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ABSTRACT

THE INFLUENCE OF SIMAZINE ON GROWTH
AND NITROGEN METABOLISM OF PLANTS

by James Arthur Tweedy

Research was conducted with several plant species to determine
the factors responsible for the increase in nitrogen content and
growth from applications of 2-chloro-h,6-bis-(ethylamino)-s-triazine
(simazine). Attempts to find a biological assay for this response
with algae and apple seedlings were not successful. Corn proved to
be a good test species for this reSponse when grown in nutrient cul-
tures under low temperature and low available nitrate levels.
Neither the dry weight nor the nitrogen content of corn increased
from simazine applications if nitrate levels or temperatures were
optimum for growth, or if the ammonium ion was substituted for
nitrate in the culture solution.

Simazine did not alter the respiration rate of excised roots
from barley, cucumber, or corn seedlings germinated under low and
high temperatures. Higher nitrate reductase activity was measured in
simazine treated corn plants grown under low temperatures and low
nitrate levels. The leaves of corn plants grown under the same con-
ditions, and subjected to short exposures of I["602 during photosyn-
thesis, contained higher levels of I“c -labeled aspartic and glutamic

acids.

THE INFLUENCE OF SIMAZINE ON GROWTH

AND NITROGEN METABOLISM OF PLANTS

By

James Arthur Tweedy

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Horticulture

I966

ACKNOWLEDGEMENTS

The author expresses his sincere appreciation to Dr. S. K. Ries
for his criticisms and suggestions on the research, and his guidance
and assistance during the preparation of this thesis. Appreciation
is also expressed to Drs. D. R. Dilley, A. L. Kenworthy, H. M. Sell,
and N. E. Good for their guidance and suggestions in editing the
manuscript.

Acknowledgement is also given to Dr. N. E. Tolbert and Dr.

John Hess of the Biochemistry Department for suggestions and use of
laboratory facilities in the photosynthesis experiments. Appreciation
is due to Dr. Philip Filner of the AEC Plant Research Laboratory for
his suggestions and use of laboratory facilities in the nitrate
reductase study.

Special appreciation is due to my wife, Mary, for her encourage-
ment and sacrifices throughout the period of graduate study, and
especially during the preparation of the manuscript.

This research was partially supported by PHS Grant l-ROl-ESOOAB.
The financial support of the Geigy Agricultural Chemical Company is

also gratefully acknowledged.

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS. . . . . . . . . . . . . . . . . . . . . . . . ll
LIST OF TABLES. . . . . . . . . . . . . . . . . . . . . . . . . v
LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . vii

INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . . . l

REVIEW OF LITERATURE. 3
Physical Properties of Simazine. 3
Mode of Action . 3
Metabolism of Simazine . 6

Formation of Hydroxysimazine by Plants . 7

Degradation Studies on Triazine Ring and Alhyl
Side Chain of Simazine. . . . . . . . . . . . . 8
Micro-Organism Metabolism of Simazine. . . . . . . . . l0
Influence of Simazine on Metabolism in Plants. . . . . . . l2
Nitrogen Nutrition . . . . . . . . . . . . . . . . . . . . IS
Summary of Literature Review . . . . . . . . . . . . . . . 18
MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . 20
Preparation of Simazine Stock Solutions. . . . . . . . . . 20
General Analytical Procedures. . . . . . . . . . . . . . . 20
Germinating Seedling Studies. . . . . . . . . . . . . . . 20
ReSpiration Studies. . . . . . . . . . . . . . . . . . . . Zl

Algae Studies. . . . . . . . . . . . . . . . . . . . . . . 22

Growth Studies on Corn and Apple Seedlings . . . . . . . . 23

General Plant Growing Procedures . . . . . . . . . . . 23
Apple Seedling Study . . . . . . . . . . . . . . . . . 2h
Corn Study . . . . .

Table of Contents (Cont.) Page

Nitrate Reductase Study on Corn. . . . . . . . . . . . . . 26
‘hCOZ Fixation Studies on Corn Treated With Simazine . . . 28
Experimental Design and Statistical Analysis . . . . . . . 31
RESULTS AND DISCUSSION. . . . . . . . . . . . . . . . . . . . . 32
Germinating Seedlings Studies. . . . . . . . . . . . . . . 32
Respiration Studies. . . . . . . . . . . . . . . . . . . . 32
Algae Studies. . . . . . . . . . . . . . . . . . . . . . . 37
Growth Studies With Apple Seedlings. . . . . . . . . . . . 39
Growth Studies With Corn . . . . . . . . . . . . . . . . . Ah
Nitrate Reductase Studies With Corn. . . . . . . . . . . . 53
I“(202 Fixation Studies With Corn . . . . . . . . . . . . . 53
SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
APPENDIX. . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

L|TERAmRE C|TEDO O O O O O O O O O O O O 0 O O O I O O C O O 0 61+

Table

I0.

II.

LIST OF TABLES

Dry weight and nitrogen content of corn germinated
at different temperatures in simazine. . . .

Dry weight and nitrogen content of cucumbers ger-
minated at different temperatures in simazine.

Oxygen uptake by intact barley roots as influenced
by three levels of simazine. . . . . . . . . .

Effect of three levels of simazine on oxygen uptake
of excised barley root sections. . . . . . .

Oxygen uptake by corn seeds germinated in three
levels of simazine . . . . . . . . . . . .

Oxygen uptake by excised corn root sections in
simazine solutions . . . . . . . . . . . . . . . .

Influence of simazine on oxygen uptake of germinating
corn seeds measured at two temperatures. . .

Dry weight and nitrogen content of algae grown in a
culture solution containing three concentrations
of simazine. . . . . . . . . . . . . . . .

Influence of simazine on the dry weight and nitrogen
content of algae grown at two concentrations of
ammonium nitrate . . . . . . . . .

Dry weight of algae grown at ISo C in culture
solutions containing simazine. . . . . . . .

Dry weight of algae grown at two temperatures and
treated with simazine at three concentrations.

Dry weight of apple seedlings grown at two tem-
peratures and two nitrogen and simazine
concentrations . . . . . . . . . . . . . . . . . .

Nitrate reductase levels in etiolated corn seedlings
grown in low nitrate levels and treated with
simaZine O O O O O O O 0 O O O O O O 0 O O

Page

33

33

3k

3k

36

36

36

38

38

#2

1.2

1‘3

5h

Table

IA. Dry weight, milligrams nitrogen, and nitrate re-
reductase activity of corn plants grown in
nurtient culture with #2 ppm nitrate nitrogen,
at temperatures of 22. 5° C day and l7. 00 C night,
and treated with two levels of simazine. . . . .

I5. Nitrate reductase activity of corn plants grown in
nutrient culture with 42 ppm nitrate nitrogen,
at temperatures of 22. 5° C day and l7. 00 C night,
and treated with three levels of simazine. . . .

l6. Percent 14C distribution of products in the me-
thanol-water soluble fraction formed by leaves
from simazine greated corn plants during photo-

synthesis in C02 . . . . . . . . . . . .

vi

Page

SA

55

56

Figure

LIST OF FIGURES

The rgsponse of algae to simazine when grown at
'5 Co 0 o o o o o o o

The influence of temperature on the response of corn
plants to simazine grown in low nitrogen solutions

The increase in growth at low temperature of corn
treated with 0.08 ppm simazine when NO is the
source of nitrogen compared to NH“ as he source
of nitrogen. . . . . . . . . . .

The increase in milligrams of nitrogen at low tem-
perature of corn treated with 0.08 ppm simazine
when N03 is the source of nitrogen compared to
NH“ as the source of nitrogen. . . . . . . .

Typical cogn plants grown in low levels of nitrate
at 22.5 c day and 17.0° C night temperatures.

The linear increase in mg of nitrogen with increas-
ing concentrations of simazins in corn plants
grown at 22.5° c day and l7.0 c night with
#2.0 ppm nitrate as the nitrogen source. . . . .

The linear increase in dry weight of corn plants
with increasing concentrations of simazine in
corn plants grown at 22.50 C day and 17.00 C
night with h2.0 ppm nitrate as the nitrogen
source . . . . . . . . . . . . . . . . .

vii

Page

AD

45

47

#8

#9

SI

52

INTRODUCTION

Simazine (2-chloro-h,6-bis(ethylamino)-s-triazine) was first
synthesized by Hofinmwiin l885 (59). Cast _t.gl. (33, 3h) reported
herbicidal activity for this chemical seventy years later. Simazine
is characterized by high toxicity to an extensive range of plant
species when applied to soil, and its persistence in the soil makes
it valuable for long term non-selective weed control.

Since its introduction in I955, simaZine has become one of the
most important selective herbicides for controlling weeds in many
perennial horticultural crops such as fruit trees, brambles, and
woody ornamentals. Research on the effectiveness of simazine as a
herbicide in fruit tree plantings by Ries 33 21- (72) led to the
observation that it induced higher leaf nitrogen and increased growth
compared to fruit trees where the weeds were controlled by hand hoe-
ing or black plastic mulch.

High nitrogen levels in fruit trees may cause poor fruit qual-
ity, thus the observed increased nitrogen level from applications of
simazine to fruit trees was of practical importance.

An extensive review of the literature disclosed no reports of
other herbicides which had a direct effect on increasing plant growth
and nitrogen content when used at recommended rates. IndoIe-3-acetic
acid (IAA) was reported to increase the growth of dwarf pea seedlings

(82), intact rice (Oryza sativa L.) seedlings (53), a silkless mutant

of maize (3i), and cucumber (Cucumus sativus L.) seedlings (50).

 

Tordon (h-amino-3,5,6-trichloropicolinic acid), an auxin type herbi-
cide resulted in shoot elongation in intact cuttings of Mung bean

(Phaseolus aureus L.) (22).

