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United States
Air Quality Criteria for
Ozone and Related
Photochemical Oxidants
(Second External Review
Draft)
Volume III of
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EPA/600/R-05/004CB
August 2005
Air Quality Criteria for Ozone and Related
Photochemical Oxidants
Volume
National Center for Environmental Assessment-RTF Office
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC
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DISCLAIMER
This document is a second external review draft for review purposes only and does not
constitute U.S. Environmental Protection Agency policy. Mention of trade names or commercial
products does not constitute endorsement or recommendation for use.
PREFACE
National Ambient Air Quality Standards (NAAQS) are promulgated by the United States
Environmental Protection Agency (EPA) to meet requirements set forth in Sections 108 and 109 of
the U.S. Clean Air Act (CAA). Sections 108 and 109 require the EPA Administrator (1) to list
widespread air pollutants that reasonably may be expected to endanger public health or welfare;
(2) to issue air quality criteria for them that assess the latest available scientific information on
nature and effects of ambient exposure to them; (3) to set "primary" NAAQS to protect human
health with adequate margin of safety and to set "secondary" NAAQS to protect against welfare
effects (e.g., effects on vegetation, ecosystems, visibility, climate, manmade materials, etc); and
(5) to periodically review and revise, as appropriate, the criteria and NAAQS for a given listed
pollutant or class of pollutants.
In 1971, the U.S. Environmental Protection Agency (EPA) promulgated National Ambient
Air Quality Standards (NAAQS) to protect the public health and welfare from adverse effects of
photochemical oxidants. The EPA promulgates the NAAQS on the basis of scientific information
contained in air quality criteria issued under Section 108 of the Clean Air Act. Following the
review of criteria as contained in the EPA document, Air Quality Criteria for Ozone and Other
Photochemical Oxidants published in 1978, the chemical designation of the standards was changed
from photochemical oxidants to ozone (O3) in 1979 and a 1-hour O3 NAAQS was set. The 1978
document focused mainly on the air quality criteria for O3 and, to a lesser extent, on those for other
photochemical oxidants (e.g., hydrogen peroxide and the peroxyacyl nitrates), as have subsequent
revised versions of the ozone document.
Ill-ii
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To meet Clean Air Act requirements noted above for periodic review of criteria and
NAAQS, the O3 criteria document, Air Quality Criteria for Ozone and Other Photochemical
Oxidants, was next revised and then released in August 1986; and a supplement, Summary of
Selected New Information on Effects of Ozone on Health and Vegetation, was issued in January
1992. These documents were the basis for a March 1993 decision by EPA that revision of the
existing 1-h NAAQS for O3 was not appropriate at that time. That decision, however, did not take
into account some newer scientific data that became available after completion of the 1986 criteria
document. Such literature was assessed in the next periodic revision of the O3 air quality criteria
document (completed in 1996) and provided scientific bases supporting the setting by EPA in 1997
of an 8-h O3 NAAQS that is currently in force together with the 1-h O3 standard.
The purpose of this revised air quality criteria document for O3 and related photochemical
oxidants is to critically evaluate and assess the latest scientific information published since that
assessed in the above 1996 Ozone Air Quality Criteria Document (O3 AQCD), with the main focus
being on pertinent new information useful in evaluating health and environmental effects data
associated with ambient air O3 exposures. However, some other scientific data are also presented
and evaluated in order to provide a better understanding of the nature, sources, distribution,
measurement, and concentrations of O3 and related photochemical oxidants and their precursors in
the environment. The document mainly assesses pertinent literature published or accepted for
publication through 2004.
The present Second Draft O3 AQCD (dated August 2005) is being released for public
comment and review by the Clean Air Scientific Advisory Committee (CASAC) to obtain
comments on the organization and structure of the document, the issues addressed, the approaches
employed in assessing and interpreting the newly available information on O3 exposures and
effects, and the key findings and conclusions arrived at as a consequence of this assessment. Public
comments and recommendations will be taken into account making any appropriate further
revisions to this document for incorporation into the final version of the document to be completed
and issued by February 28, 2006. Evaluations contained in the present document will be drawn on
to provide inputs to associated PM Staff Paper analyses prepared by EPA's Office of Air Quality
Planning and Standards (OAQPS) to pose options for consideration by the EPA Administrator with
regard to proposal and, ultimately, promulgation of decisions on potential retention or revision, as
appropriate, of the current O3 NAAQS.
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Preparation of this document was coordinated by staff of EPA's National Center for
Environmental Assessment in Research Triangle Park (NCEA-RTP). NCEA-RTP scientific staff,
together with experts from other EPA/ORD laboratories and academia, contributed to writing of
document chapters. Earlier drafts of document materials were reviewed by non-EPA experts in
peer consultation workshops held by EPA. The document describes the nature, sources,
distribution, measurement, and concentrations of O3 in outdoor (ambient) and indoor environments.
It also evaluates the latest data on human exposures to ambient O3 and consequent health effects in
exposed human populations, to support decision making regarding the primary, health-related O3
NAAQS. The document also evaluates ambient O3 environmental effects on vegetation and
ecosystems, man-made materials, and surface level solar UV radiation flux and global climate
change, to support decision making on secondary O3 NAAQS.
NCEA acknowledges the valuable contributions provided by authors, contributors, and
reviewers and the diligence of its staff and contractors in the preparation of this draft document.
Ill-iv
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Air Quality Criteria for Ozone and Related
Photochemical Oxidants
(Second External Review Draft)
VOLUME I
Executive Summary E-l
1. INTRODUCTION 1-1
2. PHYSICS AND CHEMISTRY OF OZONE IN THE ATMOSPHERE 2-1
3. ENVIRONMENTAL CONCENTRATIONS, PATTERNS, AND
EXPOSURE ESTIMATES 3-1
4. DOSIMETRY, SPECIES HOMOLOGY, SENSITIVITY, AND
ANIMAL-TO-HUMAN EXTRAPOLATION 4-1
5. TOXICOLOGICAL EFFECTS OF OZONE AND RELATED
PHOTOCHEMICAL OXIDANTS IN LABORATORY ANIMALS
AND IN VITRO TEST SYSTEMS 5-1
6. CONTROLLED HUMAN EXPOSURE STUDIES OF OZONE AND
RELATED PHOTOCHEMICAL OXIDANTS 6-1
7. EPIDEMIOLOGICAL STUDIES OF HUMAN HEALTH EFFECTS
ASSOCIATED WITH AMBIENT OZONE EXPOSURE 7-1
8. INTEGRATIVE SYNTHESIS: EXPOSURE AND HEALTH EFFECTS 8-1
9. ENVIRONMENTAL EFFECTS: OZONE EFFECTS ON
VEGETATION AND ECOSYSTEMS 9-1
10. TROPOSPHERIC OZONE EFFECTS ON UV-B FLUX AND
CLIMATE CHANGE PROCESSES 10-1
11. EFFECT OF OZONE ON MAN-MADE MATERIALS 11-1
III-v
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Air Quality Criteria for Ozone and Related
Photochemical Oxidants
(Second External Review Draft)
(cont'd)
VOLUME II
CHAPTER 2 ANNEX (ATMOSPHERIC PHYSICS/CHEMISTRY) AX2-1
CHAPTER 3 ANNEX (AIR QUALITY AND EXPOSURE) AX3-1
CHAPTER 4 ANNEX (DOSIMETRY) AX4-1
CHAPTER 5 ANNEX (ANIMAL TOXICOLOGY) AX5-1
CHAPTER 6 ANNEX (CONTROLLED HUMAN EXPOSURE) AX6-1
CHAPTER 7 ANNEX (EPIDEMIOLOGY) AX7-1
VOLUME III
CHAPTER 9 ANNEX (ENVIRONMENTAL EFFECTS) AX9-1
Ill-vi
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Table of Contents
Page
List of Tables ni-xii
List of Figures III-xiv
Authors, Contributors, and Reviewers III-xvi
U.S. Environmental Protection Agency Project Team for Development of Air
Quality Criteria for Ozone and Related Photochemical Oxidants III-xviii
U.S. Environmental Protection Agency Science Advisory Board (SAB) Staff Office
Clean Air Scientific Advisory Committee (CASAC) Ozone Review Panel III-xxi
Abbreviations and Acronyms III-xxiv
AX9. ENVIRONMENTAL EFFECTS: OZONE EFFECTS ON VEGETATION
AND ECOSYSTEMS AX9-1
AX9.1 METHODOLOGIES USED IN VEGETATION RESEARCH AX9-1
AX9.1.1 Introduction AX9-1
AX9.1.2 Methods Involving Experimental Exposures to Ozone .... AX9-2
AX9.1.2.1 "Indoor", Controlled Environment,
and Greenhouse Chambers AX9-2
AX9.1.2.2 Field Chambers AX9-4
AX9.1.2.3 Plume Systems AX9-7
AX9.2.2.4 Comparative Studies AX9-9
AX9.1.2.5 Ozone Generation Systems AX9-12
AX9.1.2.6 Experimental Exposure Protocols AX9-12
AX9.1.3 Methods Involving Exposures to Ozone in
Ambient Air AX9-13
AX9.1.3.1 Air-Exclusion Systems AX9-14
AX9.1.3.2 Natural Gradients AX9-14
AX9.1.3.3 Use of Chemical Protectants AX9-15
AX9.1.3.4 Biomonitoring AX9-18
AX9.1.3.5 Calibrated Passive Monitors AX9-27
AX9.1.4 Numerical/Statistical Methodologies AX9-27
AX9.1.5 Improved Methods for Defining Exposure AX9-30
REFERENCES AX9-31
AX9.2 SPECIES RESPONSE/MODE-OF-ACTION AX9-39
AX9.2.1 Introduction AX9-39
AX9.2.2 Mechanisms of Ozone-Induced Plant Alterations AX9-42
AX9.2.2.1 Changes in Metabolic Processes:
Current Theories AX9-43
AX9.2.2.2 Modifications of Plant Physiological
Processes AX9-44
AX9.2.3 Ozone Uptake by Leaves AX9-45
AX9.2.3.1 Possible Reactions Within the Leaf AX9-53
AX9.2.3.2 Toxicants Within the Wall Space AX9-56
AX9.2.3.3 Products of Ozone AX9-59
AX9.2.3.4 Antioxidants Within the Apoplastic
Space AX9-65
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Table of Contents
(cont'd)
Page
AX9.2.4 Wounding and Pathogen Attack AX9-75
AX9.2.4.1 Peroxidases AX9-77
AX9.2.4.2 Jasmonic Acid and Salicylic Acid AX9-79
AX9.2.4.3 Stress-Induced Alterations in Gene
Expression AX9-81
AX9.2.5 Primary Assimilation by Photosynthesis AX9-84
AX9.2.5.1 Photooxidation: Light Reactions AX9-84
AX9.2.6 Alteration of Rubisco by Ozone: Dark Reactions AX9-85
AX9.2.7 Carbohydrate Transformations and Translocation AX9-88
AX9.2.7.1 Lipid Synthesis AX9-90
AX9.2.8 Role of Age and Size Influencing Response to Ozone . . . AX9-93
AX9.2.9 Summary AX9-95
REFERENCES AX9-97
AX9.3 MODIFICATION OF FUNCTIONAL AND GROWTH
RESPONSES AX9-106
AX9.3.1 Introduction AX9-106
AX9.3.2 Genetics AX9-109
AX9.3.2.1 Genetic Basis of Ozone Sensitivity ... AX9-110
AX9.3.3 Environmental Biological Factors AX9-112
AX9.3.3.1 Oxidant-Plant-Insect Interactions AX9-113
AX9.3.3.2 Oxidant-Plant-Pathogen Interactions .. AX9-116
AX9.3.3.3 Oxidant-Plant-Symbiont Interactions .. AX9-121
AX9.3.3.4 Oxidant-Plant-Plant Interactions:
Competition AX9-122
AX9.3.4 Physical Factors AX9-124
AX9.3.4.1 Light AX9-125
AX9.3.4.2 Temperature AX9-128
AX9.3.4.3 Humidity and Surface Wetness AX9-132
AX9.3.4.4 Drought and Salinity AX9-133
AX9.3.5 Nutritional Factors AX9-137
AX9.3.6 Interactions with Other Pollutants AX9-139
AX9.3.6.1 Oxidant Mixtures AX9-140
AX9.3.6.2 Sulfur Dioxide AX9-140
AX9.3.6.3 Nitrogen Oxides, Nitric Acid Vapor,
and Ammonia AX9-140
AX9.3.6.4 Hydrogen Fluoride and Other
Gaseous Pollutants AX9-143
AX9.3.6.5 Acidic Deposition AX9-144
AX9.3.6.6 Heavy Metals AX9-145
AX9.3.6.7 Mixtures of Ozone with Two or
More Pollutants AX9-145
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Table of Contents
(cont'd)
Page
AX9.3.7 Interactions with Agricultural Chemicals AX9-146
AX9.3.8 Factors Associated with Global Climate Change AX9-147
AX9.3.8.1 Ozone-Carbon Dioxide-Temperature
Interactions AX9-148
AX9.3.8.2 Ozone-UV-B Interactions AX9-171
AX9.3.8.3 Interactions of Ozone with Multiple
Climate Change Factors AX9-173
AX9.3.9 Summary - Environmental Factors AX9-174
REFERENCES AX9-178
AX9.4 EFFECTS-BASED AIR QUALITY EXPOSURE INDICES AX9-196
AX9.4.1 Introduction AX9-196
AX9.4.2 Summary of Conclusions from the Previous
Criteria Document AX9-197
AX9.4.3 Evaluation of Various Exposure Indices for
Describing Ambient Exposure-Response
Relationships AX9-199
