Glyphosate: Herbicidal Effects, Mode of Action and Degradation in Soil

Author(s): Paweł Kafarski, Piotr Wieczorek, Iwona Bartela, Jadwiga Dabrowska and Barbara
Ottenbreit
Source: The American Biology Teacher, Vol. 50, No. 5 (May, 1988), pp. 296-299
Published by: University of California Press on behalf of the National Association of Biology
Teachers
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 lished that reduced levels of aromatic
amino acids resulted from inhibition
How-To-Do-It of a single enzyme of shikimate
pathway (one of the two metabolic
pathways producing aromatic com-
pounds in plants and bacteria; this
pathway does not operate in animals)
Glyphosate:HerbicidalEffects,Mode of namely 5-enolpyruvylshikimate-3-
phosphate synthetase. The molecular
Action and Degradationin Soil basis of this inhibition is now well un-
derstood (Steinrucken & Amrhein
1984).
Pawel Kafarski HerbicidalEffect
PiotrWieczorek of Glyphosate
Iwona Bartela The following investigation pro-
JadwigaDabrowska vides some insight into the effects of
BarbaraOttenbreit various concentrations of herbicide
glyphosate on the growth of common
Herbicides are part of modern agri- number of internationalapplications. weed Lepidium sativum.It was chosen
cultural production systems and It is the active ingredient of as a test plant because of its low sensi-
therefore contribute significantly to Roundup?, Monsanto's herbicide for- tivity on planting conditions (fixed
the economy of agriculturalproducts. mulation consisting of glyphosate iso- light and temperature are not neces-
In the United States, losses caused by propylamine salt and surfactantsolu- sary during the experiments) and fast
weed competition with majorcrops is tion in water. Roundup? has devel- development of mature plants.
approximately $8.9 billion, while the oped into an extremely important 1.) Preparationof herbicidesolu-
total expenditures for herbicides used herbicidesince its introductionin 1971
is about $2.1 billion and the cost of ap- (Bairdet al. 1971;Franz1985)and now tions
plying these herbicides, $938 million is marketed in more than 100 coun- For all experiments describedin this
annually (Chandler 1985). Without tries. paper the pure active ingredient (mo-
current technology to control the Glyphosate has a relativelylow mo- lecular weight 169.08, obtained from
weeds, the losses would be doubled lecular weight and high water solu- Monsanto Co.) or any of the commer-
or tripled. bility, factorswhich aid in its rapid ab- cially available Roundup? formula-
At the same time, herbicidesare po- sorption and translocation by plant tions (molecularweight of isopropyla-
tent and specific inhibitors of plant tissues. Once inside the plant, gly- mine salt of glyphosate is equal to
metabolism and may therefore be po- phosate does not breakdown, nor is it 229.18) containing from 41 percent to
tentially useful as valuable tools in metabolized to a significantdegree. In 61 percent (per weight) glyphosate
basic plant physiologicalresearch.Un- soils, however, the compound is salt may be used.
fortunately,few herbicides are known strongly absorbed (preventing Prepare 5 mm solution of glypho-
to inhibit plant growth by direct inhi- leaching) and is rapidly degraded by sate by dissolving 845 mg of the com-
bition of enzymes involved in meta- microorganisms to to non-toxic or- pound in I L of distilled water or by
bolic pathways. Thus, most literature ganic products (see Figure 1) which
dealing with herbicidal effects on are then broken down to ammonia,
plants describes secondary and ter- water and carbon dioxide (Hoagland Pawet Kafarskiis an assistant professorof
tiary effects (Duke 1985a) such as chemistry at the Institute of Organic and
& Duke 1981). Moreover, glyphosate Physical Chemistry, Technical University
chlorosis. is non-toxic to insects and vertebrates of Wroclaw, 50-370 Wroclaw, Poland,
The environment has been as- and does not accumulate in animal where he earned his M.S. in 1971 and
saulted with a variety of chemical tissues (Newton et al. 1984; Sullivan Ph.D. in 1976. He also teaches organic
agents, among which herbicides con- 1985). chemistry at Pedagogical University of
tribute significantly. Some of these In early studies on the mode of ac- Opole. Piotr Wieczorek, an assistant pro-
have appeared to be relatively innoc- tion of glyphosate (Jaworski 1972; fessor of chemistry at the Institute of
uous, while others are quite haz- Roisch & Lingens 1974), it was found Chemistry, Pedagogical University of
ardous. Thus, the biodegradabilityof that the growth inhibitory effects on Opole, 40-052 Opole, Poland, received his
a herbicidemay be a factorin the deci- both Rhizobiumjaponicumand duck- M.S. in 1978 and Ph.D. in 1982 from the
sion to use it. Institute of Organic and Polymer Tech-
weed (Lemnagibba) could be com- nology, Technical University of Wroclaw.
In this paper we describe the useful- pletely reversed by addition of the ar- He teaches organic chemistry and chem-
ness of a relatively novel herbicide- omatic amino acids: phenylalanine, istry. Iwona Bartela is a primary school
glyphosate-for a laboratorydemon- tyrosine and tryptophane. teacherin Radlow, 46-331Radlow, Poland.
strationof herbicidalactivity, mode of In later studies, in which profiles of She holds an M.S. in chemistryfrom Peda-
action and stability in soil. A series of free pools of aromatic amino acids gogical Universityof Opole (1986).Jadwiga
laboratoryexercises conducted by stu- from higher plants were examined, Dabrowska teaches secondary school in
dents is used to demonstrate several levels of aromatic amino acids were Brzeg, 49-300 Brzeg, Poland. Dabrowska
properties of this herbicide. found to be greatly reduced in com- received an M.S. in chemistry from Peda-
Glyphosate, N-(phosphonomethyl) gogical University of Opole in 1986. Bar-
parison to other amino acids (Duke bara Ottenbreit earned her M.S. in chem-
glycine (see Figure 1), is an extremely 1985b). istry from PedagogicalUniversityof Opole
effective, non-selective, post-emer- Studies by Amrhein (Steinriicken& in 1986 and is a primaryschool teacher in
gence herbicide with an increasing Amrhein 1984; Duke 1985b) estab- Slawice, 45-851Slawice, Poland.

