EPA-600/3-76-031
March 1976
Ecological Research Series
EFFECTS OF OZONE ON
NITROGEN FIXATION IN LADINO CLOVER
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Corvallis, Oregon 97330
-------
RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into five series. These five broad
categories were established to facilitate further development and application of
environmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal
species, and materials. Problems are assessed for their long- and short-term
influences. Investigations include formation, transport, and pathway studies to
determine the fate of pollutants and their effects. This work provides the technical
basis for setting standards to minimize undesirable changes in living organisms
in the aquatic, terrestrial, and atmospheric environments.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
-------
EPA-600/3-76-031
March 1976
EFFECTS OF OZONE ON NITROGEN FIXATION
IN LADING CLOVER
Udo Blum and Michael Letchworth
North Carolina State University
Raleigh, North Carolina 27607
EPA-IAG-D5-0416
Project Officer
Dr. L. C. Raniere
Ecological Effects Research Division
Corvallis Environmental Research Laboratory
Con/all is, Oregon 97330
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
CORVALLIS ENVIRONMENTAL RESEARCH LABORATORY
CORVALLIS, OREGON 97330
-------
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.
-------
ABSTRACT
Research was performed to determine the effects of ozone on ladino
clover growth, nodulation, nitrogen fixation, nitrogen content, phenol
content, energy value and root exudates. Plants were exposed once or
twice to carbon-filtered air, 30 or 60 pphm ozone for 2 hrs. The effects
of ozone on growth, nitrogen fixation, % nitrogen, total nitrogen and
nodulation varied with age of plant and ozone concentration. Caloric
content of tops (plants 4 weeks old when exposed) was reduced with two
exposures of 30 or 60 pphm ozone. Root caloric content was not affected.
Phenol content of tops and roots (plants 6 weeks old when exposed) was
reduced with two 30 pphm ozone exposures. Clover exudates were not
detectably modified by ozone exposure.
This report was submitted for limited distribution in partial
fulfillment of an Interagency Agreement by the Agricultural Research
Service (ARS) under the sponsorship of the Environmental Protection
Agency (EPA). The research included in the report was cooperatively
sponsored by ARS, EPA and the North Carolina Agricultural Experiment
Station. Work was completed as of June 30, 1975.
-------
ACKNOWLEDGMENTS
Parts of this research were conducted in the North Carolina State
Unit of the Southeastern Plant Environment Laboratories. The help of the
staff is appreciated.
The assistance of Mr. Hans Hamann in statistical design and analysis
is gratefully acknowledged.
IV
-------
CONTENTS
Page
Abstract iii
Acknowledgements ' iv
List of Figures vi
List of Tables vii
I Introduction 1
II Conclusions 2
III Recommendations 3
IV Experimental Work 4
V References 21
-------
LIST OF FIGURES
Number Page
1 The effect of two 2-hr, ozone exposures on the
top dry weight of ladino clover plants. 7
2 The effect of two 2-hr, ozone exposures on the
root dry weight of ladino clover. 8
3 Changes in nodulation of ladino clover plants
following two 2-hr, ozone exposures. 9
4 Effects of ozone exposures on nitrogenase
activity/nodule of ladino clover. 10
5 Changes in nitrogenase activity/plant of ladino
clover following ozone exposure. 11
6 Effect of two 2-hr, ozone exposures on % Nitrogen
of ladino clover tops. 12
7 Effect of two 2-hr, ozone exposures on % Nitrogen
of ladino clover roots. 13
8 The effect of two 2-hr, ozone exposures on the
total N content of ladino clover tops. 14
9 The effect of two 2-hr, ozone exposures on the
total N content of ladino clover roots. 15
10 The effect of two 2-hr, ozone exposures on the
total plant nitrogen of ladino clover. 16
-------
LIST OF TABLES
Number Page
1 Caloric Content, Top Dry Weight, Root Dry Weight
and Nodule Number of Ladino Clover Exposed to
Ozone or Carbon Filtered Air. 17
2a Phenol Content, Nitrogen Content, Top Dry Weight,
Root Dry Weight and Nodule Number of Ladino
Clover Exposed to Ozone and Carbon-Filtered Air. 18
2b Top Dry Weight, Root Dry Weight, Nodule Number,
Nitrogen Content and Phenol Content of Ladino
Clover treated with Root Exudates. 18
3 Top Dry Weight, Root Dry Weight, and Nodule
Number of Ladino Clover exposed to ozone or
carbon^filtered Air Grown Separately or in
Combination (Design is such that plants interact
only by way of root exudates). 19
4 Root Exudates vs Rhizobium 20
-------
SECTION I
INTRODUCTION
The research presented in this report is part of a continuing coop-
erative project between the Agricultural Research Service, the Environ-
mental Protection Agency and the North Carolina Agricultural Experiment
Station. The title of the overall project is, "Effects, fates and trans-
formations of selected air pollutants in plants, microorganisms and soils."
