Ecological Research Series
INTERACTION OF  OZONE  AND HERBICIDES
                                   IN  SOYBEANS
                               Environmental Research Laboratory
                              Office of Research and Development
                             U.S. Environmental Protection Agency
                                     Corvallis, Oregon 97330

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                RESEARCH REPORTING SERIES

Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology.  Elimination  of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:

      1.  Environmental  Health Effects Research
      2.  Environmental  Protection Technology
      3.  Ecological Research
      4.  Environmental  Monitoring
      5.  Socioeconomic Environmental Studies
      6.  Scientific and Technical  Assessment Reports (STAR)
      7.  Interagency  Energy-Environment Research and Development
      8.  "Special" Reports
      9.  Miscellaneous Reports

This report has been assigned to the  ECOLOGICAL RESEARCH series. This series
describes,research on  the effects of pollution on humans, plant and animal spe-
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This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia  22161.

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                                          EPA-600/3-78-047
                                          May 1978
   INTERACTION OF OZONE AND HERBICIDES
               IN SOYBEANS
                    by
                Raj  Bahadur
   Mississippi Valley State University
       Itta Bena, Mississippi 38941
          contract no. R-803072
              Project Officer
              David T. Tingey
        Terrestrial Ecology Branch
Corvallis Environmental Research Laboratory
          Corvallis, Oregon 97330
CORVALLIS ENVIRONMENTAL RESEARCH LABORATORY
    OFFICE OF RESEARCH AND DEVELOPMENT
   U.S.  ENVIRONMENTAL PROTECTION AGENCY
          CORVALLIS,  OREGON 97330

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                                 DISCLAIMER

     This report has been reviewed by the Corvallis Environmental Research
Laboratory,  U.S. Environmental Protection Agency,  and approved for publication.
Approval does not signify that the contents necessarily reflect the views and
policies of the U.S. Environmental Protection Agency,  nor does mention of
trade names or commercial products constitute endorsement or recommendation
for use.
                                     11

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                                 FOREWORD
Effective regulatory and enforcement actions by the Environmental Protection
Agency would be virtually impossible without sound scientific data on pollu-
tants and their impact on environmental stability and human health.  Respon-
sibility for building this data base has been assigned to EPA's Office of
Research and Development and its 15 major field installations, one of which
is the Corvallis Environmental Research Laboratory (CERL).

The primary mission of the Corvallis Laboratory is research on the effects
of environmental pollutants on terrestrial, freshwater, and marine eco-
systems; the behavior, effects and control of pollutants in lake systems;
and the development of predictive models on the movement of pollutants in
the biosphere.

This report investigated the possibility of herbicides increasing ozone
toxicity.  An increased toxicity could significantly affect farming
practices.
                                   111

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                                  ABSTRACT
     The purpose of this investigation was to identify potentially  damaging
herbicide-ozone interactions on soybean so that injury could be reduced by
judicious selection of herbicides and tolerant cultivars.  The involvement of
herbicide influence on stomatal aperture affecting ozone fumigation injury
was studied.

     Soybean cultivars with varying sensitivity to ozone were planted in the
greenhouse in soil containing residual levels of atrazine, normally recommend-
ed levels of metribuzin or nontreated soil.  The herbicide bentazon was also
studied, but was applied to foliage when the soybeans were 3 weeks old.  After
the soybean plants were in the second trifoliolate  leaf stage or 3 to 7 days
after bentazon treatment they were fumigated with 0, 0.3, or 0.4 ppm ozone.
Transpiration rates were monitored to evaluate potential interactions.

     Atrazine, metribuzin, and bentazon all affected transpiration.  The
transpiration of the ozone tolerant Hood cultivar was not affected by bentazon
alone but after ozone treatment, transpiration increased significantly.  The
presence of atrazine residues in the soil decreased transpiration after ozone
fumigation.  In the ozone-susceptible Dare cultivar, bentazon inhibited
transpiration, but following ozone fumigation there were significant differ-
ences.  The cultivar Tracy proved to be very susceptible to metribuzin injury
and was intermediate in sensitivity to ozone injury.  It responded like Dare
to bentazon alone, and like Hood following ozone fumigation.

