil­

ity of carbohydrates and the stage of growth of the plants.
In considering the possible effects of herbicides on the
nitrate content of plants, it may be desirable to consider firs
some of the factors affecting the absorption and assimilation
of nitrates.

The order in which these are treated should not

be taken as the order of importance as it is impractical if
not impossible to assume that any one is of more importance
than another.

Availability of Nitrate to the Plant
That there is a correlation between the nitrate content
of plants and the amount of available nitrate in the soil has
been recognized by agronomists for a number of years.

Some of

the more improved methods of fertilizer application are based
on the fact that a low plant nitrate level usually indicates
a low level of soil nitrate.

Bradley et aJL. (*+) investigated

the causes of high nitrate level in crop plants and found that
soils with an abundanse of nitrogen produced crops with a high
level of nitrate.

While toxic concentrations of nitrate in

6

corn appeared to be exceptional, where nitrate was available
to the plants it was absorbed and represented a considerable
fraction of the dry weight (56).

Corn plants growing on very

fertile plots of soil previously used as animal enclosures,
were found to have accumulated nitrate to such an extent that
it constituted 18.8 percent of the dry weight of the plants
(*+9 )•

Moisture Supply
Most of the severe losses of livestock as a result of ni­
trate poisoning have occurred in the drier sections of the coun­
try.

Studies conducted with plants having abundant moisture

indicate that these plants could very well accumulate toxic
levels of nitrate if grown under drought conditions (56).
Grasses, in contrast to many other plants, were found to con­
tain only traces of nitrate, which was thought to result from
the removal of excess nitrate from the plant by guttation.
Maintaining droughty soil conditions, such as keeping the soil
moisture as low as 15 percent, resulted in a much higher con­
centration of nitrate in the plants.

Whitehead and Olson (5^-)

found that as the soil moisture increased there was a corre­
sponding decrease in nitrate until the soil moisture reached
a level of 25 percent.
level.

No differences were found above this

7

Temperature
The effects of temperature are for the most part indirect.
The nitrifying ability of soil microorganisms is affected by
the temperature.

Jones and Greaves (23) found that of the nine

soils tested for nitrifying ability, maximum nitrification oc­
curred in six of the soils at 20°, and at 30° in the remaining
three.

Active absorption of ions is accelerated by p. rapid

rate of respiration in the roots.

Temperatures which retard

root respiration therefore inhibit the uptake of nitrate and
other ions.
The rates of photosynthesis and respiration, which are
enzymatically regulated processes, are affected by tempera­
ture..

This has a direct bearing on the availability of carbo­

hydrates which has,been shown to affect nitrate metabolism in
plants.

pH of the Nutrient Medium
Soil pH is known to affect the number of soil microorgan­
isms and the rate of nitrification.

In addition, the soil pH

affects the availability of various ions directly or indirectly
involved in nitrate uptake and assimilation.

Davidson and

Shive (7) observed that peach trees absorbed and utilized am­
monium nitrogen more readily than nitrate nitrogen at pH 6
while the reverse was true when the medium was maintained at
pH If..

8

Oxygen Supply
The level of available oxygen in a nutrient substrate was
found by Gilbert and Shive (l*f) to be a factor in the rate of
nitrate absorption and assimilation.

They, and others (57)?

found that the greater the oxygen tension the greater was the
nitrate absorption by roots.

Shive (^fl) demonstrated that ni­

trate assimilation could be inhibited by oxygen and that nitrate
utilization was inversely proportional to the oxygen level.
It was found that nitrogen deficiency occurred in plants cul­
tivated in a nitrate solution when the oxygen level was as high
as 8 to 16 parts per million in the solution (13)•

Nance (30)

observed that oxygen did not inhibit active nitrate absorption
but that the assimilation of previously absorbed nitrate was
inhibited.

Effects of Light
Light may affect the assimilation of nitrate in a number
of ways.

Light is required for the synthesis of carbon com­

pounds necessary for the formation of organic nitrogen and for
the synthesis of carbohydrates which, when oxidized, probably
provide

the energy for nitrate reduction in the dark.

There

is, in addition, considerable evidence that light plays a more
direct role in nitrate reduction.

Plants kept In continuous

light have been found to contain less nitrate than other plants
(Jlf) while shading resulted in an increased nitrate content (V7 ).

9

Burstrom (5) found that wheat leaves did not assimilate ni­
trate in the dark but did in the light and that the rate of
assimilation increased with an increase in light intensity*
Excised wheat roots were found to be capable of reducing ni­
trate in the dark at the expense of respired carbohydrates*
Nitrate reduction in wheat leaves appeared to be induced by
light' and was independent of respiration*
Mendel and Visser (28), using

and green leaf disks,

carried out experiments similar to those of Burstrom and,, in
addition, investigated the effects of respiration inhibitors
on nitrate reduction in light and in darkness*

Respiration

inhibitors completely inhibited nitrate reduction in darkness
but had no effect on nitrate reduction in the light.

Van Niel

et al*, C*f8) obtained experimental evidence which supports the
contention that photochemical reduction of nitrate represents
a process in which nitrate acts directly as an alternate hydro­
gen acceptor in photosynthesis*.

Molybdenum Supply
Certain experiments indicate that molybdenum functions
as a biological catalyst in symbiotic-nitrogen fixation and
in the reduction of nitrate in plants.

Plants starved for

molybdenum have been found to accumulate considerable nitrate
in the petiolar tissue (20)*

When nitrate is the source of

nitrogen, the molybdenum requirement is greater than when am­
monia serves as the source (5*+,29)*

Plants deficient in

10

molybdenum were found to have a depressed content of sugars,
chlorophyll and organic nitrogen and a greatly increased con­
tent of nitrate (1,29).

Nitrate reductase activity has been

shown by a number of workers to be decreased in molybdenum de­
ficient plants.

Nicholas et al. (32) have shown that the ni­

trate reductase activity of some microorganisms deficient in
molybdenum was reduced to one-tenth to one-thirtieth of that
of the controls.

Plants provided with ammonia as the sole

source of nitrogen may still show signs of molybdenum defi­
ciency indicating that this element may play some role in
plant metabolism other than in nitrate reduction.

Since the

energy required for nitrate reduction in darkness in plants
must be supplied by dehydrogenation reactions in respiration,
it is possible that molybdenum, as a part of one or more de­
hydrogenases , indirectly affects nitrate reduction by enzym­
atically catalyzing certain dehydrogenation reactions (9).
Very minute amounts of molybdenum are required for ni­
trate reduction while much greater quantities are required for
symbiotic-nitrogen fixation.

It has been reported that nod­

ules from alfalfa contain five to fifteen times as great a
concentration of molybdenum as roots of the same plant (22).
There is some evidence for considering the possibility
of a role of nitrate reductase in nitrogen fixation as well
as In nitrate assimilation (10).

11

Other Ions
A number of nutrient ions directly or indirectly affect
the absorption and utilization of nitrate in plants.
sium ions are necessary for nitrate reduction.

Potas­

A deficiency

of potassium results in an accumulation of both nitrate and
carbohydrates.