 

The initial phases in these investigations were designed to
study the environmental conditions and nutrient levels necessary for
simazine to increase plant growth and nitrogen level in plants.
Several plant species were grown under various simazine concentra-
tions, environmental conditions, and nutrient levels.

The objective of this research was to determine the mechanism
by which simazine increases plant growth and nitrogen levels in
plants grown in a media which contains non-phytotoxic concentrations

of simazine.

REVIEW OF LITERATURE
Physical Properties of Simazine.

Simazine was one of the first of several triazine compounds
which showed herbicidal activity. The physical characteristics are
an important factor in the herbicidal properties of this substance.
Simazine is colorless, non-combustible, non-explosive, non-corrosive,
molecular weight of 20I.66 and melting point of 225-227°c (55). Its
solubility in water varies from 2.0 ppm at 0°C, and 5.0 ppm at 20°C
to 8A ppm at 85°C. Since simazine has a low water solubility it is
not readily absorbed and translocated out of leaves in the phloem.
0n the other hand, compounds which have relatively high solubilities
often have limited selectivity (l8).

From the viewpoint of the researcher, the solubility of sima-
zine in organic solvents is important for metabolic studies and
quantitative determinations. Its solubility in methanol, ether, and

chloroform is h00, 300, and 900 ppm reSpectively at 20°C.
Mode of Action.

Simazine moves readily through the roots and upward into the
leaves (I9, 68, 77). Almost no absorption occurs through the intact
leaf unless the cuticle is broken (l9). Davis _£”al. (l9) reported
that simazine moved from the roots upward into the leaves of cucumber

in l/2 hour. Sheets (77) detected '“c distributed throughout seedling

oats (Avena sativa L.) within three hours following root application

of lb'C-Iabeled simazine. The same author found that both absorption

of simazine from solution and the upward translocation were greater

at 37° C than at 260 C. He also found that if temperatures were

held constant, the rate of absorption and translocation increased as
the relatively humidity was decreased. This indicates that the rate of
translocation is dependent on the tranSpiration rate.

(When simazine entered the stele of the root it migrated to the
apoplast and moved in the xylem (l8). The movement continued upward
through the xylem into the area of the leaf. When simazine reached
the leaf it apparently accumulated around the edges of wide leaves and
in the tips of grass leaves (19). After it is translocated into the
outer edge of the leaf and begins to accumulate, it enters the living
cells of the chlorenchyma and drastic metabolic changes occur in the
cells of plants that are exposed to light. Plant cells exposed to
light which have been treated with toxic levels of simazine lose
their ability to oxidize water to molecular oxygen (25, 26, 36, 37,
39. 63. 6h).

Exer (26) determined that 7.l0 x lo.7 M concentration of simazine
gave a 50 percent inhibition of the Hill Reaction of isolated corn (Zea
may; L.) and Spinach (Spinacia oleracea L.) chloroplasts. Moreland
ggflgl. (63) found that simazine reduced the photochemical activity of
isolated barley (Hordeum vulgare L.) chloroplasts by 50 percent at h.6
x I0"6 5, The exact mechanism by which simazine inhibits the oxida-

tion of water and prevents oxygen production is not known. Good (37)

concluded that the site of inhibition cannot be chlorophyll unless

there is one uniquely situated chlorophyll molecule among hundreds or
thousands. However there may be a few uniquely situated chlorophyll
molecules which are associated with enzymes or substrates in a manner
which permits them to act as traps for excitation energy.

Various studies were conducted to determine the orientation of
the triazine molecule at the reactive site(s) within the chloroplast
(36, 6A). These sites of reaction may involve the chlorine attached
to the number-two carbon of the ring and the imino hydrogen of the
alkylamlno radicals attached to the number-four and number-six carbon
atoms. Moreland and Hill (6h) found that replacement of the chlorine
by a methoxy radical weakened the inhibitory action expressed against
the Hill Reaction. The 50 percent inhibitory values were increased

6

from 5.9 x IO- '5 for simazine to l.75 x IO'5 M for methoxy sima-
zine. They postulated that the electronegative chlorine atom formed
a hydrogen bond at or near an active site within the chloroplast, and
the methoxy radical probably did not participate in hydrogen bond
formation.

The role of the alkylamino groups in herbicide activity of
simazine was also studied (36, 6h). The replacement of one or both
of the imino hydrogens with an alkyl group resulted in a loss of in-
hibitory activity (6h). This indicated the participation of the imino
hydrogens in the formation of hydrogen bonds with constituents at or
near the reactive sites. However, Good (36) attempted to relate in-

hibitor potency to the bonding tendency of the imino hydrogens and

was only partially successful. He concluded that the role of hydrogen

bonding in the mechanism of inhibition remained uncertain.

Ashton _£_§l. (3, h) made a detailed study on the structural
changes in Phaseolus vulgaris L. induced by atrazine (2-chloro-h-
ethylamino—6-isopropyl-s-triazine) which has a mode of action similar
to simazine. They found disintegration of the chloroplasts of devel-
oping leaves and mature primary leaves in plants kept in the light
which had been treated with atrazine. The chloroplasts of plants
which were treated with atrazine but maintained in the dark were un-
affected. The following changes occurred in the chloroplasts of
atrazine treated plants: (a) they became spherical rather than
discoid; (b) starch disappeared from the lamellar system; (c) the
frets or parts of frets were destroyed, leading to the disorganiza-
tion of the general arrangement; (d) the compartments of the grana
swelled; and (e) the envelope and the swollen compartments ultimately
disintegrated. They propose that these changes are brought about by

the formation of a toxic substance or substances involving the inter-

action of atrazine and light in the presence of chloroplasts.

Metabolism of Simazine.

Once it had been established that simazine was an effective
herbicide, studies were initiated to determine the nature of its selec-
tivity. Both resistant and susceptible plant species absorbed con-
siderable quantities of simazine applied to their roots (I9, 69). As

t al. (l9) and Sheets (77) selectivity to simazine

indicated by Davis

was not a consequence of differences in absorption and translocation;

rather selectivity resulted from the ability of some plant species to
convert the substance to a non-phytotoxic metabolite. Several detoxi-
fication mechanisms have been proposed, and recent studies have indi-
cated mechanisms differing from early studies as to how this herbicide
is metabolized in the plant. The following discussion presents the
present knowledge of simazine metabolism by plants. Reference is
made occasslonally to studies with atrazine, which is degraded by a
process similar to simazine.

Formation of Hydroxysimazine by Plants

In I957, two years after the introduction of simazine, Roth (73)
reported that freshly pressed juice of resistant maize deactivated
simazine, whereas that of susceptible wheat (Triticum aestivum L.)
was not effective. He concluded that the resistance of corn to sima-
zine was due to an enzyme system which changed the herbicide into a
non-biologically active product. Later studies established that sev-
eral plant species converted simazine to hydroxysimazine (2-hydroxy-h,
6-bis(ethylamino)-s-triazine) which was not phytotoxic (2, IS, 16, 27,
Ah, #5, #6, 6l, 65, 66, 75). Roth and Knusli (75) isolated a cyclic
hydroxamate which occurred naturally in some plants and catalyzed the
formation of hydroxysimazine from simazine. This substance was 2,4-
dihydroxy-7-methoxy-I,h-benzoxazine-3-one, or its glucoslde.

Studies, using inbred lines of corn which vary in their tolerance
to simazine, indicated that the conversion to hydroxysimazine cata-
lyzed by the benzoxazinone compound was not a basic factor in toler-

ance (2, #4). The levels of benzoxazinone derivatives in the inbred

line of corn and in wheat were comparable, but corn was more resistant
to simazine than wheat. In a study by Palmer _£_§l. (66) with two
isogenlc corn lines, one susceptible to simazine and atrazine and one
resistant, both roots and shoots contained the benzoxazinone com-
pound. The content in roots was about equal for the two lines and
decreased with age. They concluded the resistance or susceptibility
of these two species was not due to absence or presence of the cyclic
hydroxamate. Hamilton (#5) could not show direct correlations between
resistant species and the presence of benzoxazinone derivatives even
though the content of this substance was directly related to the abil-
ity of excised roots to form hydroxysimazine.

Degradation Studies on Triazine Ring and Alkyl Side Chain of

Simazine

The previous discussion indicated that, in resistant species,
simazine is first degraded in 21x2 to the 2-hydroxy derivative by a
cyclic hydroxamate. However, some species of sorghum that are moder-
ately resistant to simazine contained no benzoxazinone compounds and
did not form hydroxysimazine (#5). Wheat and rye (Secale cereale L.)
contained benzoxazinone compounds and yet were extremely susceptible
to simazine.

th ring- and chain-labeled

Several studies were conducted using
simazine. Funderburk and Davis (3) reported '“c metabolites present
in plants supplied with ring- or chain-labeled simazine. They found

that corn, cotton (Gossypium hirsutum L.), and soybeans (Glycine max L.)

yielded appreciable amounts of I“C02 when grown in solutions contain-
ing chain-labeled simazine. In another study, corn plants were
placed in a saturated solution of ring labeled simazine, and 63 per-
cent of the total radioactivity was metabolized within 3 days (39).
Ragab and McCollum (69) grew a resistant and a susceptible plant
species in ring-labeled simazine and measured considerable quanti-
ties of 1“C02 released by the plants. The importance of ring cleav-
age ln detoxifying simazine is not known.