296 THE AMERICAN BIOLOGY TEACHER, VOLUME 50, NO. 5, MAY 1988

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appropriate dilution of Roundups Percent of the control is calculated as Mechanismof
with distilled water. In this case, the the ratio of mean length of stems or
final solution should contain 1.15 g of roots in the presence of certain herbi- GlyphosateAction
isopropylamine salt of glyphosate per cide concentration to the corre- The following exercise is a modified
1 L. The weight of Roundups used for sponding lengths of control x 100. version of Jaworski's (1972) experi-
dilution is calculated from the equa- The data could be also given graphi- ment showing that the effect of gly-
tion cally as dose-response curves phosate is partially reversed by sup-
1.15 x 100 (Fedtke 1982). Figure 2 represents an plying Lepidiumsativumwith phenylal-
X=
y example of such a curve constructed anine and/or tyrosine in its growth
using a semilogarithmic scale. Using medium. Supplying this plant with
where Y means the percentage con- these curves IDm, i.e. concentration
centration (per weight) of this salt in non-aromaticamino acids does not af-
causing the shortening of stems or the fect the herbicidalaction. We have not
commercialformulation of the herbi- concentration causing shortening of
cide. received satisfactory results using
roots by half, can be found (see Fig- tryptophane since the plants develop
2.) Procedure ure 2). infections by fungi.
Sow Lepidium sativumon wet cotton-
wool placed on six 10 cm Petri dishes
and spray with water (distilled or tap H2N-CH2COOH
water can be used). Maintain the
proper wetness of the cotton-wool glycine
until first leaves occur. Then spray the COOH
plants with 10 ml portions of solutions I
of glyphosate at concentrations of: 0 H2N-CH2- P03H2
CH24
(control) ,uM, 5 ,uM,50 p.M,500 ,uM, I (aminomethyl)-phosphonic
1.5 mM and 5.0 mM. NH acid
Grow the plants until they mature
(usually seven days after germination) CH2 H3C-NH- CH2- P03H2
keeping the cotton-wool wet during
the experiment. Note and record P03H2 (N-methyl-aminomethyl)-
changes in the general appearanceof glyphosate < - phosphonic acid
the plants on each Petri dish. Then re-
move 15 to 20 plants from each dish HO- CH2- PO3H2
(be careful not to destroy the roots)
and measure the length of their roots ( hydroxymethyl )-phosphonic
and stems. acid
3.) Data collectionand presenta- Figure 1. Chemicalcomposition of glyphosate and its main biodegradationproducts.
tion.
Preprinteddata forms greatly facili-
tate data collection, we suggest the
following format: 1001
herbicide
concentration 0 0.5 5.0 50 150 500
(p.M) (control)
80-
meanstem
length
hala
%of the
control 100 60-
meanroot
length
ar
%of the
control 100 40 -
Aa stands for standard deviation cal-
culated as follows
i - 1)
+
n-1 20 -
where: li-actual length of stem or
root
n-number of measurements
(numberof plants) 0.5 5 plD50 50 500
1-mean length of stems or
roots; calculatedas follows:
sativumexpressed as
Figure2. Concentrationdependence of the lengths of roots of Lepidium
the percentageof lengths of roots of control.
n
GLYPHOSATE 297