AX9.4.4 Identifying Exposure Components That Relate to
Vegetation Effects AX9-205
AX9.4.4.1 Role of Concentration AX9-205
AX9.4.4.2 Role of Duration AX9-208
AX9.4.4.3 Patterns of Exposure AX9-209
AX9.4.4.4 Frequency of Occurrence of Peak
Concentrations AX9-213
AX9.4.4.5 Canopy Structure AX9-213
AX9.4.4.6 Site and Climate Factors AX9-214
AX9.4.4.7 Plant Defense Mechanism-
Detoxification AX9-214
AX9.4.5 Ozone Uptake or Effective Dose as an Index AX9-215
AX9.4.5.1 Models of Stomatal Conductance AX9-216
AX9.4.5.2 Nonlinear Response and Developing
Flux Indices AX9-219
AX9.4.5.3 Simulation Models AX9-221
AX9.4.6 Summary AX9-221
REFERENCES AX9-224
AX9.5 OZONE EXPOSURE-PLANT RESPONSE RELATIONSHIPS AX9-230
AX9.5.1 Introduction AX9-230
AX9.5.2 Summary of Key Findings/Conclusions from
Previous Criteria Documents AX9-231
AX9.5.3 Ozone Indices and Ambient Exposure AX9-241
AX9.5.4 Effects of Ozone on Annual and Biennial Species AX9-260
AX9.5.4.1 Effects on Growth, Biomass, and
Yield of Individual Species AX9-261
Ill-ix
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Table of Contents
(cont'd)
Page
AX9.5.4.2 Effects on Plant Quality AX9-267
AX9.5.4.3 Effects on Foliar Symptoms AX9-268
AX9.5.4.4 Other Effects AX9-269
AX9.5.4.5 Scaling Experimental Data to
Field Conditions AX9-270
AX9.5.4.6 Summary of Effects on Short-
Lived Species AX9-273
AX9.5.5 Effects of Ozone on Long-Lived (Perennial) Species . . . AX9-275
AX9.5.5.1 Herbaceous Perennial Species AX9-275
AX9.5.5.2 Deciduous Woody Species AX9-280
AX9.5.5.3 European Critical Levels AX9-287
AX9.5.5.4 Summary of Effects on Deciduous
Woody Species AX9-287
AX9.5.5.5 Evergreen Woody Species AX9-288
AX9.5.5.6 Summary of Effects on Evergreen
Woody Species AX9-292
AX9.5.5.7 Scaling Experimental Data to
Mature Trees AX9-292
AX9.5.6 Studies with the Chemical EDU AX9-296
AX9.5.7 Summary AX9-298
REFERENCES AX9-302
AX9.6 EFFECTS OF OZONE EXPOSURE ON NATURAL
ECOSYSTEMS AX9-313
AX9.6.1 Introduction AX9-313
AX9.6.2 Case Studies AX9-321
AX9.6.2.1 Valley of Mexico AX9-321
AX9.6.2.2 San Bernardino Mountains AX9-322
AX9.6.2.3 Sierra Nevada Mountains AX9-325
AX9.6.2.4 Appalachian Mountains AX9-330
AX9.6.2.5 Plantago Studies in the United
Kingdom AX9-331
AX9.6.2.6 Forest Health in the Carpathian
Mountains AX9-331
AX9.6.2.7 Field Exposure System (FACE),
Rhinelander, Wisconsin AX9-333
AX9.6.3 Landscape Condition AX9-333
AX9.6.4 Biotic Condition AX9-337
AX9.6.4.1 Ecosystems and Communities AX9-337
AX9.6.4.2 Species and Populations AX9-343
AX9.6.5 Organism Condition AX9-347
AX9.6.6 Ecosystem, Chemical, and Physical Characteristics
(water, soil) AX9-355
III-x
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Table of Contents
(cont'd)
Page
AX9.6.6.1 Nutrient Concentrations, Trace
Inorganic and Organic Chemicals AX9-355
AX9.6.7 Ecological Processes AX9-355
AX9.6.7.1 Energy Flow AX9-355
AX9.6.7.2 Material Flow AX9-358
AX9.6.8 Hydrological and Geomorphological AX9-361
AX9.6.9 Natural Disturbance Regimes AX9-361
AX9.6.10 Scaling to Ecosystem Levels AX9-362
AX9.6.10.1 Scaling from Seedlings to
Mature Trees AX9-363
AX9.6.10.2 Surveys, Growth Correlations and
Stand-Level Modeling AX9-365
AX9.6.11 Summary of Ecological Effects of Ozone Exposure
on Natural Ecosystems AX9-372
REFERENCES AX9-376
AX9.7 ECONOMIC EVALUATION OF OZONE EFFECTS
ON AGRICULTURE, FORESTRY AND NATURAL
ECOSYSTEMS AX9-394
AX9.7.1 Introduction AX9-394
AX9.7.2 The Measurement of Economic Information AX9-395
AX9.7.3 Understanding of Air Pollutants Effects on the
Economic Valuation of Agriculture and Other
Vegetation in the 1996 Criteria Document AX9-397
AX9.7.3.1 Agriculture AX9-398
AX9.7.3.2 Forests (Tree Species) and Natural
Ecosystems AX9-400
AX9.7.4 Studies Since 1996 of Ozone Exposure Effects
on the Economic Value of Agriculture, Forests,
and Ecosystems AX9-402
AX9.7.5 Limitations of Scientific Studies and Economic
Information AX9-404
AX9.7.6 Conclusions AX9-407
REFERENCES AX9-408
Ill-xi
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List of Tables
Number Page
AX9-1 Advantages and Disadvantages of Protective Chemicals Used in
Assessment of O3 Effects on Plants AX9-19
AX9-2 Advantages and Disadvantages of Bioindicators Used to Study O3
Plant Effects AX9-20
AX9-3 Advantages and Disadvantages of Cultivar Comparisons Used in
Assessment of O3 Effects on Plants AX9-24
AX9-4 Advantages and Disadvantages of Various Dendrochronological
Techniques Used in Assessment of O3 Effects on Plants AX9-26
AX9-5 Advantages and Disadvantages of Modeling Techniques Used in
AX9-6
AX9-7
AX9-8
AX9-9
Assessment of O3 Effects on Plants
The Flow of Ozone into a Leaf and Possible Reactions
Some Rates of Reaction of Ozone with Critical Biochemicals
Superoxide Dismutase Isozymes and Isoforms
Gene Families and cDNA Clones Used as Probes for SAR
(WardetaL 1991)
AX9-29
AX9-50
AX9-55
AX9-72
AX9-76
AX9-10 Proteins Altered by Ozone as Measured by Molecular Biological
Techniques as mRNA Level or Other Gene Activity Rather than
Enzyme Activity AX9-82
AX9-11 Interactions Involving O3 and Plant Pathogens AX9-117
AX9-12 Effects of Increased Carbon Dioxide on Ozone-Induced Responses of
Plants at the Metabolic, Physiological, and Whole-Plant Levels AX9-150
AX9-13 Summary of Ozone Exposure Indices Calculated for 3- or 5-Month
Growing Seasons from 1982 to 1991 AX9-233
AX9-14 Ozone Exposure Levels (Using Various Indices) Estimated to Cause at
Least 10% Crop Loss in 50 and 75% of Experimental Cases AX9-235
AX9-15 SUM06 Levels Associated with 10 and 20% Total Biomass Loss for
50 and 75% of the Seedling Studies AX9-238
AX9-16 Summary of Selected Studies of Ozone Effects on Annual Species AX9-243
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List of Tables
(cont'd)
Number Page
AX9-17 Summary of Selected Studies of the Effects of Ozone on Perennial
Herbaceous Plants AX9-247
AX9-18 Summary of Selected Studies of Ozone Effects on Deciduous Trees
and Shrubs AX9-249
AX9-19 Summary of Selected Studies of Ozone Effects on Evergreen Trees
and Shrubs AX9-254
AX9-20 Ethylene Diurea Effects on Vegetation Responses to Ozone AX9-257
AX9-21 Ozone Exposures at 35 Rural Sites in the Clean Air Status and Trends
Network in the Central and Eastern United States From 1989 to 1995 AX9-275
AX9-22 Essential Ecological Attributes for Natural Ecosystems Affected by O3 AX9-315
AX9-23 Case Studies Demonstrating the Ecological Effects of O3 AX9-324
AX9-24 The Most Comprehensively Studied Effects of O3 on Natural Ecosystem
are for the San Bernardino Mountain Forest Ecosystem AX9-326
AX9-25 Effects of Ozone, Ozone and N Deposition, and Ozone and Drought
Stress on Finns ponderosa andPinus jeffreyi in the Sierra Nevada
and the San Bernardino Mountains, California AX9-327
AX9-26 Summary of Responses ofPopiiliis tremuloides to Elevated CO2
(+200 |imol mol"1), O3 (1.5 x ambient), or CO2+O3 Compared with
Control During 3 Years of Treatments at the Aspen FACE Project AX9-334
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List of Figures
Number
AX9-1
AX9-2
AX9-3
AX9-4
AX9-5
AX9-6
AX9-7
AX9-8a,b
AX9-9
AX9-10
AX9-11
AX9-12
AX9-13
AX9-14
AX9-15
Ozone uptake from the atmosphere
Absorption and transformation of O3 within the leaf
The uptake of O3 into the leaf
The microarchitecture of a dicot leaf
The change in the O3 concentration inside a leaf with time
Possible transformations of O3 within a leaf
Possible reactions of O3 within water
The Crigee mechanism of O3 attack of a double bond
Varied ESR radicals, trapped and not, generated by O3 under somewhat
physiological conditions
Pathogen-Induced Hypersensitivity
The interaction of H2O2 and Ca2+ movements with AB A-induced
stomatal closure
The reaction of ascorbate within the apoplasm of the cell wall and its
ultimate reduction/oxidations
The pathway leading from phospholipids to jasmonic and traumatic acid . .
The production of Rubisco and its Calvin Cycle pathway reactions
Linkage of senescence with hypersensitivity reactions and the first event
of O, attack of plants
Page
AX9-40
AX9-42
. . AX9-47
. . AX9-47
AX9-49
AX9-51
. . AX9-52
. . AX9-54
. . AX9-57
. . AX9-60
. . AX9-63
. . AX9-67
. . AX9-80
AX9-86
AX9-93
AX9-16 Diagrammatic representation of several exposure indices, illustrating how
they weight concentration and accumulate exposure AX9-200
AX9-17 Trends in May-September 12-h SUM06, peak 1-h ozone concentration
and number of daily exceedances of 95 ppb for Crestline in 1963-1999
in relation to trends in mean daily maximum temperature for Crestline
and daily reactive organic gases (ROG) and oxides of nitrogen (NOX)
for San Bernardino county AX9-207
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List of Figures
(cont'd)
Number Page
AX9-18 The number of hourly average concentrations between 50 and 89 ppb
for the period 1980 to 2000 for the Crestline, San Bernardino,
CA monitoring site AX9-209
AX9-19 Distribution of biomass loss predictions from Weibull and linear
exposure-response models that relate biomass to O3 exposure AX9-237
AX9-20 A conceptual diagram of processes and storage pools in sources and
sinks that are affected by O3 exposure AX9-335
AX9-21 Common anthropogenic stressors and the essential ecological
attributes they affect AX9-373
III-xv
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Authors, Contributors, and Reviewers
CHAPTER 9 ANNEX (ENVIRONMENTAL EFFECTS)
Principal Authors
Dr. Jay Garner—National Center for Environmental Assessment (B243-01), U.S. Environmental
Protection Agency, Research Triangle Park, NC 27711 (retired
Dr. Timothy Lewis—National Center for Environmental Assessment (B243-01),
U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
Dr. William Hogsett—National Health and Environmental Effects Research Laboratory,
U.S. Environmental Protection Agency, Corvallis, OR
Dr. Christian Andersen—National Health and Environmental Effects Research Laboratory,
U.S. Environmental Protection Agency, Corvallis, OR
Dr. Allen Lefohn—ASL and Associates, Helena, MT
Dr. David Karnosky—Michigan Technological University, Houghton, MI
Dr. Michael Nannini—Ilinois State Water Survey, IL
Dr. Nancy Grulke—USDA Forest Service, Riverside, CA
Dr. Richard Adams—Oregon State University., Corvallis, OR
Dr. Robert Heath—University of California, Riverside, CA,
Dr. Victor Runeckle—Vancouver, B.C., CN (retired?)
Dr. Arthur Chappelka—Auburn University, School of Forestry, Auburn, AL
Dr. William Massman—USDA Forest Service, Ft. Collins, CO
Dr. Robert Musselman—USDA Forest Service, Fort Collins, CO
Dr. Peter Woodbury—Cornell University, Ithaca, NY (former USDA Forest Service)
Contributors and Reviewers
Dr. Boris Chevone—Department of Plant Pathology, Virginia Technological University,
Blacksburg, VA 24061
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Authors, Contributors, and Reviewers
(cont'd)
Contributors and Reviewers
(cont'd)
Dr. Alan Davison—School of Biology, Newcastle University, Newcastle on Tyne,
United Kingdom, NE1 7RU
Dr. Bruce L. Dixon—Department of Agricultural Economics, University of Arkansas,
Fayetteville, AR 72701
Dr. David Grantz—Kearney Agricultural Center, University of California at Riverside,
Parlier, CA 93648
Dr. Allen S. Heagle—1216 Scott PL, Raleigh, NC 27511
Dr. Robert Horst, Jr.—121 Thorwald Dr., Plainsboro, NJ 08536
Dr. John Innes—Forest Sciences Centre, Department of Forest Resources, University of British
Columbia, Vancouver, BD, Canada V6T 1Z4
Dr. Hans-Jiirgen Jager—Heinrich-Buff-Ring 26-32, Institute of Plant Ecology, Justus-Leibig
University, Gessen, Germany D35392
Dr. Robert Kohut— Tower Road, Boyce Thompson Institute, Rm 131,Cornell University,
Ithaca, NY 14853
Dr. Sagar Krupa—1519 Gortner Ave., Department of Plant Pathology, University of Minnesota,
St. Paul, MN 55108
Dr. William Manning—203 Morrill, Department of Microbiology, University of Massachusetts,
Amherst, MA 01003
Dr. Howard Neufeld—Rankin Science Bldg., Appalachian State University, Boone, NC 28608
Dr. Maria-Jose Sanz—Fundacion CEAM, c/Charles Darein, 14-Parque Te Valencia, Spain
Dr. James Short!e—Department of Ag Econ, Armsby, Pennsylvania State University,
University Park, PA 16802
Dr. John Skelly—Department of Plant Pathology, Pennsylvania State University,
University Park, PA 16803
[Note: Any inadvertently omitted names of authors/reviewers will be inserted in the final draft
of this O3 AQCD, as will more complete addresses for all authors/reviewers.]