     This report was submitted in fulfillment of Contract No. R-803072 by
Mississippi Valley State University.  Under the sponsorship of the U.S.
Environmental Protection Agency.  This report covers the period December 1,
1974, to March 31, 1977, and work was completed as of April 30, 1977-
                                     IV

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                                  CONTENTS

                                                                Page


Foreword                                                        iii

Abstract                                                         iv

Acknowledgements                                                 vi
SECTION 1
  Introduction                                                    1

SECTION 2
  Conclusions                                                     2

SECTION 3
  Recommendations                                                 3

SECTION 4
  Materials and Methods                                           4

SECTION 5
  Results and Discussion                                          6

References                                                       18

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                              ACKNOWLEDGMENTS
     The constant guidance of Dr.  David T.  Tingey,  Plant Physiologist,
Terrestrial Ecology Branch of the  Corvallis Environmental Research Laboratory
Environmental Protection Agency,  Corvallis, Oregon is gratefully acknowledged.

   .  I am very sincerely indebted  to Dr. Donald Penner, Plant Physiologist
and Professor of Crop and Soil Sciences Department, Michigan State University,
East  Lansing, Michigan for his herbicide sprayer design and data analysis
assistance.  This investigation could not have been undertaken without the
sustained and active support of Dr.  Clyde Bishop,  Jr.,  Research and Develop-
ment  Office of Monitoring & Technical Support, Minority Institutions Research
Support Program, U.S. Environmental  Protection Agency,  Washington, D. C.
                                     VI

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                                 SECTION 1

                                INTRODUCTION

     A number of soybean  (Glycine max  (L.) Merr.) production areas in the
midwest and the south are located near large population centers and may
experience exposure to ozone pollution.  Ozone is the principal toxicant in
photochemical smog and the most damaging air pollutant affecting vegetation
in the United States  (1).  Soybeans and navy beans are relatively sensitive
to ozone injury and such  injury has been reported in the field due to acute
ozone exposure because of inversion layers (2).

     The vast majority of land  ( 2,103,625  acres in Mississippi alone)
currently in soybean production, especially in the south, is treated with
preplant, preemergence, early post-emergence and/or late-directed herbicides.
Up to four or five herbicides may be applied because of the long and favorable
season for growth of  crop and weed plants in the south.  Although these
herbicides may be selective, subtle effects of certain of these compounds on
tolerant crops may occur.  This is typified by the effect of atrazine (2-
chloro-4-(ethylamino)-6-(isopropylamino)-j3-triazine) on the stomatal closing
similar to antitranspirants which have been tested for this use (3).  It is
believed that ozone enters into the leaves through stomata to cause injury
(4). - Thus, compounds affecting transpiration could either increase or antag-
onize ozone injury.   Conversely, ozone has been shown to alter metabolism
rate of the herbicide diphenamid (N,N-dimethyl diphenylacetamide) in plants
(5).  In the United States over 40,000,000 acres of soybeans are planted
annually.  Even a slight  decrease in yield as a result of ozone damage or an
interaction of ozone  and  herbicides would be manifested in tons of protein
which could otherwise be  used for human and animal consumption.

     Numerous research papers have reported the interactions of the factors
such as pesticides, temperature, soil moisture, fertilizer types, cultivar
differences, and agronomic practices in altering the use of herbicides.  An
environmental factor which has received only recent attention in relation to
cultivar differences  and  herbicides interactions is ozone (6).  The purpose
of this proposed research was to study the interactions of herbicides and
acute levels of ozone as  they might alter the sensitivity of soybean cultivars,
Soybean cultivar differences in response to acute ozone dosage were demon-
strated by Tingey and et  al. (2).  Ozone under laboratory conditions has been
reported to induce stomatal closure (1).  Stomatal closure in ozone treated
oats depressed photosynthesis and reduced transpiration.  The purpose of
this investigation was to:

     (A)  Assess the extent of ozone-herbicide interaction and thus reduce
          damage to soybeans by selecting herbicides and tolerant cultivars
          and;

     (B)  Study the behavior and resistance of the stomata as port of entry
          of ozone and consequent effect on transpiration and growth of
          soybean plants.

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                                 SECTION 2

                                CONCLUSIONS
     Although ozone enters through the stomata it did not appear to affect
transpiration three days after exposure.  Thus, measuring transpiration for
several days after ozone fumigation is not the most sensitive way of evaluat-
ing ozone injury.  However, the herbicides bentazon, atrazine, and metribuzin
all affected transpiration and in the case of bentazon, subsequent ozone
fumigation increased transpiration, indicating a possible bentazon-ozone
interaction.  The three cultivars studied did not accrue leaf area at exactly
the same rate,  consequently evaluation of transpiration was more accurate
when the rates were based on the pretreatment rate and expressed as a percent-
age of that rate.  The duration of the experiments was too short to use
changes in leaf area as a criteria for either ozone or herbicide injury.