The primary objectives of this cooperative program are to understand
the impact of air pollutants on plants, microorganisms and soils that are
of importance to agriculture, and to assist other agencies in relation to
their mission of protecting the agricultural segment of the environment.
The research thrust is directed at comparative studies on vegetation
effects under phytotron, greenhouse and field conditions. Emphasis is
on: (1) dose-response curves; (2) the interaction of various factors on
the response of the whole plant to air pollutants; (3) assessing the impact
of controlled pollutant additions and ambient pollution on plant biomass,
yield and quality in the greenhouse and field, and on pollutant uptake and
transformations in the greenhouse; (4) acute and chronic screens; and,
(5) varietal responses.
Research reported here contains, as its major thrust, part of the
phytotron (controlled environment) portion of the foregoing cooperative
program. It was determined that ladino clover should be intensively
studied under controlled conditions. The results should then be verified
under greenhouse and field conditions and using selected other plant
species. Ladino clover was selected as one of the most important forage
species of the Southeast. Its use is increasing, especially in conjunction
with certain grass species. Ladino has chronic problems under field
cultivation that are not understood - air pollution may be a factor.
Ladino is a legume and thus one of the important nitrogen fixing crop
species. These factors make it an ideal test species.
The specific objectives for this research are given in Section IV.
-------
SECTION II
CONCLUSIONS
Effects of ozone, on Ladino clover growth and nodulation varies with
age of plant and concentration of ozone.
Nitrogen content of plants exposed to 60 pphm of ozone was reduced
for all exposure ages." Nitrogen content of plants exposed to 30 pphm of
ozone varied with exposure age.
Caloric content of tops (plants 4 weeks old when exposed) was reduced
by two ozone exposures of 30 or 60 pphm. Caloric content of roots was not
affected.
Phenol content and nitrogen content of tops and roots (plants 6 weeks
old when exposed) were reduced with two 30 pphm ozone exposures.
Clover root exudates were not detectably modified by ozone exposure
of clover.
-------
SECTION III
RECOMMENDATION(S)
Present indications are that the reductions of ladino clover (a
forage crop) growth, nodulation, nitrogen content, energy content, and
phenol content, observed as a result of ozone exposures, are closely re-
lated to plant energy fixation and plant energy allocation. The effects
of ozone on energy fixation and distribution need to be explored further,
The following is recommended:
1. determination of ozone effects on carbohydrate content.
2. determination of ozone effects on metabolic pathways -
ie glycolysis, carbon cycle, and monophosphate shunt.
3. determination of ozone effects on translocation.
-------
SECTION IV
EFFECTS OF OZONE ON NITROGEN FIXATION IN LADING CLOVER
Most soils continually lose nitrogen and thus require added nitrogen
to maintain soil fertility. Leguminous crops are an important source of
such nitrogen. The amount added by legumes depends on plant growth and
vigor as well as certain edaphic factors (ie. nutrient levels, water
availability, pH and temperature). Exposure of soybean, pinto bean, and
ladino clover to ozone reduces growth and nodulation (1-3). How these
relate to quantitative and qualitative crop production is uncertain.