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U.S. Environmental Protection Agency Project Team
for Development of Air Quality Criteria for Ozone
and Related Photochemical Oxidants
Executive Direction
Dr. Lester D. Grant (Director)—National Center for Environmental Assessment-RTF Division,
(B243-01), U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
Scientific Staff
Dr. Lori White(Ozone Team Leader)—National Center for Environmental Assessment
(B243-01), U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
Dr. Joseph Pinto—National Center for Environmental Assessment (B243-01),
U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
Ms. Beverly Comfort—National Center for Environmental Assessment (B243-01),
U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
Dr. Brooke Hemming—National Center for Environmental Assessment (B243-01),
U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
Dr. James S. Brown—National Center for Environmental Assessment (B243-01),
U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
Dr. Dennis Kotchmar—National Center for Environmental Assessment (B243-01),
U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
Dr. Jee-Young Kim—National Center for Environmental Assessment (B243-01),
U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
Dr. David Svendsgaard—National Center for Environmental Assessment (B243-01),
U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
Dr. Srikanth Nadadur—National Center for Environmental Assessment (B243-01),
U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
Dr. Timothy Lewis—National Center for Environmental Assessment (B243-01),
U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
Dr. Jay Garner—National Center for Environmental Assessment (B243-01), U.S. Environmental
Protection Agency, Research Triangle Park, NC 27711
-------
U.S. Environmental Protection Agency Project Team
for Development of Air Quality Criteria for Ozone
and Related Photochemical Oxidants
(cont'd)
Scientific Staff
(cont'd)
Dr. William Hogsett—National Health and Environmental Effects Research Laboratory,
U.S. Environmental Protection Agency, Corvallis, OR
Dr. Christian Andersen—National Health and Environmental Effects Research Laboratory,
U.S. Environmental Protection Agency, Corvallis, OR
Mr. Bill Ewald—National Center for Environmental Assessment (B243-01), U.S. Environmental
Protection Agency, Research Triangle Park, NC 27711 (retired)
Mr. James Raub—National Center for Environmental Assessment (B243-01),
U.S. Environmental Protection Agency, Research Triangle Park, NC 27711 (retired)
Technical Support Staff
Ms. Nancy Broom—Information Technology Manager, National Center for Environmental
Assessment (B243-01), U.S. Environmental Protection Agency, Research Triangle Park, NC
27711
Mr. Douglas B. Fennell—Technical Information Specialist, National Center for Environmental
Assessment (B243-01), U.S. Environmental Protection Agency, Research Triangle Park, NC
27711
Ms. Emily R. Lee—Management Analyst, National Center for Environmental Assessment
(B243-01), U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
Ms. Diane H. Ray—Program Specialist, National Center for Environmental Assessment
(B243-01), U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
Ms. Donna Wicker—Administrative Officer, National Center for Environmental Assessment
(B243-01), U.S. Environmental Protection Agency, Research Triangle Park, NC 27711 (retired)
Mr. Richard Wilson—Clerk, National Center for Environmental Assessment (B243-01),
U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
-------
U.S. Environmental Protection Agency Project Team
for Development of Air Quality Criteria for Ozone
and Related Photochemical Oxidants
(cont'd)
Document Production Staff
Ms. Carolyn T. Perry—Manager, Computer Sciences Corporation, 2803 Slater Road, Suite 220,
Morrisville, NC 27560
Mr. John A. Bennett—Technical Information Specialist, Library Associates of Maryland,
11820 Parklawn Drive, Suite 400, Rockville, MD 20852
Mr. William Ellis—Records Management Technician, InfoPro, Inc., 8200 Greensboro Drive,
Suite 1450, McLean, VA 22102
Ms. Sandra L. Hughey—Technical Information Specialist, Library Associates of Maryland,
11820 Parklawn Drive, Suite 400, Rockville, MD 20852
Mr. Matthew Kirk—Graphic Artist, Computer Sciences Corporation, 2803 Slater Road,
Suite 220, Morrisville, NC 27560
Dr. Barbara Liljequist—Technical Editor, Computer Sciences Corporation, 2803 Slater Road,
Suite 220, Morrisville, NC 27560
Ms. Faye Silliman—Word Processor, InfoPro, Inc., 8200 Greensboro Drive, Suite 1450,
McLean, VA 22102
III-xx
-------
U.S. Environmental Protection Agency Science Advisory Board (SAB)
Staff Office Clean Air Scientific Advisory Committee (CASAC)
Ozone Review Panel
Chair
Dr. Rogene Henderson*, Scientist Emeritus, Lovelace Respiratory Research Institute, 2425
Ridgecrest Drive SE, Albuquerque, NM, 87108, Phone: 505-348-9464, Fax: 505-348-8541,
(rhenders@lrri.org) (FedEx: Dr. Rogene Henderson, Lovelace Respiratory Research Institute,
2425 Ridgecrest Drive SE, Albuquerque, NM, 87108, Phone: 505-348-9464)
Members
Dr. John Balmes, Professor, Department of Medicine, University of California San Francisco,
University of California - San Francisco, San Francisco, California, 94143, Phone:
415-206-8953, Fax: 415-206-8949, (jbalmes@itsa.ucsf.edu)
Dr. Ellis Cowling*, University Distinguished Professor-at-Large, North Carolina State
University, Colleges of Natural Resources and Agriculture and Life Sciences, North Carolina
State University, 1509 Varsity Drive, Raleigh, NC, 27695-7632, Phone: 919-515-7564 , Fax:
919-515-1700, (ellis_cowling@ncsu.edu)
Dr. James D. Crapo*, Professor, Department of Medicine, National Jewish Medical and
Research Center. 1400 Jackson Street, Denver, CO, 80206, Phone: 303-398-1436, Fax: 303-
270-2243, (crapoj@njc.org)
Dr. William (Jim) Gauderman, Associate Professor, Preventive Medicine, University of
Southerm California, 1540 Alcazar #220, Los Angeles, CA, 91016, Phone: 323-442-1567,
Fax: 323-442-2349, (jimg@usc.edu)
Dr. Henry Gong, Professor of Medicine and Preventive Medicine, Medicine and Preventive
Medicine, Keck School of Medicine, University of Southern California, Environmental Health
Service, MSB 51, Rancho Los Amigos NRC, 7601 East Imperial Highway, Downey, CA, 90242,
Phone: 562-401-7561, Fax: 562-803-6883, (hgong@ladhs.org)
Dr. Paul J. Hanson, Senior Research and Development Scientist, Environmental Sciences
Division, Oak Ridge National Laboratory (ORNL), Bethel Valley Road, Building 1062, Oak
Ridge, TN, 37831-6422, Phone: 865-574-5361, Fax: 865-576-9939, (hansonpz@comcast.net)
Dr. JackHarkema, Professor, Department of Pathobiology, College of Veterinary Medicine,
Michigan State University, 212 Food Safety & Toxicology Center, East Lansing, MI, 48824,
Phone: 517-353-8627, Fax: 517-353-9902, (harkemaj@msu.edu)
-------
U.S. Environmental Protection Agency Science Advisory Board (SAB)
Staff Office Clean Air Scientific Advisory Committee (CASAC)
Ozone Review Panel
(cont'd)
Members
(cont'd)
Dr. Philip Hopke, Bayard D. Clarkson Distinguished Professor, Department of Chemical
Engineering, Clarkson University, Box 5708, Potsdam, NY, 13699-5708, Phone: 315-268-3861,
Fax: 315-268-4410, (hopkepk@clarkson.edu) (FedEx: 8 Clarkson Avenue, Potsdam, NY
136995708)
Dr. Michael T. Kleinman, Professor, Department of Community & Environmental Medicine,
100 FRF, University of California - Irvine, Irvine, CA, 92697-1825, Phone: 949-824-4765, Fax:
949-824-2070, (mtkleinm@uci.edu)
Dr. Allan Legge, President, Biosphere Solutions, 1601 11th Avenue NW, Calgary, Alberta,
CANADA, T2N 1H1, Phone: 403-282-4479, Fax: 403-282-4479, (allan.legge@shaw.ca)
Dr. Morton Lippmann, Professor, Nelson Institute of Environmental Medicine, New York
University School of Medicine, 57 Old Forge Road, Tuxedo, NY, 10987, Phone: 845-731-3558,
Fax: 845-351-5472, (lippmann@env.med.nyu.edu)
Dr. Frederick J. Miller*, Consultant, 911 Queensferry Road, Cary, NC, 27511, Phone:
919-467-3194, (fjmiller@nc.rr.com)
Dr. Maria Morandi, Assistant Professor of Environmental Science & Occupational Health,
Department of Environmental Sciences, School of Public Health, University of Texas - Houston
Health Science Center, 1200 Herman Pressler Street, Houston, TX, 77030, Phone:
713-500-9288, Fax: 713-500-9249, (mmorandi@sph.uth.tmc.edu) (FedEx: 1200 Herman
Pressler, Suite 624)
Dr. Charles Plopper, Professor, Department of Anatomy, Physiology and Cell Biology, School
of Veterinary Medicine, University of California - Davis, Davis, California, 95616, Phone:
530-752-7065, (cgplopper@ucdavis.edu)
Mr. Richard L. Poirot*, Environmental Analyst, Air Pollution Control Division, Department of
Environmental Conservation, Vermont Agency of Natural Resources, Bldg. 3 South, 103 South
Main Street, Waterbury, VT, 05671-0402, Phone: 802-241-3807, Fax: 802-241-2590,
(rich.poirot@state.vt.us)
Dr. Armistead (Ted) Russell, Georgia Power Distinguished Professor of Environmental
Engineering, Environmental Engineering Group, School of Civil and Environmental
Engineering, Georgia Institute of Technology, 311 Ferst Drive, Room 3310, Atlanta, GA,
30332-0512, Phone: 404-894-3079, Fax: 404-894-8266, (trussell@ce.gatech.edu)
-------
U.S. Environmental Protection Agency Science Advisory Board (SAB)
Staff Office Clean Air Scientific Advisory Committee (CASAC)
Ozone Review Panel
(cont'd)
Members
(cont'd)
Dr. Elizabeth A. (Lianne) Sheppard, Research Associate Professor, Biostatistics and
Environmental & Occupational Health Sciences, Public Health and Community Medicine,
University of Washington, Box 357232, Seattle, WA, 98195-7232, Phone: 206-616-2722, Fax:
206 616-2724, (sheppard@u.washington.edu)
Dr. Frank Speizer*, Edward Kass Professor of Medicine, Channing Laboratory, Harvard
Medical School, 181 Longwood Avenue, Boston, MA, 02115-5804, Phone: 617-525-2275, Fax:
617-525-2066, (frank.speizer@channing.harvard.edu)
Dr. James Ultman, Professor, Chemical Engineering, Bioengineering program, Pennsylvania
State University, 106 Fenske Lab, University Park, PA, 16802, Phone: 814-863-4802, Fax:
814-865-7846, (jsu@psu.edu)
Dr. Sverre Vedal, Professor of Medicine, Department of Environmental and Occupational Health
Sciences, School of Public Health and Community Medicine, University of Washington, 4225
Roosevelt Way NE, Suite 100, Seattle, WA, 98105-6099, Phone: 206-616-8285, Fax:
206-685-4696, (svedal@u.washington.edu)
Dr. James (Jim) Zidek, Professor, Statistics, Science, University of British Columbia, 6856
Agriculture Rd., Vancouver, BC, Canada, V6T 1Z2, Phone: 604-822-4302, Fax: 604-822-6960,
(jim@stat.ubc.ca)
Dr. Barbara Zielinska*, Research Professor , Division of Atmospheric Science, Desert Research
Institute, 2215 Raggio Parkway, Reno, NV, 89512-1095, Phone: 775-674-7066, Fax:
775-674-7008, (barbz@dri.edu)
Science Advisory Board Staff
Mr. Fred Butterfield, CASAC Designated Federal Officer, 1200 Pennsylvania Avenue, N.W.,
Washington, DC, 20460, Phone: 202-343-9994, Fax: 202-233-0643 (butterfield.fred@epa.gov)
(Physical/Courier/FedEx Address: Fred A. Butterfield, III, EPA Science Advisory Board Staff
Office (Mail Code 1400F), Woodies Building, 1025 F Street, N.W., Room 3604, Washington,
DC 20004, Telephone: 202-343-9994)
*Members of the statutory Clean Air Scientific Advisory Committee (CASAC) appointed by
the EPA Administrator
-------
Abbreviations and Acronyms
ABA
ABI2
ACC
ACS
ANN
ANOVA
ANP
ACS
AOT40
AOT60
AOTX
APX
AQCD
ASat
AVG
AZO
CEC
CF
CFA
CFI
CHIP
CO2
CSTRs
CU
abscisic acid
phospho-tyrosine-specific protein phosphatase
1 -aminocyclopropane-1 -carboxylate
1 -aminocyclopropane-1 -carboxylase synthase
maximum photosynthesis rate
artificial neural network
analysis of variance
Acadia National Park
allene oxide synthase
seasonal sum of the difference between an hourly concentration at the
threshold value of 40 ppb, minus the threshold value of 40 ppb
seasonal sum of the difference between an hourly concentration at the
threshold value of 60 ppb, minus the threshold value of 60 ppb
cumulative, cutoff concentration-based exposure index
ascorbate peroxidase
Air Quality Criteria Document
photosynthetic assimilation in saturating light
1 -aminoethoxy vinyl-glycine
azoxystrobin
controlled environment chambers
charcoal-filtered
charcoal/Purafil-filtered air
continuous forest inventory
Effects of Elevated Carbon Dioxide and Ozone on Potato Tuber Quality
in the European Multiple Site Experiment
carbon dioxide
continuous stirred tank reactors
cumulative uptake
III-xxiv
-------
cyt
DG
DGDG
DHA
DMPO
DNA
ECM
EDU
EEAs
EMEP
EPA
EPO
EPR
ESPACE-wheat
ESR
ET
EU
FA
FACE
FFAs
FHM
FLAG
FPM
GHG
GR
GRSM
GSH
GSH-Px
cytochrome
diacylglycerol
digalactosyldiacylglycerol
dehydroascorbate
dimethylphrrolise 1-oxide; 5,5-dimethyl-l-pyrrolineN-oxide
deoxyribonucleic acid
ectomycorrhizal fungi
ethyl enediurea
essential ecological attributes
European Monitoring and Evaluation Program
U.S. Environmental Protection Agency
epoxyconazole
electron paramagnetic resonance; ESR
European Stress Physiology and Climate Experiment on the Effects of
Carbon Dioxide and Oxygen on Spring Wheat
electron spin resonance; EPR
ethylene
European Union
fatty acid
free-air carbon dioxide enrichment (system)
free fatty acids
Forest Health Monitoring (assessment)
Federal Land Managers' Air Quality Related Values Workgroup
Forest Pest Management
greenhouse gas
glutathione reductase
Great Smoky Mountains National Park
glutathione
glutathione peroxidase
III-xxv
-------
GSSG
H+
2HDM, 2ndHDM
HNO3
H202
HO3»
HO
HO2'
HPOT
HR
IPCC
JA
KROFEX
LAI
mAOT
MDA
MDGD
mRNA
MV
NAD+
NADH
NADP+
NADPH,
NAD(P)H
NaE
NCLAN
n.d.