There are a few reports concerning dealkylation of side chain
constituents in the metabolism of herbicides in plants. Geissbfihler
gghgl. (35) reported demethylation of monuron (3-(h—chlorophenyl)-l,
dimethylurea) by micro-organisms and plants as a major pathway for
the degradation of the herbicide. As was previously mentioned,
Funderburk and Davis (30) found th02 released from plants treated
with chain-labeled simazine. A recent study with atrazine on mature
peas (£132! sativum L.) indicated that the major process of degrada-
tion in shoots was not hydroxylation at the 2-position, but rather
the dealkylation of the ethyl side chain (78). Montgomery and Freed
(6i, 62) proposed the following mechanism for the degradation of
triazine herbicides by plants which did not involve the degradation

of the alkyl side chain.

X 0H
* Cs‘r
(A. ,/\N a w
i It I II ——9 01:02 + RI-N-d*’N'c*‘N'R2
Ri'II-* *-N-R2 Rl-N—ci c*_”_R2 l
H \/ H \/ H
N N RI'III'E*'N-g*-N-R2
H H H

Micro-Organism Metabolism of Simazine

The persistence of simazine in the soil for relatively long per-
iods and its effects on micro-organisms has concerned many investi-
gators. Early research in this area indicated that deactivation of
simazine occurred under conditions conducive to micro-organism growth,
but the rate of breakdown was fairly slow (l2, 79). Burschel (l3)
found that a decrease in temperature from 25°C to 8.5°C caused a
seven-fold reduction in the rate of decomposition. He also found
that without humus, no simazine decomposition occurred and an in-
crease in the pr0portion of humus in the soil increased the intensity
of breakdown. From this he concluded that the breakdown is due to the
activity of micro-organisms.

Kaufman _£‘gl. (SI) reported that fungi were the predominant
organisms capable of degrading simazine. Among them were Aspergillus
flavipes, Aspergillus fumigatus, Penicillium purpuragenum, Fusarium
monillforme, Fusarium oxysporum, Rhizopus stolonifer, Stachybotrys sp.,

Trichoderma viride, three species of streptomyces, and four bacteria

 

*Denotes Inc-labeled carbon.

ll

believed to belong in the genus arthrobacter. Simazine at a con-
centration of 5 ppm as the sole source of carbon in a culture solu-
tion, was almost completely degraded in l2 days by Aspergillus
fumigatus. In these studies, IQCOZ was evolved only from culture
solutions containing chain-labeled simazine. No ‘hCOZ was evolved
from culture solutions containing ring-labeled simazine.

Kearney g5 31. (52) reported that Aspergillus fumigatus Fres.
degraded 36Cl-labeled simazine to at least two metabolites. One of
these was identified as 2-chloro-h—amino-6eethylamino-s-triazine.
The second 36CI-Iabeled metabolite possessed an intact s-triazine
ring but no alkyl substituents were associated with the ring. They
proposed a new pathway of metabolism for simazine which did not in-
volve the hydroxy analog reported to occur in higher plants.

MacRae and Alexander (58) detected no 1“

C02 microbiologically
released from soil receiving I“During-labeled propazine, atrazine, or
simazine. They concluded that these compounds were not decomposed
by micro-organisms. Their data on ring-labeled triazines agree with
that of Kaufman _£.§l, (37) and Kearney'_£‘gl. (52). MacRae and
Alexander did not use the side chain-labeled material. Thus, metabo=
lism by dealkylation would not be evident from soil treated with ringw
labeled triazine herbicides.

Other studies have been conducted to determine the influence of
simazine on the growth of some species of soil fungi. Chandra (l7)

concluded that pre-emergence applications of four herbicides, includ-

ing simazine had minimum influence on subsequent microbial activities

l2

affecting plant growth. He also noted that simazine, in particular,
at a concentration of 5 ppm temporarily increased the carbon dioxide
evolution l2 to lh percent in two soil types. Burnside _£.§l, (I2)
reported that high applications of simazine did not affect the nitri-

fication process of the soil or over-all microbial activities in a

Waukegan silt loam soil.
Influence of Simazine on Metabolism in Plants.

As previously indicated, simazine was absorbed and translocated
in both resistant and susceptible plant species. This section will
be devoted primarily to the influence of simazine on plant metabolism.

In I957, two years after the introduction of simazine as a
herbicide, Bartley (9) reported observations by several research
workers that corn growing in simazine treated soil was larger in
size, greener in color and produced higher yields than adjacent plots
treated with other herbicides.

In I963, Ries._£‘gl. (72) showed that peach and apple trees,
growing in a soil sprayed with simazine had a higher leaf nitrogen
content and more terminal growth than trees grown in a weed free
environment. They suggested that the herbicide treatment applied
to the soil and not the plant affected nitrogen uptake or nitrogen
metabolism. Similar observations on fruit trees have also been

made by Karnatz (49). Hadoml (#0) observed an increase in number

and size of apple fruit when simazine had been applied.

l3

DeVries (20) studied the effect of simazine on Monterey pine and
corn as influenced by lime, bases, and aluminum sulfate. He concluded
that simazine increased top-root ratios of corn but generally decreas-
ed those of pine seedlings. Simazine increased the uptake of nitrogen
by corn In all soils, of magnesium and phOSphorus in Iimed soils, and
potassium in acidified soil.

Ries and Cast (7i) reported that the addition of simazine to
nutrient solutions of corn increased the percent and total amount of
nitrogen in one test, regardless of the nitrogen level in the solu-
tion. In a second experiment, under environmental conditions more
favorable for corn growth the total quantity of nitrogen was not in-
creased although the percent nitrogen in the shoots was increased at
the low nitrogen level. They concluded that simazine effects on corn
plants are greatest on plants grown under adverse environmental con-
ditions. Freney (29) also reported increased growth and uptake of
nutrients by corn plants treated with low levels of simazine. Sima-
zine applied at 0.06 ppm in solution culture increased the yield of
corn shoots, uptake of nitrogen, phosphorus, magnesium, and potassium.
It had no effect on the roots. He found that soil incubated with
simazine did not increase mineralization of soil organic nitrogen,
nor did it have any effect on immobilization of nitrogen.

There were several reports in the literature on the influence
of triazine herbicides on physiological processes in plants. These
included studies on carbon dioxide fixation (5, 7, 85), sucrose and

serine metabolism (6), respiration (l, 6, 2l, 7k), and light

lh

reactions in photosynthesis (32, 36, 37, 63, 7h).

Zweig and Ashton (85) and Ashton t al. (5, 7) found that fixa-

tion of ”*co2 was drastically inhibited in the light by atrazine at
concentrations of I and ID ppm. Although atrazine disrupted the
photosynthetic apparatus, it had no effect on non-photosynthetic C02
fixation. Ashton _£'al. (6) also studied the influence of atrazine
on sucrose and serine metabolism. They found that in light, with
sucrose as a substrate, atrazine caused a decrease in serine, alanine,
and glyceric acid biosynthesis with a corresponding increase in aspar-
tic acid and glutamic acid in red kidney bean leaves. However, with
untreated plants kept in darkness, serine and glyceric acid biosyn-
thesis was decreased and aspartic acid was increased. In the dark,
atrazine did not affect the metabolism of Ib’C-labeled sucrose in red
kidney bean leaves. Since darkness caused no decrease in alanine or
an increase in glutamic acid synthesis as atrazine did, perhaps other
metabolic processes entirely independent of blocking photosynthesis
are responsible for these differences.

The influence of triazines on respiration has not been clearly

established. Increases in respiration from simazine applications

have been reported in barley roots (I) and elodea (Anacharis canadensis

 

Planch.) (7h). Eastin _E El. (2l) reported that root respiration
decreased in an atrazine treated resistant corn variety and there
was no effect on respiration in a variety susceptible to atrazine.

Ashton and Uribe (6) reported that atrazine did not affect the rate

of respiration of excised red kidney bean embryos.

Simazine is a potent inhibitor of photosynthesis (25, 26, 32,
36, 37, 63, 74). Most studies of its effects on photosynthesis were
conducted on seedling plants to determine the concentration which
gave a 50 percent inhibition of the Hill Reaction. Little information
is reported concerning its influence on photosynthesis in mature re-
sistant plants when applied at non-toxic levels. In the studies where
growth was stimulated by simazine, there were no studies designed to
investigate the process of photosynthesis other than total dry weight
increases of plants. Freeland (28) reported increases in both photo-
synthesis and respiration for several hours on bean leaves by the
application ofraphthoxyacetic acid. Good (38) noted dry weight in-
creases of bean leaves floated in low concentrations of several

photosynthetic inhibitors.
Nitrogen Nutrition.

The known chemical pathway of plant nitrogen metabolism may be
divided roughly into four areas; (a) the assimilation of nitrogen,
(b) the formation and interconversion of amino acids, (c) the syn-
thesis of amides, peptides, and other simple nitrogenous substances,
and (d) the formation and degradation of proteins and nucleic acids
(83). The nitrogenous substances assimilated by plants are divided
into four major classes: organic nitrogen, ammonia nitrogen, nitrate
nitrogen and molecular nitrogen (83). This section will be limited
to a brief review of the assimilation of nitrates and ammonium salts

by plants.

l6

Most plants utilize both nitrates and ammonium salts as nitro-
gen sources. However, most of the nitrogen is absorbed in the form
of nitrates (60). When ammonium fertilizers are applied to most
agricultural soils, soil nitrification results in rapid oxidation of
the ammonium ion to nitrate. The ammonium ion is utilized readily
by plants because it is in a highly reduced form and can be incor-
porated directly into amino acids without further chemical change.
Unlike nitrate ions, ammonium ions seldom aCCUmulate in plants.