The present research was undertaken with Ladino clover to determine
the effects of ozone on: growth, nodulation, nitrogen fixation, nitrogen
content, phenol content, energy value; and root exudates.
MATERIAL AND METHODS
Experiment 1
Inoculated seeds of Trifolium repens L. c.v. Tillman were planted
in gravel and Jiffy Mix (2:1).Complete nutrient solution was added to
pots every third day. Distilled water was given at other times. Plants
were grown and exposed to ozone in the Southeastern Plant Environmental
Laboratories. Growth chamber environment was as follows: 12 hrs at
45000 lux, the night was interrupted for 3 hrs with 5500 lux, temperature
22C day and 19C night, and a relative humidity of 50% during the day and
60% at night. Plants were subjected to two 2 hr exposures (one week apart)
of either carbon-filtered air, 30 or 60 pphm ozone. Groups of plants
were exposed at two week intervals to determine age effects. The first
of 4 harvests for each exposure age occurred 1 week after the second
exposure. Dry weight of tops (TDW), roots (ROW), nitrogenase activity
and nitrogen content were determined.
Experiment 2
Plants were grown in sand and Jiffy Mix (2:1) for one month in a
greenhouse. A 1/10 normal nitrogen nutrient solution was added every
third day. Plants were exposed to carbon-filtered air, 30 or 60 pphm
ozone for 2 hrs. Half of the plants were harvested one week later. The
remaining plants were exposed again and harvested the following week.
TDW, ROW, nodule number, and caloric content of tops (TPCAL) and roots
(RTCAL) were determined in each case.
-------
Experiment 3
Plants were grown in sand for 6 weeks and then exposed to carbon-
filtered air or 30 pphiri of ozone (2 hr) twice 3 days apart in a green-
house. A 1/10 normal nitrogen nutrient solution was added every third
day. Deionized water was added at other times. Root exudates were
collected 7 days after the last exposure. One hundred ml was collected
from each pot. Exudates were collected the day before nutrient appli-
cation. This was done to minimize inorganic ion input into the exudate.
The exudate was used to water 3 week old seedlings of clover for 14 con-
secutive days. All plants were then harvested. TDW, ROW, nodule number,
nitrogen content, and phenol content (4) were determined.
Experiment 4
»
Plants were grown in sand culture with 1/10 normal nitrogen nutrient
solution as described by Kochhar (1974). Four week old plants were exposed
twice 3 days apart to carbon-filtered air, 30 or 60 pphm ozone. Plants
were harvested 4 weeks later. TDW, RDM and nodule number were determined.
Root exudates (100 ml) from each reservoir were flash evaporated and taken
up in acetone (5 ml). Acetone fraction was tested for toxicity against
Rhizobium with sensitivity disks.
All data was subjected to analysis of variance unless otherwise stated.
Least significant differences at the 5% probability level were used to
illustrate differences among treatment means.
RESULTS
Experiment 1
Growth and Nodulation--
Fig l-3iA double exposure of ozone reduced top growth, root growth,
and nodulation. Plant responses to ozone varied with age and concentration
of ozone. '
Nitrogenase activity—
Fig 4-5. Some stimulation of nitrogen activity per nodule was observed
for exposure age 28+35 and 42+49. However, these responses were not signifi-
cantly different from the controls. Nitrogenase activity per plant was
reduced for exposure age 14+21.
Nitrogen content—
Fig 6-10 [Also see Table 2a] Percent nitrogen for tops and roots was
significantly higher for exposure age 14+21 for both the 30 and 60 pphm ozone
exposures. Percent nitrogen was higher for tops with the 30 pphm exposures
and higher for the roots with*the 60 pphm exposures for exposure age 28+35.
Total plant nitrogen was reduced for exposure age 14+21 and 28+35 for both
ozone exposures. For exposure age 42+49 and 56+63 only the 60 pphm exposures
reduced total nitrogen content of clover plants. Similar trends were ob-
served for nitrogen content of tops and roots.