glutathione disulfide
hydrogen ion
second-highest daily maximum 1-h concentration
nitric acid
hydrogen peroxide
protonated ozone radical
hydroxyl radical
hydroperoxyl; hydroperoxy radical
13-hydroperoxide linolenic acid
hypersensitive response
Intergovernmental Panel on Climate Change
jasmonic acid
maximum rate of electron transport for the regeneration of RuBP
saturating light
Krauzberg Ozone Fumigation Experiment
leaf area index
modified accumulated exposure over the threshold
malonaldehyde
monogalactosyldiacylglycerol
messenger ribonucleic acid
methyl viologen
nicotinamide adenine dinucleotide
reduced nicotinamide adenine dinucleotide
nicotinamide adenine dinucleotide phosphate
reduced nicotinamide adenine dinucleotide phosphate
sodium erythorbate
National Crop Loss Assessment Network
no data
III-xxvi
-------
NDF neutral detergent fiber
NF national forest
NF non-filtered
NH3 ammonia
(NH4)2SO4 ammonium sulfate
N2O nitrous oxide
NO nitric oxide
NO2 nitrogen dioxide
NO3~ nitrate
NOX nitrogen oxides
NP national park
NPP net primary productivity
n.s. nonsignificant
O2 superoxide
O2"' superoxide radical
O3 ozone
OD outer diameter
OTC open-top chamber
p, P probability value
PAD pollutant applied dose
PAL phenylalanine lyase
PAN peroxyacetyl nitrate
PAR photosynthetically active radiation
PC phosphatidylchloline
PE phosphatidylethanolamine
PFD photosynthetic flux density
PG phosphatidyl glycerol
PGSW Plant Growth Stress Model
-------
PI
ppb
ppm
PQH2
PR
PSII
qP
r2
R2
rbcL
rbcS
RH
RNA
ROG
ROS
Rubisco
RuBP
SA
SAB
SAR
SD
SE
SHEN
SLA
SMD
SNAAQS
SO2
S042
phosphatidyl inositol
parts per billion
parts per million
plastoquinone
pathogenesis-related (protein)
Photosystem II; (|)PSII
photochemical quenching
correlation coefficient
multiple regression correlation coefficient
Rubisco large subunit
Rubisco small subunit
relative humidity
ribonucleic acid
reactive organic gases
reactive oxygen species
ribulose-1,6-P2-carboxylase/oxygenase 1
ribulose bisphosphate
salicylic acid
Science Advisory Board
systemic acquired resistance
standard deviation
standard error
Shenandoah National Park
specific leaf area
soil moisture deficit
Secondary National Ambient Air Quality Standards
sulfur dioxide
sulfate
-------
SOD superoxide dismutase
SUMOO sum of all hourly average concentrations
SUM06 seasonal sum of all hourly average concentrations > 0.06 ppm
SUM08 seasonal sum of all hourly average concentrations > 0.08 ppm
TMPO tetramethylphrrolise 1-oxide; 3,3,5,5-dimethyl-l-pyrrolineN-oxide
TNC total nonstructural carbohydrate
UDGT UDP glalactose-l,2,-diacylglycerol
UNECE United Nations Economic Commission for Europe
UNECE ICP- United Nations Economic Commission for Europe International
Vegetation Cooperative Programme on effects of air pollution and other stresses on
crops and non-woody plants (UN/ECE-Vegetation; formerly -Crops)
UNEP United Nations Environment Program
UV-B ultraviolet radiation of wavelengths from 280 to 320 nm
VOC volatile organic compound
VPD vapor pressure deficit
W126 cumulative integrated exposure index with a sigmoidal weighting
function
ZAPS Zonal Air Pollution System
III-xxix
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i ANNEX AX9. ENVIRONMENTAL EFFECTS: OZONE
2 EFFECTS ON VEGETATION AND ECOSYSTEMS
3
4
5 AX9.1 METHODOLOGIES USED IN VEGETATION RESEARCH
6 AX9.1.1 Introduction
7 The scale of investigations evaluating the direct effects of O3 on plant response ranges
8 from subcellular to cellular, organismal, population, community, and ecosystem levels, with
9 each level having its own particular experimental methodologies and specialized
10 instrumentation, equipment, facilities, and experimental protocols. These investigations generate
11 data. Other types of methodologies exist for the handling of data and statistical analysis as well
12 as the utilization of data in developing the different exposure metrics or indices used to define
13 exposure, quantitative exposure-response relationships, and computer simulation models of these
14 exposure-response relationships. The objective of this section is not to provide an updated
15 encyclopedia of all the methods that have been used but rather to focus on approaches that have
16 (1) led to an improved understanding of the quantitatively measurable growth and
development responses of plants and plant communities to O3, or
17 (2) provided information about the extent and geographic distribution of the responses
of herbaceous and woody plants, both cultivated and native, to ambient O3
exposures.
18 The first part of the objective is essential for determining dose-response functions used in
19 developing impact and risk assessments of the effects of O3; it usually involves treating plants to
20 a range of artificial O3 exposures. The second part of the objective is essential for determining
21 the geographic distribution of the risk; it usually involves subjecting plants to ambient air O3
22 exposures.
23 The types of methodologies used by biochemists, molecular biologists, or plant
24 physiologists, whose interests lie in determining effects on specific constituents or in
25 understanding the mode of action of O3, are not discussed here but are addressed in Section
26 AX9.2. Methods used to characterize the O3 content of ambient air and to define exposure and
27 exposure-response relations are discussed in Sections AX9.4 and AX9.5, respectively.
August 2005 AX9-1 DRAFT-DO NOT QUOTE OR CITE
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1 The methodologies for exposure-response studies have involved many different types of
2 exposure facilities and protocols and have employed a range of statistical approaches in the
3 analysis and interpretation of the data. Most of the studies have been conducted using major
4 agricultural crop species. The methodologies have improved over the years as a result of the
5 development, availability, or application of new or improved instrumentation, physical systems,
6 and numerical approaches to data analysis. Yet equally important to the roles played by these
7 advances has been the clearer understanding that has emerged from earlier work identifying the
8 type of experimentation needed to achieve realistic assessments of the magnitude and extent of
9 the impact of O3 on vegetation of all types. As a result, significantly increased attention is now
10 being paid to field observations and biomonitoring, particularly to the responses of forest trees
11 and native vegetation.
12 Other than in various exploratory studies that have used chamber-based steady-state
13 exposure concentrations (so-called "square-wave" exposures), the trend in experimental
14 exposure protocols has been to attempt to expose plants under conditions as natural as possible
15 to temporal profiles that simulate the real world, either by conducting experiments in the field or
16 in elaborately controlled environment facilities that provide simulated field conditions.
17 Previous Air Quality Criteria Documents for Ozone and Other Photochemical
18 Oxidants (U.S. Environmental Protection Agency, 1986, 1996) described the time course for
19 these methodological developments. Although this section provides a brief overview of the
20 methodologies used in the past and their limitations, it focuses mainly on those techniques that
21 have come into prominence over the last decade. This focus has been aided considerably
22 by several compilations of experimental methodologies and facilities, such as the earlier
23 comprehensive review for the U.S. Environmental Protection Agency/National Acid
24 Precipitation Assessment Program by Hogsett et al. (1987a,b), Manning and Krupa (1992)
25 and by more recent reviews by Musselman and Hale (1997) and Karnosky et al. (2001).
26
27 AX9.1.2 Methods Involving Experimental Exposures to Ozone
28 AX9.1.2.1 "Indoor", Controlled Environment, and Greenhouse Chambers
29 The earliest experimental investigations of the effects of O3 on plants utilized simple glass
30 or plastic-covered chambers, often located within greenhouses, into which a flow of O3-enriched
31 air or oxygen could be passed to provide the exposure. The types, shapes, styles, materials of
August 2005 AX9-2 DRAFT-DO NOT QUOTE OR CITE
-------
1 construction, and locations of these chambers were as numerous as the different investigators
2 and, in spite of providing little resemblance to real-world conditions, they yielded much of the
3 basic information on the visible and physiological effects on plants. The construction and
4 performance of more elaborate and better instrumented chambers dating back to the 1960s has
5 been well-summarized in Hogsett et al. (1987a), including those installed in greenhouses (with
6 or without some control of temperature and light intensity).
7 One greenhouse chamber approach that continues to yield useful information on the
8 relationships of O3 uptake to both physiological and growth effects employs continuous stirred
9 tank reactors (CSTRs) first described by Heck et al. (1978). Although originally developed to
10 permit mass-balance studies of O3 flux to plants, their use has more recently widened to include
11 short-term physiological and growth studies of O3 x CO2 interactions (e.g., Costa et al., 2001;
12 Heagle et al., 1994b; Loats and Rebbeck, 1999; Rao et al., 1995; Reinert and Ho, 1995; and
13 Reinert et al., 1997), and of surveys of native plant responses to O3 (Orendovici et al., 2003).
14 In many cases, supplementary lighting and temperature control of the surrounding structure have
15 been used to control or modify the environmental conditions (e.g., Heagle et al., 1994a).
16 Many investigations have utilized commercially available controlled environment
17 chambers and walk-in rooms adapted to permit the introduction of a flow of O3 into the
18 controlled air-volume. Such chambers continue to find use in genetic screening and in
19 physiological and biochemical studies aimed primarily at improving our understanding of modes
20 of action. For example, some of the ongoing studies of the O3 responses ofPlantago major
21 populations have been conducted in controlled environment chambers (Reiling and Davison,
22 1994; Whitfield et al., 1996b).
23 The environmental conditions provided by indoor chambers of any type will always
24 preclude the use of the information obtained with such chambers in predicting O3 effects in the
25 natural environment, because the environmental conditions will always be measurably different
26 from field conditions. However, highly sophisticated controlled environment chambers such as
27 those described by Langebartels et al. (1997), which are subdivided into aerial and root
28 compartments with dynamic control of light intensity and photoperiod, air and soil temperature,
29 humidity, soil moisture, wind speed, and exposure to O3, may come close to simulating specific
30 natural conditions. Such chambers have provided meaningful insights into a wide array of the
31 early biochemical responses of plants to O3. They can also minimize confounding factors that
August 2005 AX9-3 DRAFT-DO NOT QUOTE OR CITE
-------
1 make indoor chamber studies only rarely able to be extrapolated to field conditions, e.g., that
2 shoots and roots develop under different temperature regimes. The applicability of the results
3 of many chamber studies may be further limited by their use of container-grown plants. Most
4 of the concerns over the applicability of CO2 enrichment studies, as discussed in Section
5 AX9.5.7.1, may also be relevant to O3 enrichment studies, as suggested by Whitfield et al.
6 (1996a).
7 Whitfield et al. (1996a) reported significant interactive effects between O3 and soil volume
8 on the growth ofPlantago major. They noted that although container size may limit root and,
9 hence, plant growth, the reverse may also be true for single plants in large containers, which do
10 not experience typical field competition for resources. Other studies found little or no effect of
11 rooting volume on plant response to O3. Heagle et al. (1979a,b; 1983) found that four wheat
12 cultivars (Triticum aestivum L.) had similar proportional suppression of seed yield to season-
13 long O3 exposure whether plants were grown in the ground or in 3.8-L pots. Similarly,
14 proportional O3 injury and yield response of field corn (Zea mays L.) (Heagle et al., 1979a) and
15 soybean (Glycine max (L.) Merr.) (Heagle et al., 1983) was similar whether the plants were
16 grown in 15-L pots or in the ground. In a two-year experiment with soybean, the relative effects
17 of CO2 and O3 on above-ground biomass and seed yield were similar whether the plants were
18 grown in pots (15 and 21 L) or grown in the ground (Booker et al., 2005). Collectively, the
19 results suggest that while planting density and rooting environment affect plant morphology and
20 growth, the relative responses of seed yield to elevated O3 may be similar whether plants are
21 grown in pots or in the ground.
22
23 AX9.1.2.2 Field Chambers
24 Although some types of closed field chambers have largely fallen out of favor in recent
25 years, closed "Solardome" field chambers (Lucas et al., 1987; Rafarel and Ashenden, 1991) have
26 been successfully used in studies of O3 x acid mist interactions (Ashenden et al., 1995, 1996).
27 Concern over the need to establish realistic plant-litter-soil relationships as a prerequisite to
28 studies of the effects of O3 and CO2 enrichment on ponderosa pine (Pinus ponderosa) seedlings
29 led Tingey et al. (1996) to develop closed, partially environmentally controlled, sun-lit chambers
30 ("terracosms") incorporating 1-m-deep lysimeters containing forest soil in which the appropriate
31 horizon structure was retained.
August 2005 AX9-4 DRAFT-DO NOT QUOTE OR CITE
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1 In general, field chamber studies are dominated by the use of various versions of the open-
2 top chamber (OTC) design, first described by Heagle et al. (1973) and Mandl et al. (1973). Most
3 chambers are ~3 m in diameter with 2.5-m-high walls. Hogsett et al. (1987a) described in detail
4 many of the various modifications to the original OTC designs that appeared subsequently, e.g.,
5 the use of larger chambers to permit exposing small trees (Kats et al., 1985) and grapevines
6 (Mandl et al., 1989), the addition of a conical baffle at the top to improve ventilation (Kats et al.,
7 1976), a frustrum at the top to reduce ambient air incursions, and a plastic rain-cap to exclude
8 precipitation (Hogsett et al., 1985). All of these modifications included the discharge of air via
9 ports in annular ducting or interiorly perforated double-layered walls at the base of the chambers
10 to provide turbulent mixing and the upward mass flow of air.
11 Wiltshire et al. (1992) described a large OTC suitable for small trees with roll-up sides that
12 permitted the trees to be readily subjected from time to time to episodic, normal, "chamberless"
13 environmental conditions. In the 6-m-high OTCs described by Seufert and Arndt (1985) used
14 with Norway spruce (Picea abies) trees, a second zone of annular enrichment was also provided
15 between 4 and 5 m. The use of OTCs was adopted for the large European Stress Physiology and
16 Climate Experiment on the effects of CO2 and O3 on spring wheat (ESPACE-wheat), conducted
17 over 1994 to 1996 at field sites in eight countries (Jager et al., 1999). However, typical
18 European chambers have the introduction of O3-enriched air at or above canopy height. The
19 relatively low costs of fabrication, operation, and maintenance has favored OTC use in field
20 studies (Fangmeier et al., 1992; Musselman and Hale, 1997). The air supplied to the chambers
21 can be readily filtered through activated charcoal to reduce the O3 concentration, or it can be
22 enriched with O3 to provide a range of exposures.