When potassium is supplied to plants deficient

in this ion there Is a sudden increase in nitrate reduction
and protein formation accompanied, in some cases, by the tem­
porary appearance of nitrite (3*+).
Phosphate ions not only affect the absorption of nitrate
but also the utilization of nitrate already absorbed.

Eelder

(19) found that the growth rate of five to six week old corn
plants when placed in a nutrient solution deficient in phos­
phorus was inhibited and the uptake of nitrate ceased.

This

was in spite of the fact that the plants were at the stage of
growth in which nitrate absorption was still high.

Tanada (V7)

found, on the contrary, that a deficiency of phosphorus re­
sulted in abnormally large increases in nitrate content.

Phos­

phorus and nitrate appear to be mutually antagonistic with re­
spect to uptake by plants.

A high concentration of either in

the plant inhibits the absorption of the other.
Oats in manganese deficient cultures have been observed
to contain an extremely high percentage of nitrate (5*+).

This

Ion enhances the reduction and assimilation of nitrate in both
roots and leaves of wheat (30,6).

Jones et al. (2*0 found

that soy beans growing in a nitrate solution without manganese

12

turned yellow and exhibited symptoms of nitrogen deficiency*
The addition of manganese ions resulted in complete recovery.
They concluded that manganese was essential for the normal re­
duction of nitrate and the formation of amino compounds.
An absence of ions containing sulfur has been shown to
inhibit the fixation of nitrogen by nitrogen-fixing bacteria
(2).

This is an indirect effect brought about by the influ­

ence of sulfur on organic nitrogen metabolism.

Sulfur may

affect the reduction of nitrate in a similar way.

Carbohydrate Supply
A readily available supply of carbohydrates is associated
with the reduction of nitrate in plants.

When the carbohydrate

supply is limited, nitrate reduction proceeds very slowly if
at all.

Plants having a high content of carbohydrates rapidly

reduce nitrate and synthesize new protein, indicating that some
relationship exists between carbohydrate respiration and ni­
trate reduction.

A number of workers have shown that there

is a relationship between the rate of nitrate reduction and
the evolution of respiratory carbondioxide (lb,5*18).

Goksoyr

(15) determined the rate of nitrate assimilation from the amount of carbon dioxide given off and found that the quotient
of extra carbon dioxide/ nitrate reduced to be 2.85.

The em­

pirical qiiotient of carbon dioxide evolved/ nitrate reduced
was found to be 2.3b.

These workers have shown that carbon

dioxide evolution was increased during the periods of nitrate

13

reduction.

The oxygen made available by the reduction was

utilized in the oxidation of carbohydrates which supplied the
energy necessary for the reduction.
Soybean and oat plants are known to accunulat carbohy­
drates in the absence of nitrate (33)*

The addition of either

nitrate or ammonium ions was found to reduce the carbohydrate
content and in particular the soluble sugars.

Vladimerov (53)

found that the addition of nitrate to nitrogen deficient plants
resulted in an increase in citric acid while the addition of
ammonium nitrogen reduced the citric acid content.

From this

he concluded that nitrate, which is in a highly oxidized state,
created conditions conducive to the intensification of the ox­
idation process leading to the formation and storage of acids.

Stage of Development
The ability of cultivated and weedy plants to draw nutri­
ents from the soil is dependent, to a large extent, upon the
stage of growth.

Singh and Singh (b2) observed that the max­

imum value of absorption was reached in weeds at the stage when
the plants were about to enter their reproductive period.
this stage there was a gradual decrease in absorption.

After

They

also noted that the order of concentration of various elements
was the same for all of the weeds of a related group.

The larg­

est* group being that in which nitrogen is the nutrient first
absorbed in large amounts.

lb

The stage of development may also affect the form of nut­
rient ions absorbed.

According to Stahl and Shive

,*+5),

some plants, during the early stages of growth, absorbed nearly
all their nitrogen in the form of ammonium ions.

As the plants

increased in age the amount of ammonium ions absorbed decreased
while the absorption of nitrate ions increased.

Inherent Ability to Accumulate Nitrate
Different species of plants vary in their ability to ab­
sorb and store nitrate and other nutrients.

Where nitrate is

the chief source of nitrogen, fairly high concentrations must
often be maintained for normal plant growth (36).

Jacques and

Osterhout (21), for example, found that the sap of certain algae
may contain a nitrate concentration of from 500 to 2,000 times
that of sea water.
The nitrate content in the expressed saps of different
plants growing in the sane association, was found by Wilson
(56) to differ greatly.

In one association, the sap of soy­

beans was found to contain 1,000 parts per million of nitrate
while that of purslane contained 5?882 parts per million.

In

another association oats were found to contain 500 parts per
million of nitrate and Amaranthus was found to contain *f,lM-0
parts per million..

Host weed plants were found to contain a

much higher content of nitrate and other nutrients than the
common crop plants growing In the same association (V2).

15

Production of Nitrate within the Plant
Evidence that nitrate nay be synthesized by plants is
rather meager,

Vickery et al*. (50,51) have demonstrated that

the nitrate levels of excised leaves of tobacco and rhubarb
were increased when these leaves were cultured in darkness in
water or in solutions containing ammonium sulfate.

The nitrate

was assumed to have been formed by the oxidation of the ammo­
nium ions.

These workers have also shorn that nitrate-free

Narcissus bulbs developed traces of nitrate when cultivated
in either distilled water or in complete nutrient solutions
containing ammonium sulfa.te (52).

Nitrate synthesis by seg­

ments of Narcissus leaves has also been reported (37).

Herbicidal Treatment
Stahler and Whitehead (*+6) report a case in which several
hundred acres of sugar beets on seven farms were accidently
treated with 2 ,h— D*. Samples of beet leaves from the seven
farms, together with several samples from untreated fields of
adjacent farms, were analyzed for nitrate.

The average nitrate

level in the untreated beet leaves, calculated in terms of po­
tassium nitrate, was found to be 0.22 percent of the dry weight.
The nitrate levels in the treated leaves ranged from 1.8l per­
cent to 8.77 percent, all of which were considered above the
minimum lethal concentration.

The author has also observed

that 2 ,h-D has a profound effect on the nitrate content of

16

sugar beet leaves.

These results Indicate that 2 ,k-D does af­

fect the nitrate

level of beet leaves to the extent that con­

siderable losses

could beexpected if the leaves were fed to

livestock.
Lambs quarters, pigweed and smart weed when treated with

2 ,*-f—D were found by Jones (25) to be very high in nitrate while
the controls contained very little.

This was thought to be

due to the 2 ,^--D checking the assimilation of nitrates into
protein.

These weeds,

found by various

as well as sugar beet leaves, have been

other workers to accumulate nitrates in toxic

concentrations even when not treated with 2 ,k-D.
Fertig (11) studied the effect of 2,1-!--D and MCP on the
accumulation of nitrate in lambs quarters, ragweed, pig^reed
and curled dock but was unable to obtain conclusive results.

It is apparent that there are many factors, in addition
to herbicidal treatment, capable of Influencing the level of
nitrate in plants.