     Interestingly, the transpiration rate of the three cultivars appeared
to be almost identical on a leaf area basis.   Thus, the differential sensi-
tivity of the three cultivars to ozone does not appear to be related to
transpiration differences that could have reflected differences in density
of stomatal distribution.

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                                 SECTION 3

                              RECOMMENDATIONS
     The data presented in the report suggest that herbicide-ozone interaction
may occur.  Measurement of transpiration appears rather insensitive to early
evaluation of interaction responses.  Thus, future studies should be less
difficult to interpret if limited to the study of the bentazon-ozone interact-
ion and the use of a more sensitive measurement to detect the interaction
response.  Studies by Tingey e_t jl.  (7) would suggest that measurement of
ethylene release would be more sensitive and appropriate to measure the
initial response.  This should be followed by obtaining fresh weight, dry
weight, and plant height data over a time period following fumigation.  The
basis for the observed action should be pursued.  This knowledge could have
predictive value in identifying other ozone-related interactions potentially
reducing soybean production.

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                                 SECTION 4

                           MATERIALS AND METHODS

TEST PLANTS

     Three soybean (Glycine max (L.)Merr.) cultivars primarily grown in the
south were selected for this investigation.  They were Dare, maturity Group
V, selected because of its relative susceptibility to ozone damage  (2);  Hood,
maturity Group VI, selected because of its relative tolerance to ozone damage
(2); and Tracy, maturity Group VI, selected because it was relatively toler-
ant to herbicide 2,4-DB (8), a recent introduction with very desirable
characteristics, but its susceptibility to ozone damage was not known.

PLANT GROWTH CONDITIONS

     Fungicide  (spergon) treated seeds of each cultivar, one seed each per
170 g styrofoam cup were planted in 100 oven-dried loamy sand soil  that
contained 73% sand, 12.5% silt, 14.5% clay and approximately 0.4% organic
matter.  Each cup of soil was brought to field capacity moisture level by
weighing and adding water to each cup immediately after seeding.  The plants
were randomly placed in a greenhouse with temperatures of 24 4^ 5°C  during day
and 13 +_ 5°C during night with 75 + 15% relative humidity.  The greenhouse
lighting was supplemented for 6 hr/day with Sylvania grolux tubes during
short days.  The light intensity was recorded by Lambda light meter model
no. LI-170 and photometric sensor LI-2105.  The plants were checked every
day for loss of moisture by weighing and watered to field capacity.  The
plastic lids were placed on the cup around the plants after the seedling had
grown to reduce the soil surface water loss by evaporation.  The nutrients in
the soil maintained satisfactory growth of plants for the duration  of the
experiments from three to five weeks.  The plants were not fertilized to
avoid a possible interaction between nutrients and the treatments.
        ;
HERBICIDE TREATMENT AND OZONE FUMIGATION

     Three herbicides, atrazine (2-chloro-4-(ethylamino)-6-(isopropylamino)-
j3-triazine), bentazon  (3-isopropyl-lH-2,1,3-benzothiodizin-4(3H)-one 2,2-
dioxide), and metribuzin (4-amino-6-tert-butyl-3-(methylthio)-aj3-triazin-5(4H)
one) were selected for the interaction study with ozone.  Atrazine, reported
to decrease stomatal aperture and reduce the transpiration rate, w*as incorpor-
ated into the soil mix at 0,  .07, .14, .28, .56, or .84 kg/ha to  simulate
residues that might be present in a soybean-corn rotation.  Bentazon, a
postemergence herbicide, was  applied at 0, 0.56, 0.84 or 1.1 kg./ha  when the
soybeans were 3 weeks old, had three trifoliate leaves, and a Plastochron
Index (P.I.) of 3.5.  The P.I. provided a numerical evaluation of leaf age,
leaf development, and the physiological maturity of the plant useful for
comparing growth stage of plants  (Tingey, D.T., 1974 Personal communication).
Metribuzin was  incorporated into  the soil prior to planting at rates of 0,
0.14, 0.28, or  0.56 kg,/ha.  Herbicide application rates were determined by
adjusting pressure on the nozzle  and speed of the conveyor belt carrying the

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plants under the stationary nozzle.