23 All field chambers create internal environments that differ from ambient air, giving rise to
24 so-called "chamber effects" with the modification of microclimatic variables, including reduced
25 and uneven light intensity, uneven rainfall, constant wind speed, reduced dew formation, and
26 increased air temperatures (Fuhrer, 1994; Manning and Krupa, 1992). Because of the constant
27 wind speed and delivery systems, OTCs can provide a more definable exposure than free-air
28 systems can due to the lack of "hot-spots", where exposures are essentially undefined, in free-air
29 systems. Nonetheless, there are several characteristics of the OTC design and operation that can
30 lead to unrealistic exposures. First, the plants are subjected to constant turbulence, which,
31 through increased uptake resulting from the consequently low boundary layer resistance to
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1 diffusion, may lead to overestimation of cause-effect relationships (Krupa et al., 1995; Legge
2 et al., 1995). However, in at least one case where canopy resistances were quantified in OTCs
3 and in the field, it was determined that gaseous pollutant exposure to crops in OTCs was similar
4 to that which would have occurred at the same concentration in the field (Unsworth et al., 1984a,
5 1984b).
6 A second concern is that the introduction of the O3-enriched air into the lower part of
7 chambers as described by Heagle et al. (1973) and Mandl et al. (1973) results in a O3
8 concentration gradient that decreases with increasing height, the converse of the situation
9 observed in ambient air in which the O3 concentration decreases markedly from above a plant
10 canopy to ground level (Griinhage and Jager, 1994; Pleijel et al., 1995, 1996). Concern that
11 studies conducted in such OTCs may somewhat overestimate the effects of O3 led to the
12 European design, which provides a decreasing downward gradient. It seems unlikely that the
13 "chamber effects" produced by the two designs will be the same. These issues are discussed
14 more fully in Section AX9.1.2.4.
15 It should also be noted that, although OTCs were originally developed for exposing row
16 crops in the field, many recent studies employing OTCs have used potted plants in order to
17 include or control edaphic or nutritional factors or water relations within the experimental
18 design. Therefore, some caution should be used when extrapolating results of pot studies to the
19 field as noted above (Section AX9.2.2.1).
20 The difficulties faced in the experimental exposure of forest trees to air pollutants in
21 chambers (e.g., Seufert and Arndt [1985]) led to the development of branch chambers such as
22 those described by Ennis et al. (1990), Houpis et al. (1991), and Teskey et al. (1991). These
23 chambers are essentially large cuvettes without temperature control and, as noted by Musselman
24 and Hale (1997), share many of the characteristics of CSTRs, i.e., transparent walls, internal
25 fans, and inlet and outlet monitors to permit the determination of O3 uptake, CO2 exchange, and
26 transpiration. Although they make it possible to expose whole branches to different O3 regimes,
27 the relevance of the data they yield in regard to the whole tree may be questionable. As noted by
28 Saxe et al. (1998), the inevitable change in environmental conditions resulting from the isolation
29 of the branch may cause different responses from those that would be obtained if the whole tree
30 had been subjected to the same environmental conditions.
31
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1 AX9.1.2.3 Plume Systems
2 Plume systems are chamberless exposure facilities in which the atmosphere surrounding
3 plants in the field is modified by the injection of pollutant gas into the air above or around them
4 from multiple orifices spaced to permit diffusion and turbulence so as to establish relatively
5 homogeneous conditions as the individual plumes disperse and mix with the ambient air. As
6 pointed out by Manning and Krupa (1992), they can only be used to increase the O3 levels in the
7 ambient air. The volume of air to be modified is unconfined, and three approaches have been
8 used to achieve desired pollutant concentrations in the air passing over the plants, producing
9 various systems that
10 (1) achieve a concentration gradient, in most instances dependent upon the direction of
the prevailing wind;
11 (2) achieve spatially uniform concentrations over a plot, dependent upon wind
direction; and
12 (3) seek to achieve spatially uniform concentrations over a plot, independent of wind
speed and direction.
13 Gradient systems created by dispensing a pollutant gas into the air at canopy level from
14 perforated horizontal pipes arranged at right angles to the prevailing wind were described
15 for SO2 studies in the early 1980s. A modified gradient system for O3 was used by Bytnerowicz
16 et al. (1988) to study effects on desert species, but there appear to have been no recent
17 applications of the method. A gradient O3-exclusion system is discussed in Section AX9.2.3.1.
18 Systems designed to achieve spatially uniform pollutant levels by ensuring that the release
19 of a pollutant is always on the upwind side of the study site were also originally described
20 for SO2 studies (e.g., Greenwood et al. [1982]). However, the adaptation of these concepts as
21 introduced by McLeod et al. (1985) in constructing a large circular field site for exposing crops
22 to SO2 led to the subsequent development of both the large-scale O3 and SO2 fumigation system
23 for forest trees in the United Kingdom in 1985 (the Liphook Forest Fumigation Project) (1992),
24 the smaller system for O3 fumigation constructed at Kuopio, Finland in 1990 (Wulff et al.,
25 1992), and the free-air carbon-dioxide enrichment (FACE) systems of gas dispersal over crops
26 (Hendrey and Kimball, 1994) and forest trees (Hendrey et al., 1999). Although originally
27 designed to provide chamberless field facilities for studying the CO2 effects of climate change,
28 large forest tree FACE systems have recently been adapted to include the dispensing of O3
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1 (Karnosky et al., 1999). Volk et al. (2003) recently described a system for exposing grasslands
2 that uses 7-m diameter plots. FACE systems discharge the pollutant gas (and/or CO2) through
3 orifices spaced along an annular ring (or torus) or at different heights on a ring of vertical pipes.
4 Computer-controlled feedback from the monitoring of gas concentration regulates the feed rate
5 of enriched air to the dispersion pipes. Feedback of wind speed and direction information
6 ensures that the discharges only occur upwind of the treatment plots, and that discharge is
7 restricted or closed down during periods of low wind speed or calm conditions. The diameter of
8 the arrays and their heights (25 to30 m) in some FACE systems requires large throughputs of
9 enriched air per plot, particularly in forest tree systems. The cost of the throughputs tends to
10 limit the number of enrichment treatments, although Hendrey et al. (1999) argued that the cost
11 on an enriched volume basis is comparable to that of chamber systems.
12 An alternative to the FACE system to free-air fumigation uses a horizontal grid system
13 through which pollutant-enriched air is discharged over the canopies of plants in field plots. The
14 original design, termed the Zonal Air Pollution System (ZAPS), was developed for studying the
15 effects of SO2 on native grasslands (Lee et al., 1975), and it was later modified by Runeckles
16 et al. (1990) by randomly dividing each of three treatment plots into four subplots, each with
17 different numbers of discharge orifices to provide various levels of O3 enrichment. With the
18 ZAPS system, changes in wind direction and speed result in varying degrees of carryover from
19 subplot to subplot, effectively resulting in 12 stochastically different seasonal exposures. The
20 system was used for studies of growth effects on field crops and 2- to 4-year old Douglas fir
21 (Pseudotsuga menziesii) saplings (Runeckles and Wright, 1996). A larger ZAPS design was
22 used by Wilbourn et al. (1995) on a grass (Loliumperenne)-c\over (Trifolium repens) mixture
23 and by Ollerenshaw et al. (1999) on oilseed rape (Brassica napus\ whereby four replicate field
24 plots were exposed to intermittent constant additions of O3 to ambient air. A ZAPS design with
25 eight spatially separated treatment plots was also developed to obtain crop response data used in
26 assessing crop losses in the Fraser Valley, British Columbia, Canada (Runeckles and Bowen,
27 2000).
28 Another recent adaptation of the FACE design was constructed to fumigate soybean
29 with CO2 and O3 in combination (Morgan et al., 2004; Rogers et al., 2004). This modified
30 FACE design was based on those of Miglietta et al. (2001) and does not force air through the
31 canopy, instead it relies on wind to disperse air across the fumigation plot.
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1 The FACE-type facility developed for the Kranzberg Ozone Fumigation Experiment
2 (KROFEX) in Germany begun in 2000 (Werner and Fabian, 2002) (Nunn et al., 2002) to study
3 the effects of O3 on mature stands of beech (Fagus sylvaticd) and spruce (Picea abies) trees is
4 more truly a zonal system that functions independently of wind direction. The enrichment of a
5 large volume of the ambient air immediately above the canopy takes place via orifices in vertical
6 tubes suspended from a horizontal grid supported above the canopy.
7 Recognizing the difficulties of modifying the aerial environments of large trees, Tjoelker
8 et al. (1994) devised a free-air system for exposing branches of sugar maple (Acer saccharum)
9 trees to O3. Near the ends of up to 10 branches, enriched air was discharged through small holes
10 in 38-cm-diameter loops of 0.635-cm-OD (outer diameter) teflon tubes positioned 20 to 30 cm
11 below the terminal foliage cluster.
12 Although plume systems make virtually none of the modifications to the physical
13 environment that are inevitable with chambers, their successful use depends on selecting the
14 appropriate numbers, sizes, and orientations of the discharge orifices to avoid hot-spots resulting
15 from the direct impingement of jets of pollutant-enriched air on plant foliage (Werner and
16 Fabian, 2002). However, because mixing is unassisted and completely dependent on wind
17 turbulence and diffusion, local gradients are inevitable even in large-scale FACE systems. Both
18 FACE and ZAPS systems have provisions for shutting down under low wind speed or calm
19 conditions and for an experimental area that is usually defined within a generous border in order
20 to strive for homogeneity of the exposure concentrations within the treatment area. They are
21 also both dependent upon continuous computer-controlled feedback of the O3 concentrations in
22 the mixed treated air and of the meteorological conditions.
23
24 AX9.2.2.4 Comparative Studies
25 All experimental approaches to the exposure of plants to O3 have shortcomings. The use of
26 laboratory, greenhouse, or field chambers raises concerns for the roles of chamber effects on
27 micrometeorology, as well as the constant turbulence over and within the plant canopy during
28 chamber operation, in modifying O3 uptake and subsequent plant response. In contrast, plume
29 systems suffer from relatively poor control of exposure levels and an inability to reduce O3
30 levels below ambient in areas where O3 is phytotoxic.
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1 Although chamber effects vary, one concern is the rise in temperature associated with
2 enclosing plants in a chamber. Still, it is not clear whether these effects are directly related to
3 temperature or are the result of temperature interactions with other environmental variables.
4 For example, Olszyk et al. (1992) undertook a 3-year study of the impact of O3 on Valencia
5 orange trees {Citrus sinesis (L.) Osbeck) in large OTCs to determine if "insidious differences in
6 microclimatic conditions could alter plant growth responses and susceptibility to pollutant
7 stress." Nonfiltered chambers were found to have somewhat lower average O3 concentrations
8 than the ambient air, and fewer hourly exceedances of 100 ppb. In cool seasons, stomatal
9 conductance was also lower, implying lower O3 uptake. However, the cumulative fruit yields
10 were doubled in the chamber trees even though photosynthetically active radiation was
11 consistently reduced by about 19% while leaf temperatures averaged more than 2 °C higher.
12 These data may be somewhat extreme, but they emphasize the need to be cautious when
13 interpreting OTC yield response data, particularly since, as in this study, no O3 enrichment was
14 involved as a complicating factor.
15 While it is clear that chambers can alter some aspects of plant growth, the question to be
16 answered is whether or not these differences affect plant response to O3. As noted in the
17 1996 O3 AQCD (U.S. Environmental Protection Agency, 1996), evidence from the comparative
18 studies of OTCs and from closed chamber and O3-exclusion exposure systems on the growth of
19 alfalfa (Medicago saliva) by Olszyk et al. (1986a) suggested that, since significant differences
20 were found for fewer than 10% of the growth parameters measured, the responses were, in
21 general, essentially the same regardless of exposure system used and chamber effects did not
22 significantly affect response. In 1988, Heagle et al. (1988) concluded: "Although chamber
23 effects on yield are common, there are no results showing that this will result in a changed yield
24 response to O3." A more recent study of chamber effects examined the responses of tolerant and
25 sensitive white clover clones (Trifolium repens) to ambient O3 in greenhouse, open-top, and
26 ambient plots (Heagle et al., 1996). For individual harvests, greenhouse O3 exposure reduced the
27 forage weight of the sensitive clone 7 to 23% more than in OTCs. However, the response in
28 OTCs was the same as in ambient plots. Several studies have shown very similar yield response
29 to O3 for plants grown in pots or in the ground, suggesting that even such a significant change in
30 environment does not alter the proportional response to O3, at least as long as the plants are well
31 watered (Heagle, 1979; Heagle et al., 1983).
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1 Recent evidence obtained using free-air exposure systems and OTCs supports results
2 observed previously in OTC studies (Table AX9-16, Figure AX9-1). Specifically, a series of
3 studies undertaken using free-air O3 enrichment in Rhinelander, WI (Isebrands et al., 2000,
4 2001) showed that O3-symptom expression was generally similar in OTCs, FACE, and
5 ambient-O3 gradient sites, supporting the previously observed variation among trembling aspen
6 clones (Populus tremuloides L.) using OTCs (Karnosky et al., 1999). The FACE study
7 evaluated the effects of 3 years of exposure to combinations of elevated CO2 and O3 on growth
8 responses in mixture of five trembling aspen clones (Isebrands et al., 2000, 2001). Height,
9 diameter, and stem volume (diameter2 x height) were decreased by elevated O3. On average for
10 all clones, stem volume was decreased by 20% over the 3 years in the elevated O3 treatment as
11 compared with the 1 x-ambient treatment. This FACE facility study is important, because it
12 confirms responses reported previously with the same clones grown in pots or soil in OTCs
13 without the alterations of microclimate induced by chambers. Currently, this is the only U.S.
14 study using this technology to have examined the effects of O3 under these conditions. This
15 study is also significant, because the elevated O3-exposure pattern used was intended to
16 reproduce the 6-year average pattern from Washtenaw County, Michigan (Karnosky et al.,
17 1999).