Some of these factors may possibly be mod­

ified by the application of sprays to the plants.

Applications

of 2,1+-D and related compounds are known to have a consider­
able effect on the metabolism and growth process of plants.
This effect could bring about changes in the rates of photo­
synthesis, root growth, nutrient absorption and transpiration.
Because of the numerous factors Influencing the uptake and
assimilation of nitrates, considerable variations in the ni­
trate levels should be expected, not only In plants growing

17

under normal conditions, but also in those herbicidally
treated plants*

MATERIALS AIID PROCEDURES

The species of weeds used in this work were selected on
the basis of availability, occurrence in pastured areas, and
reports concerning the ability of certain of the weeds to ac­
cumulate nitrate.

All of the species were found■growing on

or bordering the muck farm used for weed control investigations.
The following species were selected:
1* Amaranthus retroflexus L.
2 . Ambrosia elatior (L.) Descourtils
3* ChenoDodium alburn L.
Cirsium arvense (L.) Scop.
5*
Eunatorium macula turn L.
6 . Imnatiens biflora Walt.
7. Po a nratensis L.
8 • Polygonum Convolvulus L.
9*
Polygonum Persicaria L.
10. Prunus virginiana L.
11* Sambucus canadensis L.
12. Solanum Dulcamera L.
13* Spiraea alba Du Roi
Thalictrum dioicum L.
Some of the more commonly used herbicides were selected
for this work.

Included in these were herbicides which were

believed to offer differences in physiological activity.

The

herbicides selected were:

1.
2.
3.

. ester of 2,h— dichlorophenoxyacetic acid
Butyl ether ester of 2 ,*f,5-trichlorophenoxyacetic
acid (2,k,?-T)
Sodium salt of 2-methyl-1*-chlorophenoxyacetic acid
(MCP)

Alkanolamine salt of dinitro-o-sec-butyl-phenol (DEEP)
5. Isopropyl-N-(3-chlorophenyl) carbamate (CIPC)
6 .. Diethanolamine salt of maleic hydrazide (MH)

19

The weeds were treated with sub-lethal dosages of herbi­
cide applications.

This was done to prevent excessive dete­

rioration or death of the plants during the period in which
analyses were to be made..

With a few exceptions the spray

mixtures were applied in a volume of 80 gallons of water per
acre*.

The exceptions were treated with a volume of *4-0 gal­

lons per acre.

It was known that the period of greatest ac­

cumulation of nitrate was during the stage of growth immedi­
ately preceding the reproduction stage.

For that reason all

herblcidal applications were timed so that the period of anal­
ysis coincided as closely as possible to the stage preceding
reproduction.
Samples for analysis were obtained at 2b hour intervals,
for four days following treatment*
made two weeks after treatment-

An additional analysis was

By sampling in this manner,

both rapid and slow accumulations of nitrate could be detected.
Sampling was done early in the morning to eliminate, as much
as possible, the effect of sunlight on the nitrate accumulated
during the dark hours.
The samples were taken immediately to the laboratory where
extracts were prepared from duplicate samples of the fresh mate­
rial.

The extracts were then analyzed for nitrate content us­

ing a colorimetric method which involved the nitration of 3 >^
1 p
xylenol. ? This method was reported to have been successfully
^Analytical method"supplied by Dr. G.H. Ellis, Head of
Chemical Division of United Co-operatives, Inc., Ithaca New York.
2 See appendix for analytical method and standard curve.

20

used at the U. S. Plant Soils and Nutrition Laboratory, Ithaca,
New York,

Preliminary work indicated that the method gave con­

sistent and reproducible results and that it was much less time
consuming than the Kjeldahl method of nitrate determination,
A standard curve for use in comparing the results of the nitrate
determinations was obtained by analyzing solutions containing
known quantities of analytical grade potassium nitrate.

All

of the potassium nitrate values in the tables represent the
average of two determinations,
Prior to the summer in which the bulk of this work was
performed, a preliminary survey was made on several weed species.
These analyses were made two weeks following treatment.
results are presented in Table 1*

The

EXPERIMENTAL RESULTS

In the preliminary study, ten we_>ds were trea.ted with the
various chemicals and the effect on the nitrate accumulation
determined.

The results (Table 1 ), indicated that nitrate ac­

cumulation, if it occurred, did not persist for the two-week
period foil.owing application of the chemicals.
Based on the data obtained, the weeds sprayed in the later
experiments were divided into four groups.

These groups are

as followst

1 . Weeds which showed no significant differences between
either chemical treatments or days
Chenouodium album
Cirslum arvense
Primus vlr.giniana
Thalictrum dioicum
2.. Weeds in which there were differences only between
chemical treatments
Eunatorium maculaturn
Imnatiens biflora
Sanbucus canadensis
Solanum Dulcamara

3 . Weeds in which there were significant differences be­
tween chemical treatments and between days on which
analyses were made
Amaranthus retroflexus
Ambrosia elatior
Polygonum Convolvulus
Polygonum Persicaria
L.

Weeds which showed significant differences
the days on which analyses were irir3.de.

only

between

22

TABLE 1
NITRATE CONTENT OF WEEDS TWO WEEKS AFTER
APPLICATION OF HSRBICIDAL SPRAYS

Mg. of KNO-^ per Gram of Dry Weight
Wood
Spe'-ies

Ambrosia
elatior
Bidens
vulsata

Chemical and pounds per acre
•
*

+
«

•
•-

•
•-

•
•

•
•

J2,>-D :2,!+,5-T: MCP : DNBP I CIPC : MH :Control
•
•
•
•
•
#
*
•
•
1A lb; 1/1+ lb. :1A lb; 1/2 lbs 1 lb. 1 lb.
9-30

10.37

9.30

10.37

9.15

9.15

9A O

.59

•*71+

.1+1+

.51

.51

.59

.66

11.67 12.10

11.76

12.10

Chenopodium
album

12 A c

Eupatorium
maculaturn

1.39

1.57

1.57

2.11

1.93

1.8^

1.81+

Impatiens
biflora

3.72

5.99

6.26

6 .7b

6.50

6.7“+

7.78

Poa
pratensis

1.86

1A 2

1.35

.97

1.05

1.20

2.09

Po Ivp:onurn
Persicaria

3.31

*+.13

3.65

1.98

3.81

3.65

3A 7

18.81)-

17.66

12.lf-0 12.*+0

Prunus
vireiniana

15.08 1^+.6^

Spiraea
alba

1.29

1.35

I .23

1.23

1 .A6

1.23

lAO

Thalictrum
dioicum

3.A

3.27

1.71

1.10

1 .8U-

1.71

3.27

Group One
The nitrate levels of Chenopodium album (Table 2) and
-QjLf-Sium arvense (Table 3) were found to be quite high, while
the nitrate levels of Prunus vimeiniana (Table *+) and Thalictrum dioicum (Table 5) were relatively low*

Statistical anal­

yses showed that variations between treatments and between days
were not large enough to be significantly different.

Group Two
Chemical treatments of Eupatorium maculaturn (Table 6),
with the exception of MCP, resulted in significant increases
in nitrate content over the control at some time during the
two week period.