     The plants of the three cultivars were exposed to ozone concentrations
of 0, 0.3, or 0.4 ppm for 2 hr in chambers previously described  (9).  Ozone
was generated by controlling the flow of dry oxygen through a Sanders' Ozoni-
zer with an output of 0.50 mg/hr.  The ozone concentration within the chamber
was measured with a Mast-ozone meter  (model 724-2) interfaced with a recorder,
During fumigation the light intensity was 35+5 klux, the temperature was
17 + 2°C, and the relative humidity was 85 + 3% inside and outside the cham-
bers in the greenhouse.  In the first study, the plants were fumigated 7 days
after bentazon application; in the second study, 3 days after bentazon
application.

TRANSPIRATION RATE MEASUREMENT

     Changes in transpiration rate were used to assess plant injury due to
ozone, the herbicides, or the combination of both.  In several studies, rates
were also compared to prevailing rates prior to herbicide and ozone treatment.
The water loss by plants was determined by weighing each cup every day and
adding enough water to the soil so as to bring it up  to the original predeter-
mined field capacity moisture level.  As the plants grew older the
predetermined weight of the cup was revised upwards periodically.

     The leaf surface area was also measured in some  experiments with leaf
area meter Lambda model no. LI-3000.  Transpiration rate and leaf surface
area provided the basis for expressing the rate of transpiration in terms of
weight/area/time.

     The plants of the three cultivars were indexed for ozone and herbicide
visual injury on a scale ranging from 0 to 100, 4 days after treatment.  The
injury estimates were made for each trifoliate leaf of the plant and then
grouped and averaged for the whole plant.  The 0 was  for undamaged leaf and
100  for dead leaves.  Cultivars Dare and Hood were treated with 1.1 kg/ha
bentazon followed with 0.4 ppm ozone fumigation after 3 days and photographed
5 days later for the visual injury scoring.

EXPERIMENTAL DESIGN AND DATA ANALYSIS

     The uniformly selected plants were placed in the greenhouse and in the
fumigation chambers according to a randomized complete block design with four
to eight replications.  The data were statistically analyzed by analysis of
variance program by computer at Michigan State University.  The significant
differences among means of the treatments were determined using Duncan's
Multiple Range Test.  Herbicide treatments were considered as the main plots,
while cultivars and ozone concentrations as the sub-sub plots with a given
number of replications in three way factorial experiments.

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                                 SECTION 5

                           RESULTS AND DISCUSSION
     It is generally agreed that ozone enters the plant leaf through the
stomata and that oxidant injury occurs first in the vicinity of penetration.
In a study to determine whether ozone influenced transpiration in the ozone
susceptible Dare cultivar, the tolerant Hood, or Tracy cultivar with or with-
out a bentazon treatment 7 days earlier, the results indicated little or no
effect of ozone on transpiration (Table 1).   Only the Tracy cultivar in the
absence of a bentazon treatment showed a slight but significant increase in
transpiration.  From these results one might speculate that modest levels of
ozone pollution do not increase moisture stress on the total plant although
the  topical  or localized effect is severe.   In this study the transpiration
data are based on the transpiration of the particular plant prior to herbicide
or ozone treatment.  No differences in transpiration were observed among the
three cultivars averaged over all other treatments.  The experiment showed
that bentazon at 1.1 kg/ha inhibited transpiration when other factors were
not considered.  This expected result confirmed the previous report of Penner
(10) that bentazon rapidly inhibits transpiration.  However, the data from
this study show two additional interesting features.  First, following ozone
treatment, the water loss picture is reversed and secondly that the cultivars
do not respond similarly.  The transpiration of the ozone tolerant Hood was
not affected by bentazon alone but after the ozone treatment, transpiration
increased significantly.  In the susceptible Dare, the bentazon inhibited
transpiration, but following ozone fumigation there were no significant
differences.  Visual injury ratings indicated a bentazon-ozone interaction
for Dare but not Hood (Figure 1).  Tracy seems to be an intermediate in
susceptibility to ozone injury and responded like Dare to bentazon treatment
alone and responded like Hood following ozone fumigation.  In a repeat experi-
ment, bentazon failed to inhibit transpiration in Dare prior to fumigation
but did stimulate transpiration again of Tracy at the 0.56 kg/ha rate.  The
response of Hood in the repeat experiment was similar to that reported in
Table 1.

     In a second study,  the application of 0.84 kg/ha of bentazon markedly
inhibited transpiration of all three cultivars.  This was still evident after
ozone fumigation (Table 2).  The Tracy cultivar showed less inhibition than
Dare or Hood.  The Hood cultivar appeared to have significantly lower
transpiration than the other two cultivars.