Since nitrate nitrogen is in a highly oxidized state, while in
amino acids and other organic compounds nitrogen is in a highly re-
duced state, it is evident that nitrate must be reduced preceding
amino acid synthesis. There is much evidence that nitrate reduction
proceeds in a stepwise manner as follows:

N03 ——> N02 ——> HNO —> NHZOH __) NH3
Evans and Nason (23, 2#) isolated enzymes from higher plants and
NeurOSpora which catalyze the reduction of nitrate to nitrite. This
reaction preferred nicotinamide-adenine dinucleotide phosphate (NADPH)
as an election donor. In another study, young corn plants placed in
complete darkness for #8 hours lost 90 percent of their nitrate re-
ductase activity (#2). The activity was quickly restored when the
plants were returned to the light. Vanecko and Varner (8l) found that
ISN-nitrite is reduced to amino nitrogen by intact leaves in both
light and darkness, and that the rate of reduction in the light was
I0 to 20 times that in darkness. Their results indicated that nitrite

is reduced to the level of ammonia and the photochemical splitting of

17

water provided the reducing power for the reaction.

Later studies have shown that light effects on nitrate reductase
are indirect (I0, #I, 56, 67, 70, 76). Beevers _t al. (I0) concluded
that light increased uptake of nitrate as a result of increased per-
meability and also increased the rate of synthesis of reducing power.

Nitrate reductase is an inducible enzyme which requires the
presence of nitrate (l0). Neither nitrite nor ammonia induced the en-
zyme. Induction of nitrate reductase was roughly prOportional to the
amount of nitrate present In the plant tissue (l0, l#). Therefore
any factors which increased the rate of absorption, or availability
of nitrate, resulted in more enzyme in the tissue.

2 Upon reduction of nitrate to ammonia, the ammonia was fixed by
three major reactions, the synthesis of glutamic acid, glutamine, and
carbamyl phosphate, respectively (8#). In some micro-organisms and
plants, alanine or aspartic acid formation was substituted for that
of glutamic acid. These compounds served in plant and microbial cells
as precursors of all the other amino acids, since the alpha-amino
group of glutamic or aspartic acid were transferred to other alpha-
keto acids by transamination.

Much of the research on nitrogen nutrition was conducted using
corn as a test crop. Corn is a warm season crop which grows better
at 27°C than at 2l°C and temperatures as low as l5°C often retards
growth, flowering and maturation (#8).

Corn plants which have been supplied nitrate as nitrogen source

and grown in light contain relatively large quantities of nitrate

l8

reductase (l0, #2, #3). Beevers _t‘gl. (l0) reported that induction
of nitrate reductase was temperature dependent, and a part of this
dependence was based on the increase in nitrate content of the tissue
with increase in temperature during induction. The maximum induc-
tion temperature for enzyme activity in corn seedlings was 38°C and

corn plants grown at 250C lost approximately 50 percent of their en-

zyme activity.

Summarygof Literature Review.

Most investigators agree that simazine inhibits the growth of
plants by blocking the light dependent oxido-reductlon reactions of
the chlorOplast, specifically the oxidation of water to molecular
oxygen.

Resistant plants can degrade simazine to a non-toxic metabolite,
although the pathway of degradation is not fully understood. It was
originally proposed that resistant species could degrade simazine to
hydroxysimazine in a reaction catalyzed by a naturally occurring
benzoxazinone compound or its derivative. In a study on peas treated
with atrazine, dealkylation of the side chain, and not conversion to
hydroxyatrazine was considered as the basic factor in tolerance.

It has also been reported that many of the common soil fungi and
bacteria dealkylated simazine by the same pathway as that proposed
for atrazine by peas. Based on these results, plant metabolism stud-
ies without considering micro-organism activity might be questioned.

Increases in growth and nitrogen content have been observed in

l9

several plant species grown in soils or nutrient cultures which contain
low levels of simazine. Little information is available about the
growth promoting properties of this material. The greatest response

is usually observed under low available nitrogen conditions. In one
study, it was suggested that plant reSponse to simazine stimulation

of growth and nitrogen content, was greatest under adverse environ-
mental conditions, but these conditions were not studied in any great
detail. No reports are available concerning the influence of sima-

zine on nitrogen nutrition in plants.

MATERIALS AND METHODS
Preparation of Simazine Stock Solutions.

Aqueous stock solutions of simazine were formulated at 5.0 parts
~per million (ppm). These solutions were prepared by dissolving 50
milligrams (mg) of simazineI per l00 milliliters (ml) of chloroform.
Aliquots of‘the chloroform-simazine solution were added to the aqueous
stock solution to give a final concentration of 5.0 ppm. The chloro-
form was evaporated from the solution by stirring over heat (approxi-
mately 80° C), and the final concentration of the solution was

determined by aliquot procedures.
General Analytical Procedures.

.-The plant tissues were dried in a forced air oven at 80° C. The
dried plant tissues were weighed on a model H Mettler analytical bal-
ance.and ground in a Wiley Mill through a #0 mesh screen. Nitrogen

was determined by the micro-Kjeldahl procedure (8).
Germinating Seedling Studies.

To determine the influence of simazine on the nitrogen content
and dry weight of seedlings, ten seeds of corn (cultivar, Gold
Cup) and 20 seeds of cucumber (cultivar, Wisconsin SMR-IB) were
placed in a single Petri dish containing 2 Whatman No. I filter papers

saturated with 7 ml of treating solution which contained simazine at

 

1/ Simazine, 99 percent active obtained from Geigy Agricultural
Chemicals, Ardsley, New York.

20

2I

concentrations of 0, 0.0l, 0.l0, and l.0 ppm formulated in a l x
lo.“ M CaCIz solution. Since there was little evaporation from
the closed Petri dishes, no additional solution was added during
the course of the investigation. The seeds were germinated in
darkness at temperatures of 2#0 C for l6 hours and IB0 C for 8 hours
in one chamber, and 2#° C for l6 hours and I30 C for 8 hours in the
other chamber. These temperature regimes were maintained for 5 days.
After the treatment period, the plants were dried, weighed and ana-
lyzed for nitrogen.

This experiment was repeated twice on corn, employing a greater
range of simazine concentrations and temperature treatments. Seven
days after treatments the seedlings were divided into shoots, roots

and seeds, measured, dried and weighed.
Respiration Studies.

Seeds of barley (cultivar, Trail) and seeds of corn (cultivar,
Michigan #00) were germinated in No. 7 Wausau quartz sand and watered

daily with a l x lo’“

,M CaClz solution containing simazine. When
the roots were approximately 5 cm long, the seedlings were washed
free of sand and respiration of seedlings, or excised roots was
determined.

Respiration rates were measured by placing the plant tissues
into a 20 ml Warburg respirometer flask containing # ml of a treating

solution and 0.2 ml l0% (w/v) KOH in the side arm for C02 absorption.

Respiration as indexed by oxygen consumption was determined

22

manometrically according to the procedure of Umbreit _t'_l. (80).
The plant tissues were dried and weighed, and the data were expressed
as micro-liters oxygen consumption/mg dry weight/unit time.

Each Warburg flask was considered a replicate. The replicates

were maintained separately throughout germination and growth periods

in order that a randomized complete block design could be utilized.

Algae Studies.

Stock and treated solutions of algae (Chlamydomonas reinhardtii)
were cultured in a nutrient solution (Appendix, Table I) in 250 ml
Erlenmeyer flasks fitted with air inlets: these were placed on a
reciprocating shaker of about 60 excursions per minute, thus providing
a gentle but thorough agitation of the loo ml of medium. The cells
were cultured in a controlled environment chamber which maintained a
temperature in the culture medium of 20° C. The chambers provided
I500 foot candles of continuous light from fluorescent and incandes-
cent bulbs. For aeration of the medium, filtered air was mixed with
C02 by bubbling both through distilled water at rates which gave a
final C02 concentration 0.5 percent.

The concentrations of simazine in the treating solutions were:
0, .Ol, .IO, and I.O ppm. These solutions were innoculated with
algae at a population in which the light transmittance at 670 milli-
microns on a Bausch and Lomb spectrophotometer was 95 percent. It
was established in preliminary experiments that the growth rate of

.algae under the conditions of these experiments was logarithmic from

23

l2 hours to 72 hours after innoculation. All harvests were made
at 68 to 7# hours after innoculation.

The algae were harvested by centrifugation in a Model HR-I
International High Speed Refrigerated Centrifuge at l0,876 x g.
After centrifugation, the supernatant was poured off and the
pellet of packed cells was washed with a small amount of water
into a tared watch glass. The cells were dried, weighed, and
analyzed for nitrogen.

The effect of simazine on algae growth at two nitrogen levels
was studied. The algae were cultured and harvested as in the pre-
vious experiment. The concentrations of simazine in the culture
solutions were: 0,and 0.05 ppm in one experiment, and 0, 0.0l,
0.05, and 0.l0 ppm in a second experiment. The concentrations of
nitrogen were I75 and 700 ppm actual nitrogen as ammonium nitrate.

Another study was initiated to determine the effect of sima-
zine on the growth and nitrogen content of algae when grown at
different temperatures. The algae were cultured, harvested and
weighed as in the first experiments. The concentrations of simazine
in the culture solutions were: 0, 0.05, 0.l0, and 0.20 ppm. The

temperatures of the solutions were l2° C and l5° C
.growth Studies on Corn and Apple Seedlings.

General plant growing procedures. Seeds of apple (Pyrus malus L.

cultivar, McIntosh) and corn (cultivar, Michigan #00) were used in

2#

determining the influence of simazine on growth and nitrogen content.
The stratified apple seeds were planted in Arcillitel and when the
cotyledonary leaves were well developed, transplanted into l0 cm clay
pots containing No. 7 Wausau quartz sand. The corn seeds were planted
embryo down in No. 7 Wausau quartz sand. When the coleoptiles were
6-8 cm tall, the seedlings were carefully washed from the sand with
running tap water and the endosperm was removed. Results from pre-
liminary studies showed that the endosperm could be removed from the
seedling without permanent damage to the plant, and by this method
nitrogen deficiency could be induced at an earner stage in growth.
The seedlings were then transplanted into l0 cm clay pots containing
No. 7 Wausau quartz sand, and watered daily with the nutrient solu-
tions at different simazine concentrations.