There was an immediate increase (2k- hours)

in nitrate concentration followed by a decline over the two
week period.

At the fourteenth day, only the plants which re­

ceived the DNBP treatment remained significantly higher in ni­
trate than the control.

There was no definite pattern of ni­

trate increase or decrease after the first day.

That is, some

treatments caused a gradual increase in nitrate followed by
a decrease while others brought about a decrease in nitrate
followed by an increase.

MCP produced significant decreases

at the second, third and fourth day intervals.
One the first day following treatment of Impatiens biflora
(Table 7), significant increases were observed for all treat­
ments except that of DNBP.

In most cases the nitrate content

2b

TABLE 2
NITRATE CONTENT OF CIIENOPCDIUM ALBUM L. FOLLOWING
APPLICATION OF HERBICIDAL' SPRAYS

Mg. of KNO, per Grara of Dry height
Chemical and Pounds
Applied per Acre

Days after treatment
m
•

1

s

2

:

1

1

b

lb

2 ,lf-D
l A lb.

38.62

36.02

A . 57

39.79

bi.57

2A,5-T
lA it>.

b2 .81

>+2.19

>+2.19

A . 8i

39. A-

lA

MCP
lb.

’+0.95

bl . 5 7

bO . 95

b2. 81

b5,2b

DNBP
1/2 lb.

A .65

A A 2

b5.2b

M+.O^

>+3.b2

M-2.86

bb.Ob

b$.2b

^2.81

30.96

1 lb.

A . 19

b5.2b

A.O^

bo .63

bo . 95

Control

b$.2b

b2.22

A . 99

i+2.19

bb . 6 2

CIPC

1 lb.
NH'

Statistical analysis showed no significant differences
between days or treatments.

25

TABLE 3
NITRATE CONTENT OF ClRSIJJll ARVENSE (L.) SCOP.
FOLLOWING APPLICATION OF HERBICIDAL SPRAfS

Mg. of KNO3 per Gram of Dry Weight
Chemical and Pounds
Applied per Acre

Days after treatment
*

1

2

28.72

29.15

36.38

35.83

37. A

? A 1—T
l A it.

39.63

35.83

35.31

38.53

39.08

MCP
l A lb.

39.08

33.70

3V.79

37. A

37.99

37.99

36.90

36.35

35.83'

36.35

VO. 15

35.31

35.31

35.83

3V.79

1 lb.

38.53

35.83

35.31

33.35

32.32

Control

39.63

33.70

39.60

36.90

37.99

2 ,5-D
l A lb.

t

3

1

V

:

IV

DNBP

1/2 lb.
CIPC

1 lb.
MIT

Statistical analysis showed no significant differences
Between days or treatments.

26

TABLE 14-

o

•

*7*

NITRATE CONTENT OF PRUNtTS VIRGINIANA L. FOLLOWING
APPLICATION OF HSRBICIDAL SPRAYS

Chemical and Pounds
Applied per Acre

KNO^

*
•-

1

:

per Gram of Dry Weight

Days after treatment
♦
2

t

3

;

b

: li+

2 j [i--D

10.88

9*92

10.38

10.10

10.10

lb.

7.8V

8.85

9.V7

9.23

8.97

MCP
l A lb.

9.22

9.60

1 0 .2 3

9.72

9*72

DNBP
1/2 lb.

9-8?

9.V6

9.96

9*72

8.97

CIPC
1 lb.

9-V8

10.10

9.61

10.88

9*98

1 lb..

10.10

9.^7

10. V6

10.16

9.73

Control

9.35

10.23

9 •8V

9*96

8.97

l A lb.

IA

MH

Statistical analysis showed no significant differences
between days or treatments.

27

TABLE 5
NITRATE CONTENT OF THALICTRUM DIOICOK L. FOLLOWING
APPLICATION OF HERBICIDAL SPRAYS
1
o

KNO^ per Gram of Dry Weight

Chemical and Pounds
Applied per Acre

Days after treatment
i

;

2

:

3

:

2, *+—D
l A lb.

6. 60

V. 21

6..21

5.06

2A,5-T
l A lb.

A 70

3.78

A85

6.70

MCP
l A lb.

b.57

3.86

>+.99

5.9+7

DNBP
1/2 lb.

^.-92

5.39

5.23

5.06

CIPC
1 lb.

7.67

5.33

h.b7

5,8b

MH
1 lb.

3.3>+

3.60

3.66

3Ao

Control

3.27

2 .A

1+-78

6 .60

•
*
s

l^f

Statistical analysis showed no significant differences
between days or treatments.

28

of sprayed plants remained relatively constant throughout the

two-week period.

The nitrate level of the control increased

constantly, with the result that at the end of the two-week
period only the MCP treated plants remained significantly high­
er in nitrate.
In no case did the spraying of Sambucus canadensis result
in a significant increase in nitrate over the control (Table 8),
The 2,^-D treatment resulted in a highly significantly lower
nitrate content for at least four days following the applica­
tion.

At the end of the two-week period however, the nitrate

level In this treatment was similar to that in the majority
of other treatments.

The nitrate level in the 2,k,5~T sprayed

plants remained normal for the first four days but was consid­
erably reduced at the end of two weeks.
Chemical treatments, with the exception of DNBP, resulted
in a significantly lower content of nitrate in Solanum Dulca­
mara (Table 9)-

Cn the fourth day following application of

the chemicals, the DNBP treated plants contained a signifi­
cantly greater amount of nitrate than the control.

However,

analyses at the end of the two-week period showed that the
nitrate content of these plants had fallen much below that of
the control.

29

TABLE 6
NITRATE CONTENT OP EUPATORIUM KACULATUH L. FOLLOWING
APPLICATION OF HSRBICIDAL SPRAYS

Mg. of KNO^ per Gram of Dry Wei gilt
Chemical and Pounds
Applied per Acre

Days after treatment
»
*■
2
s
... t
3
:
*-

2, N-D
l A lb.

13.06

17.87** 17 .56

2A,5-T
l A lb.

12.09

11.38

MCP
l A lb.

8.39

6. 8*+

DNBP
1/2 lb.

»}t^£

lb.B?

15.3^

MH
1 lb.

18.05

18.55

Control

8.86

9.17

*

if.80*

_

22.35
5j«

A . 89

8.88**

10.95

23.68** 23.38o** 16.58

CIPC
1 lb..

**

ll+
10.76

5.37

7.13** 13. A
19.26*

hi#.
23.0'2 ”

16.08

11 . A

A . 89** 10.57*
9.38

*
:

llK 89

15.66
A ,M+

Least significant difference between treatments at 5n =
3.8*+
** Least significant difference between treatments at
=
5.22
No significant difference between days

30

TABLE 7
NITRATE CONTENT ON IMPATIENS BIFLORA WALT. FOLLOWING
APPLICATION OF HERBICIDAL SPRAYS

Mg., of KNO^ per Gram of Dry Weight
Chemical and Pounds
Applied per Acre

Days after treatment
•
•

•

•-

2

:

3

b

: 11+

IP *27

12.28

12.60

11.62*

1 3 .9 6

A . 93

l A lb.