     In additional experiments with eight replications and application of
0, 0.50, and 1.12 kg/ha of bentazon to the three soybean cultivars the Hood
cultivar again had significantly less transpiration than Dare on a per plant
basis.   However, in this experiment, leaf areas were measured and conversion
of transpiration to grams water lost per cm2 per 24 hr showed Dare, Hood, and
Tracy to have values of 0.131, 0.138, and 0.131, respectively.  Thus the
decreased transpiration of Hood cultivar in Tables 2 and 3 is merely a

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reflection of less leaf area, confirming the results presented in Table  1
indicating no difference among cultivars in transpiration rate.

     Soybeans frequently follow corn in the crop rotation.  It is not unusual
for low levels of atrazine to persist into the following year if the weather
has been cool and dry during the treatment year.  Ladlie _e_t _al_. (H) showed
that low levels of atrazine stimulated, whereas higher levels inhibited
transpiration.  The data obtained in this study (Tables 3 and 4) failed  to
show the stimulation of transpiration by low rates of atrazine but did show
inhibition of transpiration at the 0.84 kg/ha rate.

     In 1975 low levels of metribuzin acted similarly to atrazine; however,
in 1976, 0.28 kg/ha of metribuzin significantly stimulated transpiration
(Table 5) .  The cultivar Tracy appeared to be much more susceptible to
metribuzin injury than the other cultivars.

     Metribuzin did not appear to interact with ozone fumigation to affect
transpiration (Table 6), whereas in another study the presence of 0.14 kg/ha
of atrazine in the soil appeared to interact with the ozone fumigation to
reduce transpiration (Table 7).  These results could have occurred if ozone
fumigation initially caused an increase in water loss resulting in increased
atrazine uptake.  The increased atrazine caused stomatal closure and conse-
quent decreased water loss of transpiration.

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Figure 1.   Top left,  Dare treated with 1.12 kg/ha bentazon and 0.0 ppm ozone.
           Visual injury 30-35%.   Bottom left, Dare treated with 1.12 kg/ha
           bentazon and 0.40 ppm ozone.   Visual injury 75-80%.  Top right,
           Hood treated with 1.12 kg/ha  bentazon and 0.0 ppm ozone.  Visual
           injury 25-30%.  Bottom right, Hood treated with 1.12 kg/ha bentazon
           and 0.40 ppm ozone.   Visual injury  35-40%.

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TABLE 1.  THE INFLUENCE OF BENTAZON, CULTIVARS, AND OZONE LEVELS ON
          TRANSPIRATION OF 3-WEEK-OLD SOYBEAN




Bentazon Cultivar Ozone Level
Rate (kg/ha) (ppm)
Transpiration3
March

6 days after 3 days after o-
bentazon treatment zone fumigation
(% of base rate) (% of base rate)
0





.56





1.12






0
.56
1.12

0
.56
1.12
0
.56
1.12
0
.56
1.12
Dare
Dare
Hood
Hood
Tracy
Tracy
Dare
Dare
Hood
Hood
Tracy
Tracy
Dare
Dare
Hood
Hood
Tracy
Tracy
Effect of bentazon



Effect of bentazon
Dare


Hood


Tracy


0
.4
0
.4
0
.4
0
.4
0
.4
0
.4
0
.4
0
.4
0
,4
99 cd
90 abed
88 abc
93 abed
92 abed
97 bed
92 abed
90 abed
94 abed
83 a
100 cd
101 d
84 ab
83 a
92 abed
82 a
84 ab
85 ab
139 bed
124 abc
116 ab
138 bed
110 a
142 bed
131 abed
142 bed
138 bed
130 abed
155 d
149 cd
143 bed
136 abed
151 cd
142 bed
150 cd
142 bed
averaged over cultivar and ozone levels



x varieties









93 b
93 b
85 a
averaged over ozone levels
94 be
91 ab
84 a
90 ab
88 ab
87 ab
95 be
101 c
84 a
128 a
141 b
144 b

132 ab
137 abc
139 abc
127 a
134 abc
147 be
126 a
152 c
146 be
                                                    (continued)

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Table 1 (continued)
                                                    Transpirations
                                         March	
Bentazon     Cultivar     Ozone level   6 days after     3 days after ozone
Rate (kg/ha)                 (ppm)      bentazon treatment  fumigation
                     	(% of base rate) (% of base rate)

          Effect of ozone averag^ over cultivars and bentazon levels

                              0               92 a              137 a
                             .4               89 a              138 a
          Effect of cultivar averaged over all bentazon and ozone levels

              Dare                            90 a              136 a
              Hood                            89 a              136 a
              Tracy                           93 a              141 a
Cleans within columns with similar letters are not significantly different
 at the 5% level by Duncan's Multiple Range Test.
                                     10

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TABLE 2.  THE INFLUENCE OF BENTAZON, CULTIVARS, AND OZONE LEVEL ON WATER LOSS
          FROM 3-WEEK-OLD SOYBEAN.