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 1.) Preparation
of solutions
Preparea 1 percent solution of gly-
phosate by dissolving 10 g of herbicide
in 1 L of water (or by appropriatedilu-
tion of Roundupg). Divide this solu-
tion into 100 ml portions and to five
add: 2 g of tyrosine to the first solu-
tion, 2 g of phenylalanine to the
second, 2 g of glycine to the third, 2 g
of lysine to the fourth and 1 g of tyro-
sine and 1 g of phenylalanine to the
fifth portion.
2.) Studies on glyphosate biode-
gradationusing Lepidiumsativum
as a bioindicator
Prepare seven 10 g samples of the
soil (three of them sterilized) and
place them in covered Petri dishes.
Add 10 ml of 0.1 percent solution of
glyphosate to the three dishes con-
taining non-sterilizedsoil and to three
dishes containing sterilized soil. Add
10 ml of distilled water to the seventh
dish (control).
Keep the samples of soil at room
temperature for three weeks, main-
taining the proper wetness of the soil,
then sow Lepidiumsativum and allow
it to grow for 7 to 9 days. Determine
the mean lengths (see the first experi-
ment) of stems and roots of plants on
each Petri dish and present the results
as in the first experiment.

2.) Procedure
Sow Lepidium sativumon wet cotton-
wool placed on seven 10 cm Petri
dishes and maintain the appropriate
wetness of the cotton-wool during the
experiment. When the first leaves ap-
pear, spray the plants accordingto the A~ -B
following scheme: dish 1 with water
(control), dish 2 with 1 percent gly-
phosate solution, dish 3 with glypho-
sate supplemented with tyrosine, dish
4 with glyphosate solution containing
piienylalanine, dish 5 with herbicide
solution containing glycine, dish 6
with glyphosate and lysine solution, 0 00
and dish 7 with herbicide solution
containingboth phenylalanine and ty- 0 0
rosine. After 5 to 7 days note the
changes in the general appearanceof
plants in each Petri dish, and then de-
termine the mean lengths (see pre-
ceding experiment)of the plant's roots
and stems. The data may be present in
a similarformat to the first exercise.

Biodegradationof Glyphosate
in Soil
The following experiment provides
evidence that glyphosate is easily and
quickly degraded by soil microor-
ganisms.
2 3 l . 2 3 4 5
1.) Collectionand preparationof
soil samples 0 0
Place a 100 g sample of soil in a
polyethylene bag or in a covered jar 0
(to maintainthe naturalwetness of the
soil). Sterilize half of this portion by
autoclaving or by refluxing in 1 L of
ethanol for 20 min; refluxing may be
done by filtering the ethanol and
washing the sample with distilled
water alternately.Sterilizationkills the
microorganismsliving in the soil, and
thus makes biodegradationof glypho- Figure3. Thin layer chromatographicstudies on the fate of glyphosate in soil after:3 hours
sate impossible. On the other hand, (plate A), 2 weeks (plate B) and a month (plate C) from the beginning of the experiment.
the sterilized soil still remains capable The plates were spotted with: (aminomethyl)phosphonicacid (1), glycine (2), glyphosate
of degrading the herbicidechemically. (3), sample extractedfrom sterilized soils (4) and non-sterilizedsoils (5).

298 THE AMERICAN BIOLOGY TEACHER, VOLUME 50, NO. 5, MAY 1988

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 3.) Thefate of glyphosatein soil
Place three sterilized and three non-
sterilized 10 g soil samples in 50 ml Er-
lenmayer flasks (with covers). Add 10
ml of 0.1 percent glyphosate solution
containing 0.5 g of sodium bicar-
bonate to each flask and keep the
samples at room temperature main-
taining the proper soil wetness.
Study the composition of biodegra-
dation products after three hours,
after two weeks and after a month
from the beginning of the experiment.
Carefully add 20 ml of hot 5 percent
hydrochloric acid to the flask con-
taining sterilized and non-sterilized
soil. Mix this solution well and filter
with suction. Wash the samples sev-
eral times with 5 ml portions of the
acid on the funnel. Combine filtrates
and washings from each sample sepa-
rately, and evaporate each to 1 ml
under reduced pressure using a rotary
evaporator. Study the composition of
this solution by means of thin layer
chromatography using any of the
commerciallyavailable plates covered
with silica gel and phenol-water-
acetic acid (20:7:3)as eluent (we sug-
gest that the instructor prepare the
eluent). Visualize the spots spraying
the plates with 1 percent ninhydrin
solution in ethanol. To find out how to
make thin layer chromatography,con-
sult any available chemical laboratory
manual (for example, Brewster, Van-
derwerf, McEven 1970). Use glypho-
sate, (aminomethyl) phosphonic acid
and glycine as reference compounds.
Figure 3 illustrates one of the ex-
amples of the results obtained by our
students. Note that after a month no
ninhydrin-positive compounds were
found in the soil. This indicates that
glyphosate was degraded to inorganic
products, while still present in steril-
ized soil.
Our students obtained various re-
sults from this experiment. The signif-
icant differences in the observed time
of total degradation of glyphosate, as
well as the major path of this process
(via glycine or (aminomethyl) phos-
phonic acid), reflect the variations in
microbiological composition of the
collected soil samples.
Acknowledgments
The authors wish to thank Mon-
santo Co. for a generous gift of
N-(phosphonomethyl) glycine and
Roundup?.
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