12.60*

2 A,5-T
l A lb.

13.58** 13.96*

MCP
l A lb.

22.98** 23.35** 16.73

11.30

:

2 3 .3 6 ** 19.50

DNBP
1/2 lb.

10.61+

12.93

11.62*

1 1 .9 5

12.93

CIPC
1 lb.

12.60*

13.96*

16.02

1 2 .2 7

12.28*’

MH
1 lb.

13.96** 18 A7** 15.31

A . 28*

15.31

11.62

16.37

Control

9.67

1 1 .3 0

A . 28

L*S.D. between treatments at 5% m 2.J0
** L.S.D. between treatments at 2.% - 3**+0
No significant difference between days

sfts|

31

TABLE 8
NITRATE CONTENT 0? SAILBUCUS CANADENSIS L. FOLLOWING
APPLICATION OF KERBICIDAL SPRAYS

Mg. of KNO3 per Gran of Dry Weight
Chemical and Pounds
Applied per Acre

Days after treatment
*
2
:
1
:
V
:
*

1

•

*
•
:

IV

2 ,V-D
1/V lb.

A . 30** 15.22** 13.67** 16.11** 2b. 20*'

2,1+, 5-T
i A it).

26.3V

23.50

2V.81

25.6!+

16.79*'

2V.20

23.85

25.51

25.9b

2V..81*

22.00

26.29

2 5•9V

26.3V

25.51*

21..?k*

23.50

22.63

28 .37

25.6V

1 lb.

25.90

26.3!+

2V.20

25.25

26.29

Control

25.51

25.9V

23.85

28.76

29.17

MCP

l A lb..
DNBP

1/2 lb..
CIPC

1 lb.
MH

* L..S*D* between treatments at 5% - 3.60
* L.S.D. between treatments at
» V.89
No significant difference between days

TABLE 9
NITRATE CONTENT OF 30LANUH DULCAMARA L. FOLLOWING
APPLICATION OF HERBICIDAL SPRAYS
— . him,

niw^wii

Mg., of KNO^ per Gram of Dry Wei-gbt
Chemical and Pounds
Applied per Acre

Days af ter trea fcment
•
•

1

:

•
•

2

:

•

8

:

1+

!

l ^ f

2,*f-D
l A lb.

13 AS** 13*08

2A*5~T
l A lb.

11A3** 10.5?** 10.36** 11.95** 13 A 6**

MCP
l A it.

*

#

+

*

10.92

*

*

A . 62** 11.78**

sfc S|c

„

11.07** 10.91** 11.78

12.8?

21.90

22.92

16.03

DNBP

1/2 lb.

s k ?k

13.91

22.61

23.89

*

*

CIPC
1 lb.

A . A * * 12.1+9

16.27** 16.77** 16.01**

MH
1 lb.

13.95** 16.01

A . A * * 16.25** A . 36**

22.92

22.21

Control

*

22.00

*

L.S.D. between treatments at
m 2*32
** L.S.D. between treatments at 1% = 3.-16
No significant difference between days

20.55

22.21

33

Group Three
The nitrate content of this group differed significantly
between treatments as well as between the days following treat­
ment .
At no time did the herbicidal treatment of Ambrosia elatior
(Table 10) result in an increase in nitrate content above that
of unsprayed plants.

The 2,*-:— D treatment appeared to be most

effective in reducing the nitrate level.

Two weeks following

the spraying, nitrate levels were reduced we13. below both that
of the control and the levels observed 2k- hours after treatment.
The results of treating Amaranthus retroflexus (Table 11)
indicate that on the first day following the application of
the herbicides, the nitrate content of all treatments, except
that of CIPC, was significantly lower than that of the control*
The nitrate level of all lots showed an increase on the second
and third days-

At the fourth day the nitrate content decreased

as compared to the control and at two.weeks the level of all
treatments was considerably below that observed on the first
day..

Analyses on the fourteenth day showed the DNBP and MH

sprayed plants to be significantly higher in nitrate than the
control.

All other differences between the treated and un­

treated plants on different days were due to the lower nitrate
content in the sprayed plants.
Unlike most of the weeds worked with, there was not a consistant upward or downward trend in the nitrate content of Pol­
ygonum Convolvulus (Table 12).

The nitrate levels varied

3*1-

considerably from day to day but the pattern was not consist­
ent for the different treatments#.

The 2,)+-D treated plants

contained a significantly higher nitrate level than the con­
trol on the first, second, third and fourteenth days, follow­
ing spraying#.

The 2 , h r5~T treated lot contained a higher ni­

trate level than the untreated plants on the fourth and four­
teenth days, while' plants which were treated with MCP contained
more nitrate on the second, third and fourth day Intervals.
With the exception of the 2,^-,5-T1lot, the increases in nitrate
were not significantly higher than in the control plants.
The nitrate levels of Polygonum Persicaria (Table 13)
varied considerably on the first day following treatment,, but
all compounds except 2,WD brought about significant reductions#
The nitrate content increased on the second, third and fourth
days but the increases were not significantly different from
the control plants.

At the fourteenth day only 2,^-D had failed

to cause a highly significant decrease in the nitrate content
of this weed.

Group Four
Poa nratensis (Table l*f) was found to have a very low ni­
trate content throughout the period in which analyses were made.
There were significant differences in nitrate content between
the 2b hour periods in which analyses were made.

However, these

were due to the downward trend in nitrate content which occurred
in all Poa samples.

35

TABLE 10

NITRATE CONTENT OF AMBROSIA ELATIOR (L..) DESCOURTILS
FOLLOWING APPLICATION OF HERBICIDAL SPRAYS

Mg*, of KNOo per Gram of Dry Weight
Chemical and Pounds
Applied per Acre

Days after treatment
#
•

1

:

2

:

•
*

•
•

•

3

:

L f..

.

A

2,5-D
l A lb.

20.29** 17.99** 22.00** 21.60** 13.86**

l A it.

27.07*

MCP
l A lb.

20.96** 23.28** 25.22'** 23.63

2k.22** 25.27**

25.55** 12.32**
.

*

*

sjs sfr:

13.59

DNBP
1/2 lb.

26.98*

26.31*

27.O7** 26.69** 10.38**

CIPC
1 lb.

23-95

2>+.29

27.88** 25.55** 11.09

MH
1 lb.

25.96** 26.60*

Control
^ L*S.D.
L.S.D.
L.S-D*
L*S.D#.

5j< *

28.63

29.06

28.63*

25.55** 13.52**

31.26

32.16

between treatments at 5% = 2.31
between treatments at 1% = 3*1?
between days at 5%3 1«96
between days at 1% ■ 2.66

18.01

36

TABLE 11
NITRATE CONTENT OF AMARAITTITIJS RETROFLEXQS L. FOLLOWING
APPLICATION OF HERBICIDAL’SPRAYS

Mg. of KNO^ per Gram of Dry Weight
Chemical and Pounds
Applied per Acre

Days after treatment

a

1

lh

2 5*4— D

l A lh.

35.37** 37.63*

37.57** 37.63** 19.2*+**

2,A5-t
l A lb.