Bentazon Rate Cultivar
(kg/ha)
0 Dare

Hood

Tracy

.84 Dare

Hood

Tracy

Ozone Level
(ppm)
0
.4
0
.4
0
.4
0
.4
0
.4
0
.4
Water Loss (3 day average)
after ozone fumigation3
(gin/plant)
4.5 d
4.4 cd
4.0 be
3.9 b
4.4 cd
4.8 d
2.4 a
2.7 a
2.7 a
2.5 a
3.6 b
3.6 b
        Effect of bentazon averaged over cultivars and ozone levels

        0                                                   4.3 b
        ,84                                                  2.9 a
        Effect of cultivars averaged over bentazon and ozone levels

                      Dare                                  3.5 b
                      Hood                                  3.2 a
                      Tracy                                 4.1 c
        Effect of bentazon x cultivars averaged over ozone levels
0
.84
0
.84
0
.84
Dare

Hood

Tracy

4
2
3
2
4
3
.5 d
.5 a
.9 c
.6 a
.6 d
.6 b
  LMeans within columns with similar letters are not significantly different
  at the 5% level by Duncan's Multiple Range Test.
                                     11

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TABLE 3.  THE INFLUENCE OF SIMULATED ATRAZINE RESIDUE LEVELS ON THE TRANSPIRA-
          TION OF THREE CULTIVARS OF 3-WEEK-OLD SOYBEAN.	

 Atrazine Rate                     Cultivar                Transpiration3
     (kg/ha)                                                 (gm/plt/24 hr)
       0                             Dare                         8.7  d
                                     Hood                         8.2  be
                                     Tracy                        8.5  cd

       .07                            Dare                         8.8  d
                                     Hood                         8.0  ab
                                     Tracy                        8.7  d
,14 Dare
Hood
Tracy
8.6 d
7.8 a
8.5 cd
            Effect of atrazine averaged over all  three  cultivars

       0                                                          8.5  a
       .07                                                         8.5  a
       .14                                                         8.5  a
             Effect of  cultivars averaged over all atrazine  three  levels

                                     Dare                         8.7  b
                                     Hood                         8/0  a
                                     Tracy                        8.5  b
 cleans within  columns with  similar  letters are not  significantly  different  at
 the 5% level  by Duncan's Multiple  Range Test.
                                      12

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 TABLE 4.  THE INFLUENCE OF ATRAZINE ON THE TRANSPIRATION  AND  LEAF  AREA OF
	THREE CULTIVARS OF  3-WEEK-OLD  SOYBEAN	


                                          Transpirational
Atrazine Rate           Cultivar           Water Loss3           Leaf  Area
     (kg/ha)	(g/plt/24 hr)          (cm2/plt)
0 Dare
Hood
Tracy
.28 Dare
Hood
Tracy
.56 Dare
Hood
Tracy
.84 Dare
Hood
Tracy
10
10
10
10
9
10
10
10
9
9
9
9
.0
.1
.2
.0
.8
.2
.3
.0
.7
.5
.4
.4
cde
cde
de
cde
bed
de
e
cde
abc
a
a
a
81
87
83
86
89
85
78
82
69



.1
.2
.2
.6
.9
.4
.4
.4
.0
-
-
—
a
a
a
a
a
a
a
a
a



                Effect  of atrazlne  averaged over  all  three  cultivars

       0                                        10.0 b               83.9  a
      .28                                       10.0 b               97.3  a
      .56                                       10.0 b               76.6  a
      .84                                        9.4
         within columns  with similar letters  are  not  significantly  different
   at  the 5%  level  by Duncan's  Multiple  Range Test.
                                      13