-------
Experiment 2
Energy Content--
Table 1. Caloric content was significantly reduced for the tops with
2 ozone exposures of 30 or 60 pphm. Caloric content of roots was not
significantly reduced.
Experiment 3
Phenol content--
Table 2a+b. Exposures of ladino clover to 30 pphm of ozone significantly
reduced phenol content of tops and roots. Nitrogen content was also deter-
mined here.
Nitrogen of tops and roots was significantly reduced from controls.
Experiments 3 and 4
Root Exudates—
Tables 2b, 3, 4. Exudates from ozone exposed plants did not inhibit
the growth or nodulation of ladino clover seedlings when compared to con-
trol plant exudates. Exudates from ozone exposed plants were not inhibi-
tory to Rhizobium when compared to controls.
-------
o:
o
Q_
o
20
18
16
14
12
10
8
6
4
2
14+21
Figure 1.
CONTROL^' .
30 PPHM 03
60.PPHM 03
iii , LSD-0.05=li27|; J
28+35 42+49
EXPOSURE AGE (DAYS)
56+63
The effect of two 2-hr, ozone exposures on the top
dry weight of ladino clover plants.
A.-significant difference between control and test.
B.-significant difference between 30 and 60 pphm ozone.
-------
3.0
2.0
CO
CD
1.0
o
§
14+21
28+35 42+49
EXPOSURE AGE (DAYS)
56+63
Figure 2. The effect of two 2-hr ozone exposures on the root dry
weight of ladino clover.
A.-significant difference between control and test.
B.-significant difference between 30 and 60 pphm ozone.
8
-------
600
500
400
EC
LU
CQ
i 300
Q
O
200
100
0
14+21
28+35 42+49
EXPOSURE AGE (DAYS)
56+63
Figure 3. Changes in nodulation of ladino clover plants following
two 2-hr, ozone exposures.
A.-significant difference between control and test.
-------
.0050 .
LU
_l
O
0.0040
S
i.0030
LU
O
".0020
.0010
JL
CONTROL
30 PPHM 0,
60 PPHM 0,
LSD-0.05=
0.00181
14+21 28+35 42+49 56+63
EXPOSURE AGE (DAYS)
Figure 4. Effects of ozone exposures on nitrogenase activity/nodule of
ladino clover.
*LSD for analysis of first 2 exposure ages only.
10
-------
3.0
3 2.0
O.
§
o
1.0
14+21
• CONTROL
A 30 PPHM 0
* 60 PPHM 0
LSD-0.05=0.512
28+35 42+49 56+63
EXPOSURE AGE (DAYS)
Figure 5. Changes in nitrogenase activity/plant of ladino clover
following ozone exposure.
A. -significant difference between control and test.
*LSD for analysis of first 2 exposure ages only.
11
-------
1
5.0
4.0
3.0
2.0
1.0
14+21
4.
28+35 42+49 56+63
EXPOSURE AGE (DAYS)
• CONTROL
A 30 PPHM 03
• 60 PPHM 06
LSD-0.05=0.269
Figure 6. Effect of two 2-hr ozone exposures on % Nitrogen of
ladino clover tops.
(.
A.-significant difference between control and test.
• 12
-------
en
5.0
4.0
3.0
2.0
1.0
0
14+21
J_
28+35 42+49
EXPOSURE AGE (DAYS)
56+63
P CONTROL
A 30 PPHM 03
• 60 PPHM 03
LSD 0.05=0.278
Figure 7. Effect of two 2-hr ozone exposures on % Nitrogen of
ladino clover roots.
A.-significant difference between control and test.
13
-------
CO
CD
LJJ
CD
§
55
50
45
40
35
30
25
20
15
10
5
0
14+21
28+35 42+49
EXPOSURE AGE (DAYS)
CONTROL
30 PPHM 0,
60 PPHM 0
3
LSD-0.05=3.69
56+63
Figure 8. The effect of two 2-hr ozone exposures on the total N
content of ladino clover tops.