The plants were grown in controlled environment chambers. The
chambers provided a light intensity of 2,000 foot candles from fluor-
escent and incandescent bulbs for l6 hours, followed by an 8 hour dark
period.

Apple Seedling Study. The air temperatures were maintained at
20° c day and 15° c night in one chamber, and l5° c day and l0° c night
in the other chamber. A nutrient solution (Appendix, Table 2) was
applied to the plants immediately after transplanting, which contained
simazine at concentrations of 0, 0.l0, and 0.50 ppm. The plants were

harvested 28 days after the initial simazine treatments.

 

.l/ A montmorollonite clay which has been calcined at high temper-
atures, and sold under the brand name of ”Turface.”

25

Corn Study. Air temperatures were maintained at 28.00 C day

 

and 22.5° c night in one chamber, and 22.5°c day and l7.0° c night
in the other chamber. A nutrient solution (Appendix, Table 2) was
applied to the plants immediately after transplanting which con-
tained simazine at concentrations of 0, 0.05, and 0.l0 ppm. The
plants were harvested 2] days after the initial treatments.

In a second experiment, the influence of simazine on the amount
of nitrogen and dry weight of corn grown at two temperatures using
nitrate and ammonium nitrogen forms was studied. Culture solutions
(Appendix, Table 3) were formulated that contained nitrogen supplied
as nitrate ion in one, and ammonium ion in the other, with concentra-
tions of 53 and l05 ppm actual nitrogen for each solution. All plants
were grown in their respective culture solutions for l# days at a
temperature of 28.0° C day and 22.50 C night. Folling this period,
the plants had attained a height of I5-20 cm. Plants of uniform size
were selected for replicates, and treated with simazine at concentra-
tions of 0 and 0.08 ppm. The temperatures remained the same in one
chamber and were dropped to 22.5° C day and l7.0° C night in the other
chamber. The plants were harvested 8 days after the simazine treatments
were initiated.

A similar experiment was designed to study a wider range of sima-
zine concentrations on the amount of nitrogen and dry weight of corn
plants which were grown in a culture solution containing nitrate ni-
trogen at a concentration of 53 ppm (Appendix, Table 3). The environ-

mental growth conditions and time of simazine treatment were the same

26

as In the previous experiment. Simazine was applied through the
nutrient solution at concentrations of 0, 0.05, 0.l0, and 0.20 ppm.
The plants were harvested 9 days after the simazine treatments were

initiated.
Nitrate Reductase Study on Corn.

Etiolated corn seelings were produced by planting corn seeds
(cultivar, Michigan #00) embryo down in No. 7 quartz sand and watered
with l x l0.°fl,CaCl2 solution. The seeds were incubated for 36 hours
at 27° C. After 36 hours, the temperature was dropped to 22° C and
the seeds were watered with a nutrient solution (Appendix, Table 3)
containing nitrate nitrogen at #2 ppm actual nitrogen and simazine
concentrations of 0, 0.05, 0.l0, 0.20, and I.0 ppm. When the coleoptiles
were 7-8 cm long, the etiolated seedlings were placed in the light 2#
hours before enzyme extraction. Three week old corn plants which had
been grown at 22.5° C day and l7.0o C night temperatures in a culture
solution containing #2 ppm nitrogen as nitrate were treated with sima-
zine concentrations of 0. 0.I0, and 0.20 ppm 7 days before enzyme ex-
traction. Each treatment was run in triplicate, with a replicate con-
sisting of 6 etiolated seedlings in the first study, and single plants
comprised replicates in the second study.

The crudeenzyme preparations were made by homogenizing l gram
of fresh tissue in 5 ml of 0.l fl trishydroxymethylamino methane (tris)

plus 0.00I fl_cysteine pH 7.5 in a previously cooled mortar and pestle

(“5° C) In the cold room (0 to +#° C). The homogenates were centrifuged

27

in a refrigerated Sorvall ss 34 rotor (o to +u° c) at l0,000 x g for
20 minutes. The supernatant was poured into test tubes which were
immediately placed in an ice bath until they were assayed for nitrate
reductaseoactivity.

The crude enzyme preparations were assayed by the following
procedure. Five tenths ml of 0.l fi_potassium phosphate pH 7.5, 0.l

ml of O.I‘fl KNO O.I ml of deionized distilled H20, O.I ml of 0.00l fl

3.
NADH, and 0.2 ml of the enzyme extract were added to a l3 x l00 mm

test tube. The order of addition of the reagents was as listed. These
solutions were incubated in a water bath at 25° C. Duplicate samples
of each extract were run, one was stapped at 0 time, and the other

was incubated for 20 minutes in the water bath. The 0 time served as

a blank for determining the quantity of nitrite produced during the

20 minute incubation period. The reaction was stopped by adding I ml
of I percent sulfanilamide in 3.N HCI. One ml of 0.02 percent N-l-
naphthylethylenedlaminedihydrochloride was added to form the colored
azo compound. After the color had developed completely (l5 to 20
minutes) the samples were centrifuged for ID minutes in a clinical
centrifuge at I720 x g. The optical density (0.0.) was measured with

a Beckman DU spectrophotometer at a wave-length of 5#0 millimicrons.

A standard curve was run, and 0.0l3 0.0. units equaled l millimicro

mole nitrite. Protein determinations were made according to the

method of Lowry _e_g a_l_. (57).

28

11*C09 Fixation Studies On Corn Treated With Simazine.

Corn seeds (cultivar, Michigan #00) were germinated embryo down
in No. 7 quartz sand. Ten days after planting, the seedlings were
washed free of sand with running tap water and the endosperm was
removed from the germinated seedling. The plants were transplanted
into 500 ml glass jars containing a nutrient solution which had been
supplied with #2 ppm nitrogen as potassium nitrate (Appendix, Table 3).
The jars containing the plant specimens were placed into controlled
environment chambers which maintained the temperatures at 28.0° C for
a l6 hour day and 22.5° C for an 8 hour night. The light intensity
during the day was 2,000 foot candles provided by fluorescent and
incandescent light. The nutrient solutions were vigorously aerated
from a central air supply which was filtered through cotton and dis-
tributed to each jar. These solutions were changed every #8 hours
in order to maintain relatively constant nutrient levels.

Ten days after transplanting, the temperatures were dropped to
22.5° c day and l7.0° c night. The plants were treated with o.l ppm
and 0.5 ppm simazine in the nutrient solution. Five days after treat-
ment with simazine the fixation studies with ILACOZ were conducted.

An apparatus had been designed for rapid equilibration of the
atmosphere with added C02 by vacuum infiltration. This air tight
reaction chamber was constructed from plexiglas with internal dimen-
sions of II cm x l5 cm x l.7 cm. One end was open and provided with

a removal cover which had two holes I.5 cm in diameter. These holes

29

were designed to hold a stopper with a stopcock attached to a vacuum
pump in one, and another stOpper with a 60 ml separatory funnel in

the other. The separatory funnel contained 0.2 mC of Ba1°C03 (specific
activity I mC/8.l mg) and 2 ml of 5(g lactic acid was injected through
a serum cap on the top of the separatory funnel to release theyll‘CO2
before the experiment was started.

A 300 watt Champion flood lamp placed perpendicular to the plexi-
glas chamber in a ventilated hood provided #,000 foot candles of light
inside the plexiglas chamber as determined by a Weston photometer.

A water bath with 3 inches of distilled water separated the lamp from
the reaction chamber. The temperature in the chamber under the light
. was 22° c.

(Plants were removed from the growth chamber as needed. Twelve-cm
sections from the tips of the 3rd, #th, and 5th leaves were cut and
placed into the plexiglas chamber as quickly as possible. The chamber
was immediately subjected to a slight vacuum (75-90 mm Hg.) and placed
under the light. The stopcock of the funnel containing the liberated
1°°°2 was Opened, and at the same time a second operator removed the
serum cap from the separatory funnel so the 'hcoz would be swept Into
the chamber. At the end of the 1“C02 exposure period the lid to the
chamber was removed and 200 ml of boiling absolute methanol was poured
into the chamber. The length of time for the entire reaction on a
60 second exposure to the l°°°2 was less than 5 minutes. There was

no visible wilting or dessication of the leaves during this short time

30

period. The methanol and tissue were transferred to a #00 ml beaker
and boiled on a hot plate in the hood for l0 minutes. The methanol
was decanted from the leaves and 30 ml of water was added to the
leaves and boiled for 5 minutes. The methanol and water extracts
were combined and the volume adjusted to 200 ml with methanol. One-
half ml of glacial acetic acid was added to the combined extracts to
lower the pH below 5.0. Non-radioactive CO2 was bubbled through the
solution for 5 minutes to remove any 1°C02. One-tenth ml aliquots were
taken from the combined extracts and placed into l5 ml of a solution
formulated for radioactive determination of aqueous mixtures in a
liquid scintillation spectrometer (5#). The quantity of radioactive
carbon was determined in a Packard Model 3003 Liquid Scintillation
Spectrometer. The counting efficiency was determined by adding a
known quantity of thfimfluene Packard standard solution to the count-
ing solution containing O.I ml of a methanol-water plant extract.

The data were converted to disintegrations per minute. The extracted
leaf tissues were dried and weighed.