18 Chambered systems such as OTCs provide a charcoal-filtered (CF), clean-air control for O3
19 experiments, while FACE and some other plume systems do not. Depending on experimental
20 intent, a replicated, clean-air control treatment is an essential component in many experimental
21 designs. This control cannot be provided by FACE systems where ambient O3 levels are
22 phytotoxic. This is especially relevant in CO2 x O3 experiments in which phytotoxic effects of
23 ambient O3 can be suppressed due to CO2-induced reductions in stomatal conductance and O3
24 uptake (Booker et al., 1997, 2004, 2005; Heagle et al., 1998; Fiscus et al., 1997, 2002, 2005).
25 Plume systems avoid chamber effects, but because they rely solely upon diffusion and
26 natural turbulence to modify the ambient O3 concentration, they may fail to achieve homogeneity
27 of the air to which the plants are exposed and may give rise to hot spots in which the enriched air
28 jets are inadequately diluted and impinge directly on foliage. A further deterrent to their
29 widespread use is the large-scale generation of O3 needed, which has, in most cases, limited the
30 numbers of treatments that can be included in an experimental design.
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1 In spite of the various advantages and disadvantages of the two systems, there is still little
2 experimental evidence that allows a direct comparison of OTCs to the free-air plume systems or
3 a determination of the degree to which chamber effects alter plant response to O3. The evidence
4 that is available suggests that chamber effects do not fundamentally alter the response of
5 plants to O3; therefore, chambers remain a useful tool for testing species sensitivity and
6 developing O3-response relationships. However, chamber effects have the potential to alter O3
7 uptake (Nussbaum and Fuhrer, 2000), so it is important to fully characterize temperature, light,
8 turbulence, and other chamber characteristics during exposure to allow extrapolation of the
9 results.
10
11 AX9.1.2.5 Ozone Generation Systems
12 Two approaches have been used to generate the O3 needed for enrichment from air or
13 oxygen: (1) high-voltage static discharge and (2) high-intensity UV-irradiation. Using gaseous
14 oxygen as feedstock, both generate O3-enriched oxygen, free from other impurities. However,
15 the use of high-voltage discharge equipment with air as feedstock requires that the output be
16 scrubbed with water to remove appreciable amounts of the higher oxides of nitrogen (especially
17 nitric acid vapor) that form concurrently with O3 (Brown and Roberts, 1988; Taylor et al., 1993).
18
19 AX9.1.2.6 Experimental Exposure Protocols
20 A few recent chamber studies of physiological or biochemical effects have continued to
21 use square-wave exposure profiles typified by a rapid rise to, and falling off from, a steady target
22 concentration. However, during the last 20 years, most approaches into studying O3 effects on
23 plant growth and development have employed either simulations of the diurnal ambient O3
24 profile or enhancement/reduction of the ambient O3 concentrations.
25 Hogsett et al. (1985), Lefohn et al. (1986), and others have described the use in controlled
26 chambers of daily exposure profiles based on observed ambient O3 profiles. Such profiles were
27 used in the elaborately controlled chamber studies of Langebartels et al. (1997), while several
28 recent chamber studies have used simpler computer-controlled half- or full-cosine wave profiles
29 to simulate the typical daily rise and fall in ambient O3 levels (Mazarura, 1997; McKee et al.,
30 1997a,b).
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1 The early studies with OTCs involved adding constant levels of O3 to ambient air O3
2 concentrations, but all recent studies have used enrichment delivery systems that maintain
3 proportionality to, and track, ambient O3 concentrations to produce levels that more closely
4 resemble field observations. Both FACE and ZAPS studies have used proportional enrichment
5 to provide a range of treatments, although Wilbourn et al. (1995) and Ollerenshaw et al. (1999)
6 adjusted their systems manually to obtain a relatively constant target concentration during
7 exposure episodes.
8
9 AX9.1.3 Methods Involving Exposures to Ozone in Ambient Air
10 The experimental methods discussed above are largely aimed at developing quantitative
11 growth-response functions to permit the estimation of the effects of different ambient O3
12 scenarios. Because such methodologies usually involve exposures to higher than ambient O3
13 levels, the applicability of the functions obtained may, to some extent, be relevant only to
14 locations that are naturally subjected to high ambient O3 levels. Furthermore, as pointed out in
15 Section AX9.4, the response functions that they generate rarely incorporate other environmental,
16 genetic, and physiological factors, many of which can severely modify the magnitude of the
17 response to O3. The consequences of ignoring such modifications have been well stated by
18 De Santis (1999). The European level for protecting crops (based on the AOT40 index; see
19 Section AX9.4) was derived from OTC studies of O3-induced yield loss in wheat observed in
20 experiments conducted mostly in non-Mediterranean locations. However, the impact of
21 ambient O3 on wheat yields in the Po Valley of northern Italy is much less than the devastatingly
22 high loss (>60%) suggested by the seasonal exceedances of the level. On a similar note,
23 Manning (2003) has recently urged the absolute necessity of seeking "ground truth" as
24 verification of the nature and magnitude of impacts on vegetation as suggested by response
25 functions using ambient O3 monitoring data.
26 Such concerns clearly show that attention needs to be focused on incorporating
27 consideration of environmental and other factors into the response functions upon which
28 standards are based. This will require the development of improved simulation response models.
29 These concerns have also led to increasing attention being paid to seeking and developing
30 alternative approaches to assessing of impact, and the geographic extent of such impact —
31 approaches that are based on in situ exposures to ambient or sub-ambient O3 levels. Although
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1 one approach, the use of air-exclusion systems, requires experimental facilities, the other
2 approaches are generally based on simple field observations or measurements and, hence, can be
3 undertaken on a wide geographic scale.
4
5 AX9.1.3.1 Air-Exclusion Systems
6 The term, air-exclusion system, usually refers to a chamberless field system specifically
7 designed to protect plants from exposure to polluted air by blowing filtered air through their
8 canopies. Hogsett et al. (1987a,b) described several dedicated systems developed in the 1960s
9 and 1970s, but there appear to have been no recent O3-exclusion studies using systems
10 specifically designed for this purpose since those described by Olszyk et al. (1986a,b). Their
11 system, a modification of the earlier system of Jones et al. (1977), consisted of perforated 31.8-
12 cm OD inflatable polyethylene tubes laid between crop rows and supplied with CF air.
13 By increasing the size of the orifices progressively in sections along the 9-m length of the tubes,
14 an exclusion gradient was created with a progressive decrease in O3 levels in the air surrounding
15 the crop from one end of the system to the other. The system was used for studies on alfalfa
16 comparing plant response in OTCs, closed field chambers, the air-exclusion system, and ambient
17 air plots (as discussed above in Section AX9.1.2.4).
18 An air-exclusion component has also been part of the overall design of many OTC
19 experiments which added CF air or mixtures of CF and ambient air to chambers as part of the
20 overall design.
21
22 AX9.1.3.2 Natural Gradients
23 Naturally occurring O3 gradients hold potential for the examination of plant responses
24 along the gradient. However, few such gradients can be found which meet the rigorous
25 statistical requirements for comparable site characteristics such as soil type, temperature,
26 rainfall, radiation, and aspect (Manning and Krupa, 1992); although with small plants, soil
27 variability can be avoided by the use of potted plants. The use of soil monoliths transported to
28 various locations along natural O3 gradients is another possible approach to overcome
29 differences in soils; however, again this approach is limited to small plants.
30 Studies in the 1970s used the natural gradients occurring in southern California to assess
31 yield losses of alfalfa and tomato (Lycopersicon esculentum L.) (Oshima et al., 1976, 1977).
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1 A transect study of the impact of O3 on the growth of white clover and barley (Hordeum vulgare
2 L.) in the United Kingdom was confounded by differences in the concurrent gradients of SO2
3 and NO2 pollution (Ashmore et al., 1988). Studies of forest tree species in national parks in the
4 eastern United States (Winner et al., 1989) revealed increasing gradients of O3 and visible foliar
5 injury with increased elevation.
6
7 AX9.1.3.3 Use of Chemical Protectants
8 The use of protective chemicals is a relatively inexpensive, promising alternative to
9 experimental field exposures in chambers or free-air systems for determining plant response
10 to O3. These chemicals have recently been used in studies of different plant species, both in the
11 United States (Bergweiler and Manning, 1999; Kuehler and Flagler, 1999) and in Europe
12 (Bortier et al., 200la; Pleijel et al., 1999; Wu and Tiedemann, 2002), to determine if ambient O3
13 concentrations affect plant growth and productivity or are just exacerbating foliar injury.
14 Several chemical compounds (e.g., antioxidants, antisenescence agents, fungicides, pesticides)
15 have been known for many years to provide plants some protection from photochemical oxidants
16 such as O3 (Manning and Krupa, 1992). Most of these chemicals were originally used as a one-
17 time application to reduce visible injury caused by acute O3 exposures. The most widely used
18 and popular of these has been ethylenediurea (EDU). Carnahan et al. (1978) reported that EDU
19 protected pinto bean (Phaseolus vulgaris) from acute O3 injury. After this initial investigation,
20 EDU was shown to suppress visible O3 injury on several species of plants under both controlled
21 and field conditions (Brennan et al., 1987; Clarke et al., 1983). However, due to lack of a
22 commercial market for this product, its commercial manufacture was largely discontinued.
23 Other chemicals, including benomyl (Manning et al., 1974), carboxin (Rich et al., 1974),
24 ascorbic acid (Dass and Weaver, 1968), and others (Manning and Krupa, 1992), also exhibited
25 some beneficial effects in reducing visible O3 injury.
26 Several recent studies have used EDU in assessing the response of several plant species
27 to O3 to help validate the proposed critical level (AOT40 = 3000 ppb.h; see Section AX9.5) for
28 crop protection in Europe (Ball et al., 1998; Ribas and Penuelas, 2000; Tonneijck and Van Dijk,
29 2002a,b). EDU appeared to provide protection from visible foliar injury, but the results
30 regarding yield and biomass reductions were mixed. In a 3-year study over 12 sites throughout
31 Europe, Ball et al. (1998) used the ratio of EDU-treated versus non-treated white clover biomass
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1 and did not find a significant relationship between biomass reductions and AOT40 level.
2 However, an artificial neural network (ANN) model including vapor pressure deficit (VPD),
3 temperature, longitude, year, and altitude explained much more of the variance (r2 = 0.79). The
4 authors suggested that the greater sensitivity at certain sites in Germany may have been due to
5 occurrence of other pollutants. This meta-analysis indicates that EDU effects may be influenced
6 substantially by environmental factors.
7 In another study, Tonneijck and Van Dijk (2002b) assessed the relationship of visible
8 injury of subterranean clover (Trifolium subterraneum) to ambient O3 at four sites over three
9 growing seasons in the Netherlands, using EDU-treated and nontreated plants. Visible injury
10 varied by site and year, but was reduced to near zero by EDU treatment. However, no
11 relationship indicative of a protective effect of EDU with this plant species was observed for
12 biomass. Tonneijck and Van Dijk (2002a) also reported similar results with pinto bean. Both
13 EDU-treated and nontreated plants were exposed to ambient O3 at three locations in Spain over
14 one growing season (Ribas and Penuelas, 2000). Reductions in yield and biomass were
15 correlated with O3 concentration and EDU provided some protective effect, although results
16 varied by location and with meteorological conditions.
17 Chemicals have also been used to assess the effects of O3 on tree species. Border et al.
18 (200Ib) injected seedlings of an O3-sensitive poplar (Populus nigra) clone with EDU and
19 measured growth over a 1-year period at a field site near Brussels, Belgium. Over the growing
20 season, stem diameter increment was significantly higher (16%), biomass was increased (9%),
21 and foliar O3 symptoms were slightly less for the EDU-treated seedlings. Ozone levels were
22 reported to be low (AOT40 = 6170 ppb.h, May to September) during the exposure period.
23 In another study, Manning et al. (2003) applied EDU (foliar spray) and sodium erythorbate
24 (NaE) at various concentrations, biweekly for three growing seasons to loblolly pine (Pinus
25 taedd) at a field site in east Texas. After 3 years, the trees were harvested and biomass
26 measured. Neither EDU nor NaE prevented foliar O3 injury, but EDU applications at 450 ppm
27 resulted in increases in stem diameter and height, and total above-ground biomass. These
28 measures of growth also tended to slightly increase with applications of NaE, but the effects
29 were statistically nonsignificant.
30 The mechanisms by which protective chemicals, especially EDU, protect plants are poorly
31 understood. However, Wu and von Tiedemann (2002) reported that applications of two recently
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1 developed fungicides (azoxystrobin and epoxiconazole) provided protection to spring barley to
2 relatively high O3 exposures (150 to 250 ppb, 5 days, 7h/day) and resulted in increases in leaf
3 soluble protein content as well as the activity of several antioxidative enzymes (e.g., superoxide
4 dismutase, catalase, ascorbate-peroxidase, and glutathione reductase). In addition, the increase
5 in these enzymes reduced superoxide levels in the leaves.
6 Despite advances in the use of protective chemicals, a number of hurdles remain in using
7 them for assessing O3 effects. The phytotoxicity of EDU is well known, and the point has been
8 made repeatedly that for a particular species or cultivar, tests under a range of environmental
9 conditions and O3 exposures must be made to establish the efficacy of EDU for quantifying O3
10 effects (Heggestad, 1988; Kostka-Rick and Manning, 1992a). Unfortunately, although many
11 studies with EDU have been conducted in recent decades, very few have used multiple EDU
12 application levels along with multiple O3 exposures to characterize the EDU system for a given
13 plant species.
14 Recent studies have also shown that EDU does not always have greater effects at higher O3
15 exposures. For pinto bean grown in pots in studies in Spain and in the Netherlands, EDU
16 increased pod yield (Ribas and Penuelas, 2000; Tonneijck and Van Dijk, 1997). However, this
17 effect was not greater at sites with higher O3 exposure despite consistent experimental protocols
18 at all sites, including growing the same cultivar in pots with adequate water (Ribas and Penuelas,
19 2000; Tonneijck and Van Dijk, 1997). Such results suggest that it may be difficult to quantify
20 ambient O3 effects using EDU, because the amount of plant growth or yield expected at a low
21 (background) O3 concentration cannot be inferred from EDU-treated plants grown at locations
22 with higher O3 exposures.
23 Several studies suggest that EDU has effects other than its antioxidant protection and
24 phytotoxicity and show that environmental conditions affect the degree of protection afforded by
25 protective chemicals. In one study, even low concentrations of EDU (8 to 32 mg I/1 soil),
26 decreased soybean yield under low O3 exposure (7-h mean of 19 ppb) in CF OTCs (Miller et al.,
27 1994). This study also demonstrated that phytotoxicity (both foliar symptoms and growth
28 effects) can differ even in the same series of experiments, apparently due to changes in
29 environmental conditions, and that EDU can suppress yield at application rates that do not
30 always cause foliar symptoms (Miller et al., 1994). Finally, this study found that EDU altered
31 biomass partitioning by increasing vegetative growth and decreasing reproductive growth.