36.00**

3 !+.37 ** 3 9 .8^*

37.57** 22.96

37.06*

38.69

37.63** 18.06**

MCP

l A lb.

^oAl

DNBP

1/2 lb

31.32** 3^.01

38.72** 37.57*

28.37'

V2.10

25.85

CIPC
1 It).

39.8*+

MH
1 lh.

35.37** 37.06** M-2..86

39.8>+** 27.12*

39.8^

^-3Ao

C o n tro l

* L.S.D.,
L.S.D.
L.S.D..
L.S.D*

between
between
between
between

ho.M-1

1+0.99

^-2.6^

treatments at 5a 35 2.60'
treatments at 1% - 3*53
days at 5% - 8.69
days at 1a = 11.81

*+1.56

2*+. 50

37

TABLE 12
NITRATE CONTENT ON POLYGONUM CONVOLVULUS L. FOLLOWING
APPLICATION 01' HERBICIDAL SPRAYS

Mg. of KNO^ per Gram of Dry Weight
Chemical and Pounds
Applied per Acre
i

Days after treatment
•
•
*
r
2
:
1
:

Z.h-D

1/*+ lb.

_

5$;

17.87*

16.55

i

lN

19.39*

21.57

17.07

l A lb.

17.60

19.91

MCP
l A lb.

19. A

21.85** 20.68** 20.16** 16.06

*^

2A,5-T

DNBP
1/2 lb.

9.1^

CIPC
1 lb.

18.89

19. A

MH
1 lb.
Control
L.S.D.
v* L.S.D.
L.S.D.
L.S.D.

12.12** 17.10*

17.07**

13.7b** 12.00** 13.12

12.75

16.30

15.09

16.05

1>+.61+*

13.50** 12.00

1 2 .9 2

12.75

17*07

1 8 .6 ^

1^.86

A . 22

15.11

between treatments at 5% = 2.08
between treatments at
« 2.83
between days at 5y- 2.M+
between days at 1^*» 3*32

3U

TABLE 13

NITRATE CONTENT OF POLYGOTTTTM PHRSICARIA L. FOLLOWING
APPLICATION OF HShBICIDAL SPRAYS

Me. of KNO^ per Gran of Dry V/eight
Chemical and Pounds
Applied per Acre

Days after treatment
•

•

1

2

!

:

3

:

b

1

lh

l A lb.

32.3*+

37.63

38.0*+

36.58

l A lb.

29.1+3*

32-.3*+

33.80

31+.2*+* 30.88**

MCP
l A lb.

26.82** 35.19

37.63

36.55

25.77**

2*+..38** 33.29

27 .>+9** 3*+.65

32.3^**

35.16

39.17

37.63

28.20**

37.09

DNBP

1/2: lb.
CIPC
1 lb..

28.00

m-i
1 lb.

26.10

30.88*

3*+.2i+

37.85

19.97

Control

3h.2h

36. n

36.68

38.58

38.01+

L.S.D.
** L„S.D.
L.S.D.
L.S.D.

between
between
between
between

**

treatments
treatments
days at 5$
days at if

at 5% = ^f.05
at if = 5.50
“ 3.*+2
= *+.65

39

The r e s u l t s

o b t a in e d fr o m th e t r e a t m e n t o f S p ir a e a a lb a

( T a b le 1 5 ) w e re s im i la r , t o
The d i f f e r e n c e s
in

n itr a te

th o s e o b t a in e d f o r Poa n r a t e n s i s *

o b s e rv e d w e re due t o

th e c o n tin u o u s d e c re a s e

c o n t e n t o f b o th th e t r e a t e d and u n t r e a t e d p l a n t s .

*fO

TABLE 1^
NITRATE CONTENT OF POA PRATERSIS L. FOLLOWING
APPLICATION CF NERBICIDAL SPRAY'S

!
lli 1
rwrruyjaa
Mg. Of KNO^ per Gran of Dry Weight
Chemical and Pounds
Applied per Acre
1
2?1+-D
l A lb.

♦ Davs after treatment
•
•
:
2
:
*+
1
#

m

:

1*+

5.62

2.9>+

3A7

3.38

3.38

l/U- lb.

5.3*f

2.59

3.65

3.12

3.03

MCP
l A lb.

*+•*+7

2.59

if*6if

2.85

2.77

DNBP
1/2 lb.

*+.*+5

2.50

3.83

3.29

2..91+

CIPC
1 lb.

b.k-7

*+.85

3.03

2.59

2.85

MH
1 lb.

2.59

*+.28

2.33

2.68

1.55

Control

b.75

2.77

2.68

3.91

2.59

2 A? 5-1

No significant difference between treatnents
L.,S.D. between days at 5% * •85’
L.S.D. between days at 1y - 1.15

>-1-1

TABLE 15
NITRATE CONTENT OF SPIRAEA ALBA DU ROI FOLLOWING
APPLICATION OF HERBICIDAL SPRAYS

Ma. of KNO^ per Grain of Dry Weight
Chemical and Pounds
Applied per Acre

Davs after treatment
*
•

♦
*

1

:

2

:

3

:

♦
•

b

: A

2,*f-D
l A lb..

10 ..16

11A 2

10 A 6

6.63

8.19

l A lb.

11.39

12.35

9.35

9.21+

7.61+

12.77

12.35

9.35

9.80

7.76

1/2 lb.

12.25

11.56

8.58

9.01

7.18

CIPC
1 lb.

12.1+3

11.7*+

8.81+

8.71

7.29

1 lb.

12.60

9.60

8.11

7.80

7.30

Control

13 A O

12.90

11.1+2

10.61

7.76

MCP

l A lb.
DNBP

MH

No significant difference between treatments
L.S.D. between days at
« >+.30
L.S.D. between days at \% — 5*A

DISCUSSION

A number of research workers have shown that sub-lethal
dosages of 2,b— D, and other herbicides, have remarkable ef­
fects on the metabolism of the treated plants (8,3*16,*f3?26)•
Not all species of plants are affected in the same manner nor
to the same degree.

This is evident from the varying degrees

of tolerance exhibited by different species of plants to a
particular chemical.

The metabolic abnormalities, brought

about by herbicidal treatment, may cause plants to accumulate
nitrate in the same manner as is done when plants are placed
under adverse growing conditions, such as drought.
Nitrate accumulation following treatment with 2,If-D has
been reported in the case of Chenonodium album (25).

This

effect was not found in this study and may be accounted for
by differences in environmental conditions under which the
work was done.

Bradley et al. (*+) have shown that the type

of soil determined to a great extent the amount of nitrate
In plants, even when the nitrate level of the soil was high.
Wide differences In soil types and soil moisture conditions
may give rise to entirely conflicting results.
Herbicidal treatments used in this study did not affect
the nitrate content of the following species:

Chenopodiuin

album. Cirslum arvense. Prunus Virginians and Thalictruin
dioicum.