Two dimensional chromatography according to the method of Benson
,_£'gl. (ll) of these extracts was employed to determine the distri-
bution of products of the 11"C which was fixed by the excised leaves.

A l5 ml aliquot of the methanol-water extract was evaporated to 0.5 ml
on a rotary evaporator under reduced pressure at 300 C. Approximately

0.2 ml of extract, which provided a sufficient quantity of radioactivity

for chromatography, was spotted on Whatman No. l paper for chromatography

3l

and the chromatogram was developed in one direction with water
saturated phenol and in the second with a l:l ratio of butanol-
prOpionlc acid containing water (l2#6 ml butanol plus 8# ml

H20-620 ml prOpionic acid plus 790 ml H20). After the chroma-
tograms had dried for approximately l2 hours they were sprayed

with NaHC03 to form sodium salts of organic acids tc> prevent vo-
Iatilization. The chromatograms were exposed to Kodak no-Screen
X-Ray film for l# days. The X-Ray films were developed and served
as a guide for determining the radioactive spots on the chromatogram.
The radioactive spots were counted with a thin window gas flow coun-
ter using a Nuclear Chicago Scaler (Model I6IA). Helium was bubbled
through a gas dispersion tube in ethanol at 0° C to provide the gas

for the counting chamber.

Experimental Design and Statistical Analysis.

A randomized complete block design was employed in all of the
simple and factorial experiments. The data were analyzed by analysis
of variance and whenever a significant F value was obtained for main
effects or interactions, they were further partitioned into single
degree of freedom for comparison of individual means.

With certain studies, it was desirable to calculate the coeffi-
cient of correlation and the linear regression equation. The data
from these studies are presented in graph form with a line drawn based

on the regression equation.

RESULTS AND DISCUSSION

Germinating Seedlings Studies.

Simazine did not affect the dry weight or percent nitrogen of
corn and cucumber seedlings grown under two different temperature
regimes (Table l and 2). As expected, an increase in respiration
from increasing the temperature resulted in a decrease in dry weight

and an increase in the percent nitrogen.
Respiration Studies.

Further studies designed to test the influence of simazine on
respiration employing manometric techniques indicated that the amount
of oxygen taken up in 3 hours by barley seedlings was not affected,
even by high concentrations of simazine (Table 3).

A second experiment was designed to study the influence of simae
zine on oxygen uptake of excised barley root sections because Allen
and Palmer (I) used excised barley roots for their studies. Simazine
had no influence on the amount of oxygen taken up over a 2 hour period
by the excised barley roots (Table #).

The results of these studies are in contrast with those of Allen
and Palmer (l). However, in all of their studies, concentrations of
simazine equivalent to 20, 33, and l00 ppm were employed. As previously
indicated, the maximum solubility of simazine in water at 20° C is only

5.0 ppm and 8# ppm at 80° C (55). They reported differences in root

32

33

Table l. Dry weight and nitrogen content of corn germinated at
different temperatures in simazine.

 

 

Temperature regime

 

 

 

l6 hour period 2#° C 2#° C
8 hour period .I8° C - ‘ l3° C
Simazine Dry weight Percent Dry weight Percent
conc. (ppm) (mg) nitrogen (mg) nitrogen
0.00 isol/ 3.681/ 2i0-V 3.till/
0.0I I80 3.#2 I90 3.l9
O.IO I70 3.36 230 3.28
I.OO I60 3.5# 200 3.26

 

,1/ F value for difference between treatments not significant
at 5 % level.

Table 2. Dry weight and nitrogen content of cucumbers germinated
~ at different temperatures in simazine.

 

 

Temperature regime

 

 

 

l6 hour period 2#° C 2#° C
8 hour period i8° c l3° c
Simazine Dry weight Percent Dry weight Percent
conc. (ppm) (mg) nitrogen (mg) nitrogen
0.00 lzol/ 5.55-1’ itiol/ 5.001/
0.0I I30 5.63 I60 5.29
O.IO IZO 5.5I I50 5.29
l.OO I30 5.#l I20 #.99

 

l/

- F value for difference between treatments not significant at
5 %.level.

3#

Table 3. Oxygen uptake by intact barley roots as influenced by
three levels of simazine.

 

 

Simazine
(ppm) ul OZ/mg dry wt/3 hours
0.0 2.1+9l’
O.I 2.33
I.O 2.3]
5.0 2.32

 

'l/ F value for difference between treatments not significant
at 5% levels.

Table #. Effect of three levels of simazine on OXygen uptake of
excised barley root sections.

 

 

Simazine conc.

 

(ppm) ul 02/mg dry wt/2 hours
0.00 5.Ill/
0.05 5.09
O.IO #.68
I.OO #.76

 

-1/ F value for difference between treatments not significant
at 5% level.

reSpiration of barley resulting from these concentration differences,
which does not seem feasible based on the physical properties of
simazine. All of their studies were conducted in a 0.2 M ph05phate

buffer at pH 7.5. Plants in the laboratory, which were accidentally

35

watered with a 0.2 M phosphate buffer at the same pH were dead within
36 hours, and the symptoms resembled that of salt toxicity. The
specific conductance of this buffer solution was l8 millimhos per cm.
Jackson (#7) reported that only extremely salt tolerant crops grew

in a solution with a specific conductance of I6 millimhos per cm. Un-
less Allen and Palmer made errors In reporting the concentrations of
simazine and buffer solution, their conclusions are not valid.

Ries and Cast (7i) proposed the hypothesis that low levels of
simazine may increase the rate of respiration and N absorption and/or
metabolism during periods of unfavorable environment for corn growth.
There were no differences in the amount of oxygen consumed by germi-
nating corn seeds treated with 3 levels of simazine (Table 5). How-
ever, the temperatures for germination and respiration measurement
were optimum for corn.

In a second study, oxygen uptake of excised root sections from
seedlings treated with simazine was measured. Simazine had no influo
ence on the amount of oxygen taken up by the root sections (Table 6).

Respiration at two temperatures by germinating corn seeds treated
with one concentration of simazine was studied, and simazine had no
effect on respiration at either temperature (Table 7).

These results do not eliminate a possible respiration increase
of roots from mature plants grown in the presence of simazine. In a

preliminary study with excised roots from corn plants several weeks old,

36

Table 5. Oxygen uptake by corn seeds germinated in three levels of
simazine.

 

 

Simazine conc.

 

(ppm) . ul 02/mg dry wt/2 hours
0.00 ll.Il/

0.05 IO.#

O.IO Il.l

I.OO ll.2

 

1/ F value for difference between treatments not significant
at 5% level.

Table 6. Oxygen uptake by excised corn root sections in simazine
solutions.

 

 

Simazine conc.

 

(ppm) ul.02/mg dry wt/S hours
0.00 36.61/
0.06 36.8

 

1/ F value for difference between treatments not significant
at 5% level.

Table 7. Influence of simazine on oxygen uptake of germinating corn
seeds measured at two temperatures.

 

 

 

Simazine Respiration
conc. (PPm) temperature (° C) ul 02/mg dry wt/2 hours
0.00 20 8.6-”
O.IO 20 8.9
0.00 30 I8.#l/
O.IO 30 I8.9

 

l/F value for difference between treatment at each temperature
not significant at 5%.level.

37

the variability of respiration within treatments was larger than that

between simazine treatments. However, on the basis of the studies

with seedlings and excised root tissues, simazine did not affect the
root respiration rate of a resistant (corn) or susceptible (cucumber

or barley) plant Species, even at toxic concentrations.

Algae Studies.

Increased growth and nitrogen content from simazine applications

were reported on annual and perennial crops. No studies were reported

on the influence of nonphytotoxic concentrations of simazine on the

growth and nitrogen content of algae. If algae responded to simazine

as plants such as corn, apples, or peaches, several experiments with

infinitely large populations could be conducted in a short time period

to study the nature of the response. In the first study, the dry weight,

(mg nitrogen, or percent nitrogen of algae grown at 20° C in a culture

solution that contained ammonium nitrate as a nitrogen source were

not altered by simazine concentrations up to O.I ppm (Table 8). The

algae did not grow in a solution of l.00 ppm simazine.

Ries._§._1. (72) reported that growth increases of peach and
aapple trees by simazine were greatest under low nitrogen conditions.
An experiment was designed to study (the influence of simazine on algae

grown at two concentrations of ammonium nitrate with one concentration

0f: simazine. The algae did not respond to simazine at either level of

"‘Ittrogen (Table 9). The dry weight of algae was no different at either

I’Vel of nitrogen, however, the percentage of nitrogen averaged 2 percent

38

Table 8. Dry weight and nitrogen content of algae grown in a cul-
ture solution containing three concentrations of simazine.

 

 

 

Simazine Dry weight Percent Nitrogen

conc. (ppm) (mg) nitrogen mg/culture

0.00 7901/ 9.21/ 70. i-‘-/
0.0l 670 9.6 63.7
O.IO 7#O IO.3 7#.7
I.00 --- --- ---

 

1/ F value for comparison of treatments not significant at the
5%.level, not including simazine at l.00 ppm.

Table 9. Influence of simazine on the dry weight and nitrogen con-
tent of algae grown at two concentrations of ammonium nitrate.

 

,—

 

 

 

Treatmgnts
Simazine ,- Nitrogen Dry weight Percent Nitrogen
conc. (ppm) conc- (ppm) (mg) nitrogen , mg/culture
0.00 I75 260‘-’ 7.51’ i9.el’
0.05 I75 220 8.5 l8.0
0.00 700 220 9.5 20.8
0.05 700 i70 10.2 l6.#

 

1/ F value for comparison of treatments not significant at the
5% level.

39

more at the higher nitrogen level, and the algae were darker green.