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1 A study of pinto bean grown in OTCs in Germany found that EDU treatment in CF OTCs
2 significantly increased yield, while EDU had no significant effect on yield in other O3 treatments
3 (Brunschon-Harti et al., 1995). In this study, O3 significantly reduced the mass of pods, shoots,
4 and roots. EDU increased root, leaf, and shoot mass across O3 treatments. However, the only
5 statistically significant interaction occurred with O3 x root mass. This study indicates that EDU
6 can stimulate above-ground growth and/or delay senescence regardless of O3 treatment.
7 The EDU approach for assessing the impact of ambient O3 exposures is potentially useful,
8 because it provides a separate line of evidence from other methods. Before using these
9 chemicals in a field setting, preliminary investigations under controlled conditions (e.g.,
10 chambers) should be done to evaluate the methods and timing of application, as well as proper
11 application rates, so as to avoid any potential toxic effects (Manning, 2000; Manning and Krupa,
12 1992). Unfortunately, such characterization has so far been limited, although substantial
13 progress has been made for radish (Raphanus sativus L.) (Kostka-Rick et al., 1993; Kostka-Rick
14 and Manning, 1992a, 1993). Thus, it is difficult to use data from existing EDU studies to
15 develop exposure-response relationships or to quantify the effects of ambient O3 exposure.
16 Despite these limitations, the EDU studies reviewed in previous O3 AQCDs (U.S. Environmental
17 Protection Agency, 1986, 1996) and the more recent studies summarized in Table AX9-1
18 (Section AX9.5) provide another line of evidence that ambient O3 exposures occurring in many
19 regions of the United States may be reducing the growth of crops and trees.
20
21 AX9.1.3.4 Biomonitoring
22 Bioindicators
23 The use of biological indicators to detect the presence of O3 injury to plants is a
24 longstanding and effective methodology (Chappelka and Samuelson, 1998; Manning and Krupa,
25 1992). A bioindicator can be defined as a vascular or nonvascular plant exhibiting a typical and
26 verifiable response when exposed to a plant stress such as an air pollutant (Manning et al., 2003).
27 To be considered a good indicator species, plants must
28 (1) exhibit a distinct, verified response,
29 (2) have few or no confounding disease or pest problems, and
30 (3) exhibit genetic stability.
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Table AX9-1. Advantages and Disadvantages of Protective Chemicals Used in
Assessment of O3 Effects on Plants
Advantages
No chambers required. Plants exposed to ambient conditions of O3, light, temperature, etc.
Can conduct studies "in situ." Equipment needs are minimal. No "chamber effects"
A high degree of replication possible both within and among locations
Disadvantages
Exposure-response studies require inclusion of other methodologies (OTCs, etc.)
Need measurements of ambient O3 and other meteorological variables (temp, rainfall, etc)
Many are toxic; have to conduct preliminary toxicology studies to determine proper rate, timing etc.
Species response can vary; need to screen for proper species to use
Mode of action not fully understood; may alter growth and biomass partitioning
Sources: Manning and Krupa (1992); Heggestad (1988); Kostka-Rick and Manning (1992b); Miller and Pursley
(1994).
1 Such sensitive plants can be used to detect the presence of a specific air pollutant such
2 as O3 in the ambient air at a specific location or region and, as a result of the magnitude of their
3 response, provide unique information regarding specific ambient air quality. Bioindicators can
4 be either introduced sentinels., such as the widely used tobacco (Nicotiana tabacum) variety
5 Bel W3, or detectors, which are sensitive native plant species (e.g., milkweed [Asclepias
6 syriaca]). The approach is especially useful in areas where O3 monitors are not operated
7 (Manning et al., 2003). For example, in remote wilderness areas where instrument monitoring is
8 generally not available, the use of bioindicator surveys in conjunction with the use of passive
9 samplers (Krupa et al., 2001) is a particularly useful methodology (Manning et al., 2003).
10 However, the method requires expertise or training in recognizing those signs and symptoms
11 uniquely attributable to exposure to O3 as well as in their quantitative assessment.
12 Since the 1996 O3 AQCD (U.S. Environmental Protection Agency, 1996) many new
13 sensitive species have been identified from controlled exposure studies and verified in the field
14 (Flagler, 1998; Innes et al., 2001). In addition, several new uses of this methodology have been
15 demonstrated, including a national O3 bioindicator network, studies in wilderness areas, and
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1 mature tree studies. Although it has been difficult to find robust relationships between the foliar
2 injury symptoms caused by O3 and effects on plant productivity or ecosystem function, visible
3 injury correlations with growth responses have been reported (Table AX9-2) (1998) (2003)
4 (2003). One workshop on the utility of bioindicators of air pollutants led to a useful series of
5 peer-reviewed publications in Environmental Pollution (Skelly, 2003).
6
7
Table AX9-2. Advantages and Disadvantages of Bioindicators Used to Study
O3 Plant Effects
Advantages
No chambers required. Plants exposed to ambient conditions of O3, light, temperature, etc.
Relatively inexpensive. Equipment needs are minimal. No "chamber effects"
A high degree of replication possible (sentinels) both within and among locations
Disadvantages
Results are generally correlative in nature with no true control
Individuals need to be trained and experienced in O3 symptom recognition
Need adequate numbers of plants (detectors) to ensure valid results
Need preliminary tests to insure a constant symptomotology of material used
Need to use more than one indicator species (detector) per area if possible
Need to quantify site characteristics (soils, light) that may influence symptom expression
Need measurements of ambient O3 (active or passive) and other meteorological variables (temp,
rainfall, etc)
Need to ensure that cultural (sentinels) practices (soil, irrigation, fertilization, etc.) are similar
among sites
1 National network
2 The U.S. Forest Service in cooperation with other federal and state agencies developed a
3 network of O3 bioindicators to detect the presence of O3 in forested systems throughout the
4 United States (Smith et al., 2003). This ongoing program was initiated in 1994; and 33 states
5 currently participate. In a coordinated effort, a systematic grid system is used as the basis of plot
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1 selection, and field crews are trained to evaluate O3 symptoms on sensitive plant species within
2 the plots (Coulston et al., 2003; Smith et al., 2003).
3 The network has provided evidence of O3 concentrations high enough to induce visible
4 symptoms on sensitive vegetation. From repeated observations and measurements made over a
5 number of years, specific patterns of areas experiencing visible O3 injury symptoms can be
6 identified. Coulston et al. (2003) used information gathered over a 6-year period (1994 to 1999)
7 from the network to identify several species that were sensitive to O3 over a regional scale
8 including sweetgum (Liquidambar styraciflua), loblolly pine, and black cherry (Prunus
9 serotind).
10
11 Wilderness areas
12 The use of bioindicator species as detectors has proven to be an effective technique for
13 deriving a relative estimate of O3 injury in wilderness areas in both the United States and Europe
14 (Chappelka et al., 1997, 2003; Manning et al., 2002). However, to be truly effective, these
15 regional and national bioindicator studies need the inclusion of air quality data and related
16 growth studies to determine effects on productivity and ecosystem function (Bytnerowicz et al.,
17 2002; Manning et al., 2003; Smith et al., 2003). In addition, O3 often co-occurs with other air
18 borne pollutants, so it is important to consider that, in some areas, other pollutants may be
19 playing a role as well.
20 Chappelka et al. (1997, 2003) conducted surveys of foliar injury on several native plant
21 species throughout the Great Smoky Mountains National Park (GRSM), including black cherry
22 (Prunus serotind)., tall milkweed (Asclepias exaltatd), cutleaf coneflower (Rudbeckia laciniata\
23 and crownbeard (Verbesina occidentalis). Visible foliar symptoms were prevalent throughout
24 the Park, indicating that injury-producing O3 levels were widespread in GRSM.
25 Manning et al. (2002) recently summarized a multiyear (1993 to 2000) bioindicator project
26 in the Carpathian Mountain range in eastern Europe. They evaluated numerous trees, shrubs,
27 forbs, and vines for possible symptoms of O3 injury. Observations were made at plots located in
28 the vicinity of either active or passive O3 monitors (Bytnerowicz et al., 2002). Approximately
29 30 species of native plant detectors were identified as possible bioindicators, the majority of
30 which (21) were shrubs (Manning et al., 2002). Based on these observations, it was concluded
31 that O3 concentrations were sufficiently high to impact ecosystems in the region. Similar
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1 investigations regarding the sensitivity of native species have been conducted in Switzerland
2 (Novak et al., 2003) and Spain (Orendovici et al., 2003).
3
4 Mature tree detectors
5 Many studies have reported visible injury of mature coniferous trees caused by O3,
6 primarily in the western United States (Arbaugh et al., 1998) and, to a lesser extent, to mature
7 deciduous trees in eastern North America. In an effort to determine the extent and magnitude of
8 visible injury in mature tree canopies, Hildebrand et al. (1996) and Chappelka et al. (1999b)
9 conducted independent studies in the GRSM and the Shenandoah National Park (SHEN).
10 The species examined were sassafras (Sassafras albidum), black cherry, and yellow-poplar
11 (Liriodendron tulipifera L.) in GRSM and white ash (Fraxinus americana L.), black cherry, and
12 yellow-poplar in SHEN. Protocols were similar at both parks, and trees were located near O3
13 monitors at three different areas in each park. Results from both studies indicated that symptoms
14 of O3 injury were present in the trees and correlated with O3 exposure both spatially and
15 temporally. Ozone injury tended to be most severe at the highest elevation, except with yellow-
16 poplar.
17 Hildebrand et al. (1996) observed significant O3 exposure-plant response relationships with
18 black cherry. The best relationships were found between foliar injury and the cumulative
19 exposure statistics SUM06 and W126 (see Section AX9.5), indicating that higher O3
20 concentrations were important in eliciting a response in black cherry. No O3 exposure-plant
21 response relationships were found with any species tested in GRSM (Chappelka et al., 1999b);
22 but, when the data were combined for both parks, a significant correlation (r = 0.72) with black
23 cherry was found for both SUM06 and W126, and injury was the greatest (r = 0.87) at the higher
24 elevations (Chappelka et al., 1999a).
25 Based on a study in which visible symptoms of O3 injury were characterized for large,
26 mature yellow-poplar and black cherry trees in GRSM (Chappelka et al., 1999a), Somers et al.
27 (1998) compared radial growth differences among trees classified as sensitive or nonsensitive
28 based on the severity of visible foliar injury observed over a 3-year period (1991 to 1993).
29 Significantly more radial growth was observed over both a 5- and a 10-year period for the
30 nonsensitive compared to the sensitive trees. No significant relationship was found for black
31 cherry tree growth.
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1 Vollenweider et al. (2003), using data collected from continuous forest inventory (CFI)
2 plots across Massachusetts, compared growth rates among either symptomatic or asymptomatic
3 mature black cherry trees. Of the 120 trees sampled in 1996, 47% exhibited visible foliar injury.
4 Using CFI data, growth rates were compared over a 31-year period. The growth rates for
5 symptomatic trees were reduced by 28% compared with asymptomatic trees.
6 Because these studies (Somers et al., 1998; Vollenweider et al., 2003) were not controlled
7 studies and used a small sample of trees, they cannot validly be used to characterize cause and
8 effects related to the visible symptoms and radial growth they describe. However, the results
9 indicate the possibility that O3 is correlated with growth losses in some sensitive genotypes,
10 illustrating the potential usefulness of this visible O3 injury methodology in assessing effects on
11 the growth rates of mature deciduous trees.
12
13 Cultivar comparisons
14 The idea of using cultivars or isogenic lines of crop species that differed in O3 sensitivity as
15 sentinels to determine the ambient effects of O3 in the field was presented in the 1996 O3 AQCD
16 (U.S. Environmental Protection Agency, 1996). The rationale was that comparing the ratio of
17 injury scores or some measure of growth between two different cultivars varying in O3
18 sensitivity should be indicative of the relative amount of ambient stress to plants at a given
19 location. A sensitive:resistant ratio close to unity would indicate relatively low O3
20 concentrations; and a low ratio, higher O3 levels. Results from locations differing in O3
21 concentrations could be evaluated to develop exposure-response models. The original protocol
22 was derived using two isogenic lines of white clover (Trifolium repens) differing in O3
23 sensitivity (Heagle et al., 1994b, 1995).
24 This white clover model system has been used in several multi-location studies in the
25 United States (Heagle and Stefanski, 2000) and Europe (Ball et al., 2000; Bermejo et al., 2002;
26 Mills et al., 2000). Heagle and Stefanski (2000) compared results from eight sites over a 2-year
27 period with various exposure indices (SUMOO, SUM06, W126, and others) to determine a
28 best-fit regression. They found that most of the indices preformed similarly. The highest r2
29 values (0.87 to 0.93) were obtained using only the later harvests and a 6 h day"1 index (1000 to
30 1600 h). Similar multiple-comparison studies conducted in Europe using the AOT40 index (Ball
31 et al., 2000; Mills et al., 2000) yielded poorer r2 values. Factors such as air temperature, NOX
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1 (high levels at some sites), and lower O3 concentrations in Europe were suggested to account in
2 part for the differences between U.S. and European study results. Bermejo et al. (2002), in a
3 study in Spain, improved the model by comparing the biomass ratio of these white clover
4 isolines to measures of O3 uptake (flux) rather than an exposure index (AOT40). Together, these
5 studies indicate that systems such as the white clover model can help reveal O3 exposure-
6 response relationships and provide valuable information regarding ambient O3 conditions in a
7 given location. Table AX9-3 lists the advantages and disadvantages of the use of cultivar
8 comparisons in assessing O3 effects of plants.
9
10
Table AX9-3. Advantages and Disadvantages of Cultivar Comparisons Used in
Assessment of O3 Effects on Plants
Advantages
No chambers required. Plants exposed to ambient conditions of O3, light, temperature, etc.
Relatively inexpensive. Equipment needs are minimal. No "chamber effects"
A high degree of replication possible both within and among locations
Can conduct studies "in situ"
Disadvantages
Need preliminary tests to insure sensitivity and growth patterns of genotypes used are consistent
Need measurements of ambient O3 and other meteorological variables (temp, rainfall, etc)
Have to ensure cultural practices (soil, irrigation, fertilization, etc.) are similar among sites
Need to closely monitor plants for presence of other factors that may cause a misinterpretation
of results
1 Dendrochronological techniques
2 It has been difficult to determine whether O3 significantly affects tree growth and
3 productivity in the field, because O3 concentrations are omnipresent and tree response to this
4 pollutant is altered by many factors. The use of dendrochronological techniques to answer
5 questions regarding ambient O3 effects on forest growth and ecosystem function has recently
6 emerged as a very useful biomonitoring methodology (Cook, 1990; McLaughlin et al., 2002).