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TABLE 5.  THE INFLUENCE OF METRIBUZIN ON TRANSPIRATION AND LEAF AREA OF THREE
          CULTIVARS OF 3-WEEK-OLD SOYBEAN.
Metribuzin Rate Cultivar
0 Dare
Hood
Tracy
.14 Dare
Hood
Tracy
.28 Dare
Hood
Tracy
.56 Dare
Hood
Tracy
Experiment I
Transpirationa
(g/plt/24 hr)
10.0 cde
10.1 cde
10.0 cde
10.3 de
9.9 cd
10.6 e
10.0 cde
9.6 cd
8.8 b
9.6 cd
9.5 c
7.1 a
Experiment II
Leaf areaa Transpiration3-
(cm2/plt) (g/plt/24 hr)
81.1 ab
87.2 abc
83.2 abc
94.3 c
93.8 c
90.9 be
88.2 be
86.3 abc
Dead
82.8 abc
75.6 a
Dead
8.7 b
8.2 a




9.6 c
8.4 ab

8.3 ab
8.1 a

               Effect of metribuzin averaged over all three cultivars

      0                              10.0 c                          8.5 a
      .14                             10.3 c
      .28                              9.5 b                          9.0 b
      .56                              8.7 a                          8.2 a
  weans within columns with similar letters are not significantly different
  at the 5% level by Duncan's Multiple Range Test.
                                     14

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TABLE 6.  THE INFLUENCE OF METRIBUZIN AND OZONE ON THE TRANSPIRATION OF  3-
          WEEK-OLD SOYBEAN

Metribuzin Rate Cultivar Ozone Level
(kg/ha) (ppm)
0 Dare 0
.3
.4
Hood 0
.3
.4
.56 Dare 0
.3
.4
Hood 0
.3
.4
Transpiration3-
(% of irate prior to fumigation)
82.4 a
86.1 a
89.3 a
92.1 a
86.7 a
86.4 a
95.3 a
85.1 a
86.3 a
97.0 a
81.1 a
80.9 a
  i^eans with similar letters are not significantly at the 5% level by
  Duncan's Multiple Range Test.
                                      15

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TABLE 7-  THE INFLUENCE OF ATRAZINE AND OZONE ON THE TRANSPIRATION OF  3-WEEK-
          OLD SOYBEAN

Atrazine Rate Cultivar Ozone Level
(kg/ha) (ppm)
0 Dare 0
.3
.4
Hood 0
.3
.4
Tracy 0
.3
.4
.14 Dare 0
.3
.4
Hood 0
.3
.4
Tracy 0
.3
.4
Transpiration3
(% of rate prior to fumigation)
82.4 abc
85.1 abc
89.3 abc
92.1 be
86.7 abc
86.4 abc
94.6 be
70.7 a
83.6 abc
91.1 be
92.0 be
80.4 ab
101.9 c
83.6 abc
70.0 a
91.4 be
86.9 abc
78.4 ab
     Main effect of ozone averaged over cultivars and atrazine rates

                                    0                  92.3 b
                                   .3                  84.2 a
                                   .4                  81.4 a
     Main effect of cultivars averaged over ozone and atrazine rates

                  Dare                                 86.7 a
                  Hood                                 86.8 a
                  Tracy                                84.3 a
     Main effect of atrazine averaged over ozone and cultivars

       0                                               85.7 a
      .14                                              86.2 a
                                                (continued)


                                      16

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TABLE 7 (continued)
 Atrazine Rate     Cultivar     Ozone Level           Transpiration3
   (kg/ha)                          (ppm)       (% of rate prior  to  fumigation)
Atrazine-ozone interaction
0 0
.3
.4
.14 0
.3
.4
averaged over all cultivars
89.
80.
86.
94.
87.
76.
7 be
9 ab
4 abc
8 c
5 be
3 a
  Tieans with  similar  letters  are not  significantly different by Duncan's
  Multiple Range  Test.
                                      17

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                                  REFERENCES
 1.   Hill,  A.C.  and L.  Littlefield.   Ozone.  Effect of apparent photosynthesis,
          rate of transpiration,  and stomatal closure in plants.   Environ.
          Sci. Technol.  3:52-56.   1969.

 2.   Tingey,  D.T.,  R.A. Reinert and H.B.  Carter.   Soybean Cultivars.   Acute
          foliar response to ozone.   Crop Sci.   12:268-270.   1972.

 3.   Fuehring, H.D.  Effect of antitranspirants on field of  grain sorghum
          under limited irrigation.   Agron.  J.   65:348-351.   1973.

 4.   Dugger,  W.M.,  Jr., and I.P-  Ting.   Air  pollution oxidants-their  effects
          on metabolic  processes  in plants.   Annual.  Rev. Plant.  Physiol.
          21:215-234.  1970.