The hypothesis that growth of corn and fruit trees was enhanced
by simazine under adverse environmental conditions was also tested
on algae (7i). In the first study with algae grown at l5o C, the
solutions were darker green indicating more growth from simazine
(Figure I). There was a linear increase in the dry weight of the
algae, as the concentration of simazine was increased (Table l0).
In an attempt to repeat the previous low temperature study, algae
were treated with simazine, and grown at l5° and l2° C. In this
study, the dry weight was not increased by simazine, and at l5O C,
0.2 ppm simazine inhibited their growth (Table II). These studies
were repeated several times and the results from the previous tests
were not confirmed. -

The growth response observed on algae from simazine treatments
In the first low temperature study could not be explained. The
cultures in that experiment were completely randomized within repli-
cates which eliminated any temperature or light effect on the growth
«of a particular treatment. The inhibition by simazine of a pathogenic

rnicro-organism contaminant in the innoculation culture was a possibility.

(Browth Studies with Apple Seedlings.

 

The dry weight of young apple seedlings was reduced by low concen-
tl’ations of simazine, and severe injury occurred at the higher tempera-

ttll’es (Table l2). Sheets (77) reported that translocation of simazine

Figure I. The reSponse of algae to simazine when grown at l5° C.
The simazine concentrations are left to right 0.00,
0.0l, 0.05, and O.IO. The flasks are shown without
connections for aeration, and were not randomized for

the photograph.

#l

 

#2

Table l0. Dry weight of algae grown at l5° C in culture solutions
containing simazine.

 

 

 

Simazine conc. Dry weight
(ppm) (mg)
0-00 53.31/
0.05 82.7
0.l0 l07.0

 

'1/ F value for linear increase in dry weight with increase in
simazine significant at I% level. '

Table II. Dry weight of algae grown at two temperatures and treated'
with simazine at three concentrations.

 

 

 

 

Simazine conc. Temperatures (° C)
(ppm) 12 15
0.00 8l.7 al/ I98.7 all
0.05 7#.0 a l96.6 a
0.l0 82.6 a l97.3 a
0.20 67.# a l#5.0 b

 

1/ Means with unlike letters are significantly different at
the 5%,level. V I

#3

Table l2. Dry weight of apple seedlings grown at two temperatures,
and two nitrogen and simazine concentrations.

 

 

Temperatures (°C)

 

 

 

l6 hour day 20 I5

Treatments 8 hour night l5 IO
Simazine Nitrogen Dry weight Dry weight

cone. (Ppm) conc. (ppm) (9M) (9w)
0.0 #2 n.3h all I.32 abl/

O.I #2 2.52 b I.l5 b

0.5 #2 .77 c .99 b

0.0 l05 #.20 a l.#7 a

O.I IOS 2.83 b I.l2 b

0.5 I05 l.02 c .93 b

 

1/ Means with unlike letters are significantly different at
5% level.

##

increased as temperatures increased. With a more rapid translocation
rate of simazine at the higher temperature, more simazine undoubtedly
accumulated in the apple leaves, resulting in the severe injury ob-
served.

Because of the extreme sensitivity of young apple seedlings to
simazine injury, and the heterogeneity of apple seedlings, these

studies were discontinued, and further studies were conducted with corn.

Growth Studies With Corn.

A linear increase in percent nitrogen occurred from simazine
applied to corn plants grown at sub-optimum temperatures in low levels
of ammonium nitrate (Figure 2). There was no difference in the per-
cent nitrogen of plants treated with simazine grown at temperatures
optimum for corn growth, and no differences were detected between the
dry weights of the plants treated with simazine at either temperature.
The nitrogen response from simazine at low temperatures supports the
hypothesis that simazine affects nitrogen metabolism during unfavorable
environmental conditions for growth (7i). However, further studies
\Nere needed to determine environmental and nutritional parameters neces-
sary for simazine to increase both nitrogen content and dry weight of
the treated plants. I

In the previous study with corn, the seedlings were treated with
Sriinazine immediately after tranSplanting. Considerable variability

between plants that received the same treatment was observed l-2 weeks

Percent Nitrogen

#5

1.80 __
, /°

‘/
/
/
/
LJO
i— /
/
‘}3
“@337
.397

1.60_ ‘57 D" "' "U 226°C Day 17.0°c Night

3” CI"""--£J 28.o°c Day 22.5°c light

/
/
/

 

 

 

° '05 .10

Simazine Level (ppm)

Figure 2. The influence of temperature on the response of corn
plants to simazine grown in low nitrogen solutions.

#6

after treatment. To reduce this variability within replications,
simazine treatments were not made until 2 weeks after transplanting.
Uniform plants were selected for each replication prior to simazine
“treatment. In the following study nitrogen was supplied as the
ammonium ion in one treatment solution, and the nitrate ion in the
other.

There was a 25 percent increase in the dry weight of simazine
treated corn plants grown at the low concentration of nitrate nitro-
gen and at the low temperature (Figure 3). Simazine did not in-
crease the dry weight of plants if the concentration of available
nitrate was increased. When nitrogen was supplied as the ammonium
ion, there was no increase in dry weight from simazine treatments.
In this experiment, there was a l6 percent increase in mg of nitrogen
per plant treated with simazine and grown with low available nitrate
nitrogen, at the low temperature (Figure #). There was no increase
in percent nitrogen resulting from the other treatments.

In another study with corn treated with several concentrations
of simazine and grown at 22.5° C day and I7.0o C night temperatures,
under low available nitrate, plants were greener, and larger than the
<:ontrol plants (Figure 5). There was a linear increase in mg ni-
1:rogen and dry weight per plant with increasing simazine concentra-
I:ions (Figures 6 and 7).

These results indicated that corn plants treated with simazine
‘Jtilized the low level of available nitrates more efficiently if the

1temperatures for growth were sub-optimum, thus, simazine was either

Control

1‘
.

Percent o

130

120

110

100

#7

0
D— ... .43 22.5°c Day 17.0 0 Night

0—0 28.0% Day 22.5°c Night

 

 

 

 

 

 

 

D\
‘\~
i... \
‘\~
°‘° 1‘
‘\~ '0
~\“3
i__ \‘
‘\~
“\
Iii ‘\~
0 ext
D “‘1 \
"T' C}-—- -- -- -- -- -- -~ -- ‘-- ‘-- -- "-"-{j
503 E]
0% l
53 - n5

Nitrogen Level (Pam)

The increase in growth at low temperature of corn treated

with 0.08 ppm simazine when N0Q is the source of nitrogen
. J.- .

compared to NRA as the source or nitrogen.

F value for temperature x form of nitrogen x nitrogen
concentration significant at 1% level.

#8

 

 

 

 

um ._.
D\ ' c1— — —D 22.5°c Day 17.0°c Night
\ o o
H 28.ocnaar 22. can t
\ 5 8h
\\
\
110 — \
'3 \ #0
iw 3
+5 \
8 \
‘6‘ \
g \\ .
g )’ .—-D
A. win ‘_,, "' \
’-
7 / “T m.
87 —.':l
“b
5; Cl
90 I I
53 n5
Nflrqanmml(mn)
Figure #. The increase in milligrams of nitrogen at low temperature

of corn treated with 0.08 ppm simazine when N03

is the

source of nitrogen compared to NH4 as the source of ni-

trogen.

F value for temperature x form of nitrogen x nitrogen

concentration significant at I% level.

#9

Figure 5. Typical corn plants grown in low levels of nitrate at
22.5° C day and l7.0° C night temperatures. The plant

on left received 0.0 simazine, and the plant on right

0.2 ppm simazine.

 

Figure 5

1&0

120

milligrams nitrogen per plant

100

 

°\V

Figure 6.

5i

  

r-.75**

   

? - 93.02 0 ZlOX

I I

0% .10 .20

y.—

simzine (ppm)

The linear increase in mg of nitrogen with increasin con-
centrations of simazine in corn plants grown at 22.5 C day
and l7.0° C night with #2 ppm nitrate as the nitrogen source.

dry weight per plant (gm)

6.0

 

 

 

FT
3.07 I I I
0 . 05 .10 ‘ .20
simazine level (ppm)
Figure 7. The linear increase in dry weight with increasing con-

centrations of simazine in corn plants grown at 22.5° C
day, and 17.00 0 night with 42 ppm nitrate as the nitrogen
source.

53

increasing nitrate absorption, nitrate assimilation, or bbth.
Nitrate Reductase Studies With Corn.

The nitrate reductase levels of etiolated corn seedlings ger-
minated in different levels of simazine and placed in light for 2#
hours were not changed by simazine (Table I3).

Corn was grown as in previous tests where growth and nitrogen
content was increased after 7 days exposure to simazine in the nutri-
ent culture. Extracts from simazine treated plants had higher nitrate
reductase activity than control plants. This activity increased in a
linear fashion as the concentration of simazine was increased up to
0.2 ppm (Table l#). The total nitrogen content was also greater in
the simazine treated plants. In a similar experiment, a linear in-
crease in nitrate reductase activity was measured in plants exposed
to simazine at concentrations up to 0.5 PPm (Table l5).

Attempts to determine levels of nitrate in heat killed crude
enzyme extracts using a purified nitrate reductase enzyme were not
successful. Apparently, an inhibitor of nitrate reductase was

formed in the crude extracts during the heating process.

'“coz Fixation Studies with Corn.

 

 

In corn plants grown as in the previous experiments where dry
weight and nitrogen content were increased by simazine, there were

no outstanding changes in the distribution of photosynthetic products.

5#

Table I3. Nitrate reductase levels in etiolated corn seedlings
grown in low nitrate levels and treated with simazine.