A.-significant difference between control and test.
B.-significant difference between 30 and 60 pphm ozone.
14
-------
CD
§
14+21
Figure 9.
28+35
42+49
EXPOSURE AGE (DAYS)
56+63
The effect of two 2-hr ozone exposures on the total N
content of ladino clover roots.
A.-significant difference between control and test.
B.-significant difference between 30 and 60 pphm ozone.
15
-------
50
LU
CD
O
cc
Q_
40
30
20
10
14+21
•CONTROL
A30 PPHM 03
• 60 PPHM 03
LSD-0.05=4.06
28+35 42+49
EXPOSURE AGE (DAYS)
56+63
Figure 10. The effect of two 2-hr ozone exposures on the total
plant nitrogen of ladino clover.
A.-significant difference between control and test.
B.-significant difference between 30 and 60 pphm ozone.
16
-------
Table 1 [Ex 2] Caloric Content, Top Dry Weight, Root Dry Weight
and.Nodule Number of Ladino Clover Exposed to
Ozone or Carbon Filtered Air.
TDW
(gm)
ROW NODULE TPCAL RTCAL
(gm) (#) (cal/gm)
Harvest 1
Non-Exposed
30 pphm 03
60 pphm 03
Harvest 2
1.01 c**
0.62 d
0.59 d
0.35 b 137 be
0.21 cd 105 c
0.19 d 96 c
3980 be 3621 a
3951 c 3655 a
3919 c 3683 a
Non-Exposed
30
60
pphm
pphm
LSD
°3
°3
0.05
2.
1.
0.
0.
66 a
36 b
93 c
28
0.
0.
0.
0-
61 a
28 be
19 d
07
239 a
169 b
99 c
58
4136 a
4031 b
3844 d
70
3838 a
3734 a
3712 a
154 (NS)*
* non significant
**means followed by the same letter are not statistically significant
at the 5% level of probability. [N-10].
17
-------
Table 2a [Ex 3] Phenol Content, Nitrogen Content, Top Dry Weight,
Root Dry Weight and Nodule Number of Ladino Clover
Exposed to Ozone and Carbon-Filtered Air.
TDW ROW NODULE NIT* NIT NIT PH* PH PH
(gm.) (gm.) (#) TOPS ROOTS TOT TOPS ROOTS TOT.
(mg) (mg) (mg) (mg) (mg) (mg)
Non-Exposed
Plants 0.78 a** 0.30a
30 pphm 03 0.38 b 0.14b
LSD 0.05 j).16 0.05
243a 34a lla
137b 15b 5b
65.8 8.4 2
45a 168a 14a 182a
20b 85b 4b 89b
10 34 2 35
*NIT=Nitrogen
*PH=Phenol
**Means followed by the same letter are not statistically significant
at the 5% level of probability [N=10].
Table 2b [Ex. 3] Top Dry Weight, Root Dry Weight, Nodule Number,
Nitrogen Content and Phenol Content of Ladino
Clover treated with Root Exudates.
Non-Exposed
Plant
Exudate
30 pphm 0.,
Exposed
Plant
Exudate
TDW ROW NODULE NITROGEN**
(#) TOTAL
(gm) (gm) (mg.)
0.055* 0.024 51.3 0.034
0.0608 0.029 54.6 0.033
PHENOL**
TOTAL
(mg.)
0.16
0.19
*no significant difference found. [N=10]
**Data not analyzed.
18
-------
Table 3 [Ex 4] Top Dry Weight, Root Dry Weight, and Nodule Number
of Ladino Clover exposed to ozone or carbon-filtered
Air Grown Separately or in Combination (Design is
such that plants interact only by way of root
exudates).
N Ex **
N Ex + 30
N Ex + 60
60
30
30 + N Ex
60 + N Ex
LSD 0.05
0.
0.
0.
0.
0.
0.
0.
0.
TDW
(gm)
426 a*
443 a
466 a
214 b
124 c
153 be
109 c
087
0.
0.
0.
} 0.
0.
0.
0.