 5.   Hodgeson, R.H.  Alteration of diphenamid metabolism by  ozone.   Weed Sci.
          Soc. Amer. Abstr. No. 86.   1971.

 6.   Carney,  A.W.,  G.R. Stephenson,  D.P.  Ormrod,  and  G.C. Ashton.  Ozone-
          herbicide interactions  in crop  plants.   Weed Sci.  21:508-511.   1973.

 7.   Tingey,  D.T.,  C. Standley, and R.W.  Field.  Stress ethylene  evolution:
          a measure of  ozone effects on plants.  Atmospheric Environment
          10:969-974.  1976.

 8.   Hartwig, Edgar E.   "Tracy1-  A New Soybean Variety"  MAFES Mississippi
          Agricultural  and Forestry Experiment Station, December, 1973.
          Information Sheet 1227.

 9.   Heck,  W.W., J.A. Dunning, and H. Johnson.   Design for a simple plant
          exposure chamber.  National Center for Air  Pollution Control Publica-
          tion APTD - 68-6.  1968.

10.   Penner,  D.  Bentazon selectivity between soybean and Canada  thistle.
          Weed Res.  15:259-262.   1975.

11.   Ladlie,  J.S.,  W.F. Meggitt,  and D. Penner.  Effect of atrazine on soybean
          tolerance to  metribuzin.  Weed Sci.  25:115-121.  1971.
                                      18

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                                   TECHNICAL REPORT DATA
                            (Please read Instructions on the reverse before completing)
  REPORT NO.
    EPA-600/3-78-047
             3. RECIPIENT'S ACCESSI ON1 NO.
4. TITLE AND SUBTITLE
 Interaction  of  Ozone and Herbicides in Soybeans
                                                          5. REPORT DATE
                                                                  May 1978
                                                          6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
 Raj Bahadur
                                                          8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
 Mississippi Valley State University
 Itta Bena, Mississippi  38941
             10. PROGRAM ELEMENT NO.
              1AA602
             11. CONTRACT/GRANT NO.

               R-SC3072
 12. SPONSORING AGENCY NAME AND ADDRESS
 U. S. Environmental Protection Agency
 Corvallis  Environmental Research Laboratory,  EBRD,  TEB
 Corvallis,  OR  97330
             13. TYPE OF REPORT AND PERIOD COVERED
              Final   Dec  1974  to  Mar 1977
             14. SPONSORING AGENCY CODE
               EPA/600/02
 15. SUPPLEMENTARY NOTES
 16'ABSTRACTThe  purpose of this investigation was  to  identify potentially damaging  herbi-
 cide-ozone  interactions on soybean so that injury  could be reduced by judicious  selec-
 tion of herbicides and tolerant cultivars.   The involvement of herbicide influence on
 stomatal  aperture affecting ozone fumigation injury was studied.
      Soybean  cultivars with varying sensitivity to ozone were planted in the  greenhous
 in soil containing residual levels of atrazine, normally recommended levels of metri-
 buzin or  nontreated soil.  The herbicide bentazon  was  also studied, but was applied to
 foliage when  the soybeans were 3 weeks old.   After the soybean plants were in the
 second trifoliolate  leaf stage or 3 to 7 days  after bentazon treatment they were
 fumigated with 0, 0.3, or 0.4 ppm ozone.  Transpiration rates were monitored  to
 evaluate  potential interactions.
      Atrazine, metribuzin, and bentazon all  affected transpiration.  The transpiration
 of the ozone  tolerant Hood cultivar was not  affected by bentazon alone, but after
 treatment,  transpiration increased significantly.   The presence of atrazine residues
 in the soil decreased transpiration after ozone fumigation.  In the ozone-susceptible
 Dare cultivar, bentazon inhibited transpiration, but following ozone fumigation  there
 were significant differences.  The cultivar  Tracy  proved to be very susceptible  to
 metribuzin  injury and was intermediate in sensitivity  to ozone injury.  It responded
 like Dare to  bentazon alone, and like Hood following ozone fumigation.
                                KEY WORDS AND DOCUMENT ANALYSIS
                  DESCRIPTORS
                                              b. IDENTIFIERS/OPEN ENDED TERMS
                          c.  COSATI Field/Group
 ozone
 herbicides
 soybeans
 transpiration
 atrazine
 metrabuzin
 bentazon
  air pollution
  terrestrial effects
02/A,D
06/F
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                                            19
  , U.S. GOVERNMENT PRINTING OFFICE, 1978-796-778/142 REGION 10

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