 

 

 

Simazine Nitrate reductase
conc. (ppm) mu moles KNOZ per mg protein per 20 min.
0.00 53.9.l/
0.05 6I.5
O.IO 52.7
0.20 52.#
l.00 57.3

 

«l/F value for comparison of treatment not significant at 5%
level.

Table l#. Dry weight, milligrams nitrogen, and nitrate reductase
activity of corn plants grown in nutrient culture with
#2 ppm nitrate nitrogen at temperature of 22.5°c day
and l7.0°C night and treated with two levels of simazine.

 

 

Simazine Dry Weight Nitrogen Nitrate reductase
conc.(ppm) (grams) mg/plant . mu moles KNOZ per mg
protein per 20 min.

 

0.00 1.62 al/ 57.03 al/ 2.32/
0.10 1.90 a 6#.68 b, 8.8
0.20 1 9A a 6#.l0 b 21.1

 

.l/Means with unlike letters significantly different at l%.level.

Z/F value for linear increase in nitrate reductase activity
with increase in simazine concentration significant at l% level.

55

Table l5. Nitrate reductase activity of corn plants grown in
nutrient culture with #2 ppm nitrate nitrogen at tem-
peratures of 22.5°c day and l7.0°C night, and treated
with three levels of simazine.

 

 

 

Simazine Nitrate reductase
conc. (ppm) mu moles KNOZ per mg protein per 20 min.
0.00 3.761/
O.IO 6.50
0.20 7.#5
0.50 I2.68

 

11/ F value for linear increase in nitrate reductase activity
with increase in simazine concentration significant at 5%.level.

However, there were some trends that support observations from the
previous studies. The levels of aspartate increased as the concen=
tration of simazine increased at both exposure periods (Table l6).
The reason for the drop in aSpartate levels at the 90 second expos~
ure is probably because steady state levels had not been attained

at the 60 second exposure period. Glutamate levels increased at the
60 and 90 second exposure period. These differences at the 60 sec-
ond exposure were within the realm of experimental error and should
not be interpreted as true differences without conducting further
tests. The observed increases in aspartate levels,in the simazine

treated plants support the results of the nitrate reductase studies.

.ouzcme Lou
mcomumcmouc_m_p mm pummocaxo 03mm.“ vouomcuxo mo gamma: >en swam....e\nox.m mou:. \H.

 

56

mm.mm ~o.oo. cocoa. . oo.oo. oo.oo. mm.mm

 

.muOH
45.8 cm.m mo.m .~.. m..m .m.m asooco..oun_x
-- i- i- -.a mm.m m..m o.un u.cuu>.m
locomOEm
Nc.- mm.~m mm.~m mm.~m cm..~ no..m unocuam
wa.~. 5.... mm.m mm.m. .m.m~ cm.o~ nooncamoea cnmam
.. .m. mu. .1 co. co. oonto.o
m~.~ ma.. mm.. mm. -- a“. oonsatse
Nu. m... .a. m:.. a... om.. concou>.a
ii an. m_. 1.. ii 11 333:3 33. 40
m~.m um.m mm.m m..m m~.m Nm.m oc.u>.u
.m.m om.m mm.~ -.~ m~.~ ao.m oc.com
mm.~ om.a. oa.~. ~m.o. aa.m. n.... oon.nz
mm.a mc.m oo.~ mm.c .n.m mm.m oomtoam<
mm. mm. um. mm. :m. om. mooEmus.o
mw.m~ No..~ ~8.m~ .m.a. am.a. m..m oc.cm.<
muuauota
:o_umx_u
mo. x m.m me. x m.m mo. x m.c mo. x N.. mo. x m.~ mo. x ~.~ \aoox.u «cue.
m.o ..o o m.o ..o o

 

 

. liflsmav .ocou oc_~mE_m
no.coa ocamoaxo vacuum om

Asmav .ucou u:_NmE_m
uo_coaaouamoaxo vacuum om

 

 

c. m.monuc>m0uo;a mc.cae mace—q cuou voumocu oe_~me.m 60L» mo>mo_ >n noELom
co.uumcm o.n:_0m Lugosi—Ocmzuoe new c. muuavoca mo co_u:n_cum.v u:— ucoucom .m. o.nmh

57

The reduction of nitrate to ammonia would be observed first in
glutamic acid, since ammonia reacts with‘s'i keto glutaric acid through
glutamate dehydrogenase to form glutamic acid. A subsequent reac-
tion of glutamate with oxaloacetic acid through transamination re-
sults in the formation of aspartic acid.

There was more ‘“002 "fixed” by plants treated with 0.1 ppm
simazine at both exposure periods than with control plants. At 0.5
ppm there was an increase at the 90 second exposure period and a
decrease at the 60 second exposure period. Further studies to
determine photosynthetic rates should be designed, employing mano-
metric techniques or subjecting leaf tissues, carrying on steady
state photosynthesis in a known quantity of C02, to known quanti-

f ll‘coz for a fixed time. The increase in dry weight of

ties o
simazine treated plants obviously indicates that more C02 is being

fixed if respiration is not inhibited.

SUMMARY

This research encompassed studies designed to determine the
nutritional and environmental parameters and mode of action reSponsi-
ble for the observed increase in growth and nitrogen content of plant
species subjected to herbicidal concentrations of simazine.

Several plants were tested in an attempt to establish a bio-
logical assay for this effect. Elaborate attempts with algae
interacting temperature, nitrogen level, and algae population with
simazine were not successful. Young apple seedlings were not a
good test plant because of their heterogeneity, and slow growth
rate.

Corn plants proved to be a good biological test species when
grown under Specific nutritional and environmental conditions.
These conditions were: low temperature, and low available nitrate
nitrogen. If nitrate levels or temperatures were increased, or if
nitrogen was supplied as the ammonium ion, there was no increase
in nitrogen or dry weight from simazine applications. This in-
creased growth from simazine under low temperature, and low
available nitrate was linear at concentrations from 0.05 to 0.20
PPm-

Manometric studies and tests with germinating seedlings indi-
cated that Simazine did not alter respiration rates of barley,
cucumber, or corn germinated under low and high temperatures. The

nitrate reductase activity in corn was increased in plants which

58

59

contained more nitrogen from simazine applications. Similar corn
plants Subjected to short exposure periods of ll"C02 contained higher

I#

levels of C-labeled glutamic and aspartic acids.

Based on these results, it may be hypothesized that simazine
either increased nitrate absorption, or induced the synthesis of the
enzyme under adverse environmental conditions. If the latter is
true, simazine would be invaluable for studying control mechanisms
in the synthesis of the nitrate reductase enzyme.

Much of the fertilizer applied to soil as the ammonium ion is
converted to nitrate by micro-organisms. These studies could ex-
plain the growth and nitrogen response observed on simazine treated
fruit trees, since their major “flush” of growth occurs in the
spring, and often under sub-optimum temperatures for growth.

Further research should be conducted to determine if this re-

sponse is due to simazine, or a metabolite, and the relevance of this

biochemical action to the herbicidal action of triazine compounds.

60

APPENDIX

6i

Table l. Nutrient culture solution for growing algae.

 

 

 

Stock solution ml of stock solution/l
Chemical (gm/l)‘ of nutrient solution
I/
NH4N03 40.0 50.0-
MgSOh.7H20 u.0 50.0
CaCl2 0.5 50.0
thpoh 30.6 50.0
K°2P°# I7.# 50.0
NaFeg/ h.0 10.0
Minor elements
H3803 2.86
MnCl2.#H20 I.8I
ZnSOu.7H20 0.22
CuSOh.5H20 0.08
H2M004.H20 0.02 1.0

 

,«1/ Volume needed for 700 ppm nitrogen.

'g/ Sequestrene, Iron Chelate (contains I2% as metallic) Geigy
Agricultural Chemical Company.

62

Table 2. Nutrient culture solution for growing plants with ammonium
nitrate as the source of nitrogen.

 

 

 

 

Stock solution ml of stock solution/l
Chemical (gm/l) of nutrient solution
K2$0u 87.0 5.0
MgSOu‘7H20 2#6.0 2.0
Ca(H2POu)2‘H20 I2.6 l0.0
CaSOh°2H20 l.72 200.0
NHhNO3 80.0 l.Ol/
NaFe-g-l 11.0 10.0
Minor elements
MnCl2-#H20 l.8l
ZnSOh'7H20 0.22
CuSOu'SHZO 0.08 .
HzMoOh°H20 0.02 I.O
1/

Volume needed for 28 ppm nitrogen.

12/ Sequestrene, Iron Chelate (contains l2%.as metallic) Geigy
Agricultural Chemical Company.

63

Table 3. Nutrient culture solution for growing plants with ammonium
and nitrate as the source of nitrogen.

_I_._

 

ml of stock solution/I

Stock solution of nutrient solution

 

 

Chemical (gm/l) Nitrate Ammonium
KN03 101.0 2.01/ --
(1111,9250,l 132.0 -- 1.01/
MgSOh.7H20 2u6.0 2.0 2.0
CaCI2.2H20 1h7.0 5.0 5.0
KHZPOA 136.0 1.0 1.0
K250“ 87.0 5.0 5.0
KOH 100.0 0.22/ 0.22/
NaFeil u.0 10.0 10.0

Minor elements

H3803 2.86
MnCI2.#H20 I.8I
ZnSOh.7H20 0.22
CuSOh.5H20 0.08
H2M004.H20 0.02 1.0 1.0

 

,1/ Volume needed for 28 ppm nitrogen.

2/

- Volume needed to adjust final pH of nutrient solution to 6.5.

'l/ Sequestrene, Iron Chelate (contains l2%.as metallic) Geigy
Agricultural Chemical Company.

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6#~

l3.

l#.

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65

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66

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