^3

The h e r b i c i d a l t r e a tm e n t s on tw o s p e c ie s ,
Ig-tum and I n m a tie n s b i f l o r a « r e s u l t e d
n itr a te

c o n te n t..

in

r a p id

E u p a to riu m m acnin c r e a s e s i n

I n th e m a j o r i t y o f weeds t r e a t e d t h e r e was

e i t h e r a r e d u c t io n o r no change i n

n itr a te

c o n t e n t d u r in g

th e

same p e r io d .
Some w e e d s, f o r e x a m p le : A m b ro s ia e l a t i o r « A m a ra n th u s
r e t r o f 1 e xu s and P o lyg o n u m P e r s i c a r i a . w e re o b s e rv e d t o
la te

n itr a te

on th e

second, t h ir d

h e r b ic id a l tre a tm e n t.
in

n itr a te

lo w in g

le v e l o c c u rre d in
in d ic a t in g

th e f o u r t h

th e f o u r t e e n t h d a y f o l ­

b o th th e t r e a t e d p l a n t s

th a t fa c to rs

and th e

n itr a te

c o n tr o ls ,

o t h e r th a n th e h e r b i c i d a l t r e a t m e n t s
cha ng es i n

o f te m p e r a t u r e , l i g h t ,

p l a n t s fr o m

to

T hese In c r e a s e s an d d e c re a s e s i n

w e re r e s p o n s ib le f o r th e
a t io n s

d a ys f o l l o w i n g

I n th e s e weeds t h e r e was some r e d u c t io n

c o n t e n t fr o m

tre a tm e n t.

and f o u r t h

accum u­

th e n i t r a t e

le v e ls .

V a r i­

m o is tu r e and th e p r o g r e s s o f th e

one s ta g e o f g ro w th t o a n o th e r c o u ld a c c o u n t f o r

th e changes i n

th e n i t r a t e

le v e l o f b o th tr e a te d p la n ts

and

th e c o n t r o l.
I n no ca se d id a n y p a r t i c u l a r h e r b i c i d a l t r e a t m e n t r e s u l t
in

c o n s i s t e n t l y h ig h e r o r lo w e r n i t r a t e

F o r e x a m p le ,

it

I n lo w e r n i t r a t e

le v e ls

in

a ll

s p e c ie s .

was o b s e rv e d t h a t th e 2,*+-D t r e a t m e n t r e s u l t e d
le v e ls

th a n th e o t h e r t r e a t m e n t s when a p p lie d

t o A m b ro s ia e l a t i o r . Sambucus c a n a d e n s is and Poly_gonujn P e r s i ­
c a r ia .
v u lu s .

The r e v e r s e was t r u e

in

th e ca se o f P olygonum C o n v o l­

lfl+

Ten of the fourteen weeds were found to contain nitrate
levels in excess of the one and one-half percent considered
by Bradley et al. (*+) to be the maximum concentration of ni­
trate in plants which livestock could safely consume.
ten weeds were:

These

Amaranthus retroflexus. Ambrosia elatior,

Chenov odiun album, Cirslum arvense. Funatorium maculabum. Im•patiens biflora. Polygonum Convolvulus . Polygonum Persicaria.
Sambucus canadensis and Solanum Dulcamara.

In only two weeds

could the toxic level of nitrate be attributed to the effects
of herbicidal treatjuent.

The application of 2,h-D, DBBP, CIPC

and till to Euna,torium macula turn resulted in a rapid accumulation
of nitrate to the extent the the plant could, If consumed by
livestock in sufficient quantities, cause nitrate poisoning.
The application of MCP, CIPC and Mil to Imnatiens biflora re­
sulted in similar increases in nitrate concentraction.
Livestock poisoning by Solanum Dulcamara and Prunu.s vir­
gin! ana has frequently been reported.

The 2,h-D and 2,^,5^

treatments, which often have been employed in eradicatin these
two species, did not result in Increased levels of nitrate
in these plants.
The data indicate that large day to day variations in
nitrate content were common and were often of greater signif­
icance than the variations due to the chemical treatments.

SUMMARY AND CONCLUSIONS

1*.
s ix

F o u rte e n s p e c ie s o f w eedy p l a n t s w e re t r e a t e d w i t h

d i f f e r e n t h e r b ic id e s

th e n i t r a t e

2.

and th e e f f e c t o f th e t r e a t m e n t s on

c o n t e n t was d e te r m in e d .

The herbicides were applied at sub-lethal dosages

and analyses of the nitrate content were made on the first,
second, third, fourth and fourteenth days following the appli­
cation.
3*

The nitrate content of the weeds was determined by

a comparison with standard solutions of potassium nitrate us­
ing

a method involving the nitration of 3,!+ xylenol..
b.

The nitrate levels of four of

unaffected by the herbicidal application.

the treated weeds were
These weeds were:

Chenonodium album, Cirsium arvense. Prunus virginiana and
Thalictrum dioicum..
5.

Herbicidal treatment of five of the weeds resulted

in significant increases in nitrate content.

These weeds end

treatments were:
a.
b.

The DHBP
treatment of Amaranthus retroflexus
The 2,N-D, DNBP, CIPC and MH treatments of Eunatorium
maculaturn
c„ All treatments of Imnatiens biflora
d.; The 2,b— D, 2,*+,J-T and MCP treatments of Polygonum
Convolvulus
e. The DNBP treatment of Solanum Dulcamara.
6.

Treatments on Polygonum Persicaria caused significant
reductions in nitrate content.

b6

Ten o f th e f o u r t e e n weeds s t u d ie d

7.
in

s u f f ic ie n t q u a n titie s

s to c k i f

consum ed i n

to

cau se n i t r a t e

c o n s id e r a b le

c o n ta in e d n i t r a t e

p o is o n in g o f l i v e ­

q u a n t it ie s

even th o u g h no

s p ra y s h a d b e e n a p p lie d *
8 ..
to x ic

In

o n ly tw o o f th e weeds c o u ld th e a c c u m u la tio n o f

c o n c e n t r a t io n s

o f n itr a te

be a t t r i b u t e d

e f f e c t o f h e r b ic id a l tre a tm e n t.

s o le ly

to

th e

These tw o weeds w e re B u p a t-

o r itm i m a c u la tu rn and I m p a tie n s b i f l o r a ,
9*

ho h e r b i c id e

a p p e a re d t o ha ve th e

same a f f e c t on

a l l o f th e weeds t e s t e d .
10.

O f th e tw o w e e d s , Chenopodium albu m and A m a ra n th u s

r e t r o f l e x u s « p r e v io u s ly r e p o r t e d
lo w in g

tre a tm e n t w ith

2 ,*+ -D ( 2 5 ) ,

t o a c c u m u la te n i t r a t e
it

fo l­

was fo u n d t h a t C henopodium

was n o t a f f e c t e d b y a n y o f th e t r e a tm e n t s and A m a ra n th u s a c c tin u la te d n i t r a t e
11*.

D u r in g

s o i l c o n d itio n s
tra te

o n ly as a r e s u l t o f th e DNBP t r e a t m e n t .
th e c o u rs e o f t h i s

s tu d y i t

was o b s e rv e d t h a t

o f t e n had a g r e a t e r e f f e c t u p o n th e p l a n t n i ­

c o n t e n t th a n d i d

th e h e r b i c i d a l t r e a t m e n t .