0.
ROW
(gm)
144 a
133 a
135 a
077 b
032 c
047 c
033 c
028
NODULE
(#)
53.
58.
49.
26.
21.
33.
23.
18.
5
8
6
1.
9
3
3
9
a
a
ab
c
c
be
c
*means followed by the same letter are not statistically significant
at the 5% level of probability {N=10].
** N Ex - Non exposed plant grown alone.
N Ex + 30 - Non exposed plant grown with plants exposed to 30 pphm
of ozone.
\
N Ex + 60 - same as N Ex + 30 but plants exposed to 60 pphm instead
of 30 pphm.
30 N 60 - exposed plants grown alone.
30 + N Ex - exposed plants from N Ex + 30.
60 + N Ex - exposed plants from N Ex + 60.
19
-------
Table 4 [Ex 4] Root Exudates vs Rhizobium
ZONES OF INHIBITION
(nun)
N Ex Plant Exudates ** 0.096 *
N Ex + 30 Exudates 0.081
N Ex +"60 Plant Exudates 0.080
30 Plant Exudates 0.065
60 Plant Exudates 0.040
Acetone 0.15
LSD 0.05 0.10 [NS]
Prob > F 0.099 c.v. = 77%
* Non significant [N=16]
** N Ex - non exposed
30 - plants exposed to 30 pphm ozone
60 - plants exposed to 60 pphm ozone
20
-------
SECTION V
REFERENCES
1. Reinert, R.A., D.T. Tingey, and C.E. Koons. The Early Growth of Soy-
beans as Influenced by Ozone Stress. Agron. Abst. 63:148. 1971.
2. Tingey, D.T. and U. Blum. Effects of Ozone on Soybean Nodules.
J. Environ. Qual 2: 341-342. 1973.
3. Kochhar, M. Phytotoxic and Competitive Effects of Tall Fescue on
Ladino Clover as Modified by Ozone and/or Rhizoctomia So1am'.
Dissertation. 1974.
4. Andersen, R.A. and J.R. Todd. Estimation of Total Tobacco Plant Phenols
by Their Bonding to Polyvinyl Phyrrolitone. Tobacco Science 12: 107-111.
1968.
21
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/3-76-031
3. RECIPIENT'S ACCESSION>NO.
4. TITLE AND SUBTITLE
EFFECTS OF OZONE ON NITROGEN FIXATIO.N IN
LADING CLOVER
5. REPORT DATE
March 1976
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Udo Blum and Michael Letchworth
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Botany Dept.
North Carolina State University
Raleigh, North Carolina 27607
10. PROGRAM ELEMENT NO.
1AA006 ROAP 21ALR Task 24
11. CONTRACT/GRANT NO.
EPA-IAG-D5-0416
12. SPONSORING AGENCY NAME AND ADDRESS
Corvallis Environmental Research Laboratory
Ecological Effects Research Division
Office of Research and Development
U.S. Environmental Protection Agency
Corvallis, Oregon 97330
13. TYPE OF REPORT AND PERIOD COVERED
Interim Final
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Research was performed to determine the effects of ozone on ladino clover growth,
nodulation, nitrogen fixation, nitrogen content, phenol content, energy value and
root exudates. Plants were exposed once or twice to carbon-filtered air, 30 or 60
pphm ozone for 2 hrs. The effects of ozone on growth, nitrogen fixation, % nitrogen,
total nitrogen and nodulation varied with age of plant and ozone concentration.
Caloric content of tops (plants 4 weeks old when exposed) was reduced with two ex-
posures of 30 or 60 pphm ozone. Root caloric content was not affected. Phenol
content of tops and roots (plants 6 weeks old when exposed) was reduced with two 30
pphm ozone exposures. Clover exudates were not detectably modified by ozone exposure,
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
ozone
nitrogen fixation
plant growth
air pollutants
microorganisms
soils
air pollution
ozone exposure
51
B. DISTRIBUTION STATEMEN1
LIMITED
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
28
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
22
GPO 995-565
------- |