LITERATURE CITED

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2*. Anderson,,. A.J. and D.. Spencer. Molybdenum and Sulfur
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Bradley, W.B., H.F. Eppson and O.A. Death. Livestock
Poisoning by Oat Hay and Other Plants Containing Nitrates.
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5.

Burstrom, PI. Photosynthesis and Assimilation of Nitrate
by Wheat Leaves. Lantbrukshogskolans Annaler. 11: 1-50,
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2196, 19*+5.

6 . ____ ,
______ • The Nitrate Nutrition of Plants.. Lantbruks­
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Biological Abstracts.

21:

1221, 19k7-

7* Davidson, O.W. and J.W. Shive. The Influence of the
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by Peach Trees Grown in Sand Cultures. Soil Sci. 37?
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8 . Davidson, W.B., J.L. Doughty and J.L. Bolton.
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Nitrate
Canadian Jour. Comp. Med. 5?

9. Evans, H.J., E.R. Purvis and F.E. Bear. Molybdenum Nu­
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and A. Nason. Pyridine Nucleotide-nitrate^Re­
ductase from Extracts of Higher Plants. Plant Physiol.
28: 233-2 5*+, 1953-

h8

11. Fertig, S.N. Livestock Poisoning from Herbicide Treated
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12• _________ . Herbicidal Poisoning of Livestock.
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Seventh Ann*. Northeast. Weed Control Conf., bb-h77 1953*
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lb.

_______ a n d __________ The Importance of Oxygen in the
Nutrient Substrate for Plants - Relation of the Nitrate
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15*

Goksoyr, J.. On the Effect of Chlorate Upon the Nitrate
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16.

Grigsby, R.H. Some Effects of 2,k-D on Ragweed and Cer­
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17•

___ ____ and C.D. Ball* Some Effects of Herbicidal
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1953.

18. Harnner, K.C.
Effects of Nitrogen Supply on Rates of Photo­
synthesis and Respiration in Plants. Bot. Gas. 97 •7bb76k, 1936.
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Growth as a Determining
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Wetenschapp. Amsterdam. 51+s 275-286,
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Factor for the In­
K.^Nederland. Akad.
1951* Seen in
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20.

Hewitt, E.J. and E.W. Jones.. The Production of Molybdenum
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21.

Jacques, A.G., and W.J.V. Osterhout. The Accumulation of
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22.

Jensen, I-I.L. Nitrogen Fixation in Leguminous Plants.
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72: 265-293, 19^8.

1+9

23. Jones, L.W. and J.E. Greaves. The Influence of Incubation
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Jones, L.-H.^W.B. Shepardson and C.A. Peters. The Func­
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P la n t P h y s i o l .

2bt

3 0 0 -3 0 7 ,

19^9.

25.

Jones, H. Unpublished work cited by G.J. Willard. Proc.
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26.

Klingman, G.C. and G.II. Ahlgren. Effects of 2,*4~D on
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27.

Lynn, G.E. and K.C. Barron s. The Hydrocyanic Acid (IICN)
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Newsom, I . E . and E.W. Stout. Oat Hay Poisoning.
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32'.

Nicholas, D.J.D., A.. Nason aid W..D* M c E lr o y . Molybdenum
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Jour.

33. Nightingale, G.T. The Nitrogen Nutrition of Green Plants..
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3^1.

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iks

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36.

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37.

Pearsall, W.I-I. and M.C. Billimoria. The Influence of
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Die Bestimmung des
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39-

Rogers, C.F. and W.L. Boyd. Sudan Gra,ss and Other Cyanophoric Plants as Animal Intoxicants. Jour. Amer. Vet.
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1+3

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and
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The
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„_______ , __________ 3 _______
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APPENDIX

53

X y le n o l M e th o d f o r N i t r a t e

1.

D e t e r m in a t io n

R e a g e n ts

a*

0,1 f-> NaOH

b.„

112^ 0 ^

- X v o lu m e H2 0 t o 3 v o lu m e s o f c o n c e n tr a te d

H2S0^
c.

II2 S0^_ -

5 v o lu m e s o f

to

1 v o lu m e o f c o n c e n tr a te d

HgSO^
d.

2

%

a c e to n e s o l u t i o n

o f 3,*+ x y l e n o l

(V h y d r o x y 1 ,2

d im e th y lb e n z e n e )
e.

P h o s p h o tu n g s tic a c i d

f.,

A g (N H ^ ) 2 A g2 SQ^.
make t o

g.
2.

C C l^ -

^fO

%

S a tu r a t e c o n c e n t r a t e d ,
C o n c e n tr a te 60 m l.

100 m l. w i t h

to

d is tille d

b o ilin g

NH^OH w i t h

30 m l. b y b o i l i n g

and

H20

D i s t i l l o v e r CaO and f i l t e r *

P ro c e d u re
a.

E x t r a c t sam p le i n
m l. o f

^2^9

W a rin g B le n d o r ,. 1 -1 0 gram s i n

^ o r ^ m in u te s *

m ilk f i l t e r s ,

f o ld e d l i k e

Take a l i q u o t ,

20 m l * ,

2 m l.

F ilt e r

th r o u g h c o t t o n

c o n v e n t io n a l f i l t e r

add 2 m l.

p h o s p h o t u n g s t ic a c i d .

100

H2 S0^ (5 t o

p a p e r.

1 ) and

C e n t r if u g e a g a in and

t a k e an a l i q u o t f o r n i t r a t i o n .

5>+

b.

Add 1 ml. 01 xylenol and add HgSOij. (1 to 3) to 3 times
the volume of the aliquot, ie 5 ml. aliquot ancl 1? ml.
HgSOij...

Put on shaker for 15 minutes at from 35-55°C.

Caution:
tant.

The reaction temperature is extremely impor­

Vi/here total reactant volume is under 10-1? ml.,

it may he necessary to warm the tubes before the 15
minute interval has elapsed.

If the temperature goes

above 55° ? serious loss will be encountered due to
side reactions.
c.

After the 15 minute shaking, dilute with approximately
50 ml..H2O.
funnels.

Cool and transfer to 125 ml. separatory

Extract with two 15 ml. portions of CCli^..

Extract the CCI1+ phase with 50 ml. of 0.1% NaOH and
discard the CCl^ layer.

Filter the aqueous solution

and read in an Evelyn Colorimeter with a M+0 mu filter.
A standard curve is run by carding through known
quantities of KNO^ from step b in the procedure with
the blank used as 100 % transmission.
The xylenol method of determining the nitrate in plant
materials was reported by Rauterberg and Benischke (38) in

191+9 . Ellis (footnote p. 19)? in personal commumication, re­
ported making several modifications in the procedure.

100
90
80

70

Percent Transmittance

60

30

20

18

o
F ig . 1 .

.2 5

.5 0
Mg. o f KNO^ /

.75
5 m l.

1 .0 0
o f s o lu tio n

S ta n d a rd c u r v e o f t r a n s m it t a n c e o f KNO^
s o lu tio n s •

1.25