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1 The technique is useful when either instrument or passive O3 monitoring methods are used to
2 determine ambient O3 conditions.
3 Initial experiments were primarily correlative in nature and attempted to relate symptoms
4 of visible injury with growth losses as revealed by tree ring analysis (Arbaugh et al., 1998;
5 Benoit et al., 1983; Peterson et al., 1995; Somers et al., 1998; Swank and Vose, 1990). These
6 studies evaluated radial growth patterns determined by cores removed from trees in the presence
7 or absence of overt O3 injury symptoms.
8 The method has also been adapted to better understand forest ecosystem function
9 (McLaughlin and Downing, 1995; Bartholomay et al., 1997; McLaughlin et al., 2003). The
10 response of mature loblolly pine growing in eastern Tennessee to ambient O3 and moisture stress
11 was evaluated by McLaughlin and Downing (1995, 1996). They made radial growth
12 measurements from 12 to 37 times per year using dendrometer bands and determined
13 relationships between O3, moisture stress, and radial growth. Exposures to O3 concentrations
14 >0.04 ppm with high temperatures and low soil moisture resulted in short-term depression in
15 radial growth. Reductions in growth were estimated to vary from 0 to 15% per year and
16 averaged approximately 5% per year.
17 Bartholomay et al. (1997) examined white pine (Pinus strobus) radial growth in eight
18 stands throughout Acadia National Park, Maine over a 10-year period from 1983 to 1992. They
19 related growth rates to several factors, including O3 concentration. Ozone levels were negatively
20 correlated with radial growth in seven of the eight stands. Site characteristics were important in
21 the relationship: stands growing on shallow, poorly drained soils were most sensitive to O3 in
22 the late portion of the growing season, possibly due to premature senescence of foliage.
23 However, litterfall measurements were not reported. Trees growing on better sites were more
24 sensitive to O3 during the entire growing season, indicating the possibility of high O3 uptake
25 rates throughout the growing season. Although these field studies (Bartholomay et al., 1997,
26 1996; McLaughlin and Downing, 1995) did not compare the direct effects of O3 on the two pine
27 species, they indicate that potential interactions exist among O3 and other climatic and edaphic
28 factors, such as temperature and soil moisture.
29 Using both automated and manual dendrometer bands, McLaughlin et al. (2002) examined
30 the growth response of yellow-poplar trees recently released from competition. In addition to
31 measuring growth, sap flow measurements were conducted and soil moisture was measured in
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1 the vicinity of the trees. They were not able to detect O3 effects in this 1-year study. Advantages
2 and disadvantages of dendrochronology techniques for evaluating whole-tree physiological
3 responses for individual trees and forest stands are listed in Table AX9-4.
4
5
Table AX9-4. Advantages and Disadvantages of Various Dendrochronological
Techniques Used in Assessment of O3 Effects on Plants
Advantages
Provide information regarding growth effects under ambient conditions
Good historical information regarding O3 effects
Can provide data on daily and seasonal growth and O3 patterns and correlate with physiological
function
Provide information on forest function related to ambient O3 concentrations
Can link data with process-level growth models
Disadvantages
Results are generally correlative in nature with no true control
Need background O3 and meteorological data (historical records)
Need to account for other factors such as competition, in analyzing data
Individuals need to be trained in counting growth rings
Replication can be difficult (expensive and technological limitations)
Complicated statistical analyses are sometimes required
Can be expensive, especially if using automated growth (dendrometer) bands
1 The use and evolution of various dendrochronological methods in the field of air pollution
2 effects research is reviewed in detail by McLaughlin et al. (2002). Automated dendrometer
3 bands provide a powerful tool for measuring radial growth responses of trees on an hourly or
4 daily basis. Diurnal patterns of growth can be related to water use and O3 concentrations using
5 time-series analyses. The major drawbacks of the method are that it is expensive and time
6 consuming.
7
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1 AX9.1.3.5 Calibrated Passive Monitors
2 Many studies have used passive monitors in the mapping of ambient O3 concentrations,
3 especially in remote areas (Cox and Malcolm, 1999; Grosjean et al., 1995; Krupa et al., 2001).
4 Because they are cumulative recording devices, they do not record short-term variations in O3
5 concentration but only the total exposure over a given interval, usually between 1 to 4 weeks.
6 Thus, they produce a measurement that resembles the instrumentally derived exposure index
7 SUMOO. However, it is common to divide the cumulative exposure by the number of hours of
8 exposure to get an hourly average. In addition, Krupa et al. (2001, 2003) were able to estimate
9 the underlying frequency distribution of hourly O3 concentrations from passive samplers using
10 models based on a collocated O3 monitor, showing the potential for passive samplers to provide
11 estimates beyond total O3 sum.
12 Runeckles and Bowen (2000) used the ZAPS system described in Section AX9.2.2.3 to
13 subject both crops and passive monitors (Williams, 1994) to a range of exposures. Passive
14 monitors were also exposed at 16 agricultural field sites along a transect through the Fraser
15 Valley, British Columbia, Canada. Most field sites were downwind of the Greater Vancouver
16 metropolitan area. All passive monitors were replaced at weekly intervals and the data from
17 those in the ZAPS plots were "calibrated" to crop responses by means of Weibull exposure-
18 response functions. Since the meteorological conditions throughout the valley were reasonably
19 consistent from site to site, the use of these functions with data from the network passive
20 monitors as inputs permitted the estimation of crop losses at the network sites. The overall
21 method was, thus, a hybrid of several methodologies.
22 Although based on a single study, the use of passive monitors has potential for assessing
23 crop losses at sites removed from locations with known ambient O3 concentrations. Provided
24 that the network and calibration sites have similar meteorological conditions, the method yields
25 crop loss estimates that are responses to local ambient O3 levels as influenced by local
26 meteorological conditions.
27
28 AX9.1.4 Numerical/Statistical Methodologies
29 Proper experimental design strategies including replication, randomization, and
30 experimental protocols are paramount in O3-effects research. These have been discussed in
31 detail in previous O3 AQCDs (U.S. Environmental Protection Agency, 1996, 1986), as have the
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1 different statistical analytical procedures used to determine the probable significance of results.
2 However, new investigative approaches have demanded the adoption of new analytical methods.
3 For example, the use of dendrochronological techniques has led to the use of time-series analysis
4 (McLaughlin et al., 2003) and linear aggregate models (Cook [1990], as reviewed by
5 McLaughlin et al. [2002]).
6 In spite of the rigors of the analyses, many differences occur in the published literature for
7 almost any plant response to O3 stress. Differences inevitably result from different researchers
8 studying different locations, using different experimental methodologies and genetically
9 different plant material even when using a common species. The techniques of meta-analysis
10 can be used to consolidate and extract a summary of significant responses from a selection of
11 such data.
12 Despite the differences in responses in the 53 primary studies used, a recent meta-analysis
13 by Morgan et al. (2003) of the effects of O3 on photosynthesis, growth, and yield of soybean
14 showed "overwhelming evidence for a significant decrease in photosynthesis, dry matter
15 production and yield. . . .across all the reported studies on effects of chronic O3 treatment." The
16 meta-analysis defined O3 stress as exposure to -70 ppb O3 for at least 7 days and found average
17 shoot biomass and seed yield decreases of 34% and 24%, respectively. Furthermore, although
18 other stress factors such as drought and UV-B did not affect the O3 responses, elevated CO2 was
19 found to significantly decrease O3-induced losses.
20 The meta-analysis method clearly has the potential to consolidate and refine the
21 quantitative exposure-response models for many species. The majority of the reported growth
22 and physiological responses related to O3 stress are for individual plants, primarily in various
23 types of exposure chambers. It is difficult to extrapolate these responses to stand/community,
24 ecosystem, or region-wide assessments, particularly in view of the importance of the significant
25 interactions that may occur between plant responses O3 and other environmental stresses. Along
26 with the shift in effects research to a more ecological approach, these concerns necessitate a
27 move from simple regression analysis to more complex mathematical approaches to handle a
28 wider array of independent input variables than O3 exposure alone. Other independent input
29 variables that must be accounted for include air and soil temperatures, soil moisture, relative
30 humidity, wind speed, and, particularly in the case of natural systems, biotic factors such as pests
31 and pathogens, plant density/spacing, and measures of plant competition.
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1 Artificial neural network methodology was used by Balls et al. (1995) for "unraveling the
2 complex interactions between microclimate, ozone dose, and ozone injury in clover" and in the
3 study with the protectant chemical EDU, discussed in Section AX9.2.3.3 (Ball et al., 1998). The
4 multi-factor model for predicting the effects of ambient O3 on white clover developed by Mills
5 et al. (2000) utilized both ANN and multiple linear regression methods.
6 Models incorporating ANNs are of the "regression" type (Luxmoore, 1988) in contrast to
7 "mechanistic" or "phenomenological" models which have wider applicability. Process-level
8 models of either type have been developed at the organelle, individual plant (Constable and
9 Taylor, 1997; Weinstein et al., 1998), canopy (Amthor et al., 1994), and stand level (Ollinger
10 et al., 1997; Weinstein et al., 2001) and provide estimates of the rate of change of response
11 variables as affected by O3 over time. However, as pointed out in the 1996 O3 AQCD (U.S.
12 Environmental Protection Agency, 1996), mechanistic process models lack the precision of
13 regression models as well as their ability to estimate the likelihoods of responses. In their
14 extensive reviews, Kickert and Krupa (1991) and Kickert et al. (1999) summarized the
15 advantages and shortcomings of many different models and made the important point that most
16 of the models that have been described provide consequence assessments that quantify the
17 magnitudes of effects, but not risk assessments that quantify the likelihoods of such effects.
18 Descriptions of several specific models are provided in other sections of this criteria document,
19 and advantages and disadvantages of modeling techniques used in assessing O3 effects on plants
20 are summarized in Table AX9-5.
21
22
Table AX9-5. Advantages and Disadvantages of Modeling Techniques Used in
Assessment of O3 Effects on Plants
Advantages
• Provide an understanding of cause-effect relationships over time
Disadvantages
• Have to make assumptions based on a paucity of data
• Most models are very complex and difficult to understand
• Need to be evaluated for predictive validity
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1 AX9.1.5 Improved Methods for Defining Exposure
2 Ambient air quality is defined in terms of the measured O3 concentrations in the air at plant
3 height above ground level. Compilations of such concentration data have long been used as
4 surrogates of the exposures to which plants are subjected. However, as long ago as 1965, field
5 research provided evidence that plant response was a function, not of ambient O3 concentration
6 per se, but of the estimated flux of O3 to the plant canopy (Mukammal, 1965). Subsequently,
7 Runeckles (1974) introduced the term "effective dose" to define that part of the ambient
8 exposure that was taken up by a plant. Fowler and Cape (1982) later referred it as "pollutant
9 applied dose" (PAD), defined as the product of concentration, time and stomatal (or canopy)
10 conductance, with units g m"2. Such estimates of O3 uptake or flux provide a more biologically
11 relevant description of exposure than the simple product of concentration and time alone, and
12 they formed the basis of Reich's 1983 "unifying theory" of plant response to O3 (Reich, 1983).
13 However, it was not until the early 1990s that the inherent advantages of using O3 flux
14 rather than O3 concentration as a basis for determining response effects began to be widely
15 accepted, as demonstrated by the subsequent increase in publications involving flux
16 measurements and modeling (e.g., Fuhrer et al. [1997]; Griinhage and Jager [2003)]; Griinhage
17 et al. [1993; 1997]; Massman et al. [2000)]; Musselman and Massman [1999]; Pleijel [1998]).
18 A key requirement for flux determination is the measurement of stomatal or canopy
19 conductances, using established porometer/cuvette techniques or eddy correlation methods.
20 The usefulness and relevance of flux as a measure of exposure are discussed in detail in Section
21 AX9.4.
22 Efforts to develop regional-scale models of O3 deposition and stomatal uptake are currently
23 under way with a view to providing improved assessments of the risks to vegetation across
24 Europe (Emberson et al., 2000; Simpson et al., 2001, 2003).
25
26
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1 AX9.2 SPECIES RESPONSE/MODE-OF-ACTION
2 AX9.2.1 Introduction
3 The evaluation of O3 risk to vegetation requires fundamental understanding of both the
4 functioning of the vegetation and how external environmental influences can alter that function.
5 For biological organisms subjected to atmospheric O3, those alterations can be complex and
6 multiple. In addition, biological organisms have plasticity to external interactions due to their
7 complex internal, self-correcting systems, making the task of identifying their "correct"
8 functioning difficult. This section emphasizes reactions of O3 with the cell and tissue, rather
9 than the whole plant, to describe the fundamental mechanisms known to govern the response of
10 the plant to O3 exposure.
11 The many regulatory systems contained in leaves change both as a function of leaf
12 development and in response to various environmental stresses. Leaves function as the major
13 regulators of anatomical and morphological development of the shoot and control the
14 translocation of carbohydrates to the whole plant (Dickson and Isebrands, 1991). This section
15 discusses the movement of O3 into plant leaves and their biochemical and physiological
16 responses to O3.
17 The 1996 criteria document (U.S. Environmental Protection Agency, 1996) assessed the
18 information available at that time concerning the biochemical and physiological responses to the
19 movement of O3 into plant leaves. This information continues to be valid. Ozone uptake in a
20 plant canopy is a complex process involving adsorption to surfaces (leaves, stems, and soil) and
21 absorption into leaves (Figure AX9-1). However, the initial biochemical changes that result
22 within leaf cells after the entry of O3 and how these changes interact to produce plant responses
23 remain unclear. The response of vascular plants to O3 may be viewed as the culmination of a
24 sequence of physical, biochemical, and physiological events. Only the O3 that diffuses into a
25 plant through the stomata (which exert some control on O3 uptake) to the active sites within a
26 leaf impairs plant processes or performance. An effect will occur only if sufficient amounts
27 of O3 reach sensitive cellular sites that are subject to the various physiological and biochemical
28 controls within the leaf cells. Ozone injury will not occur if (1) the rate and amount of O3 uptake
29 is small enough for the plant to detoxify or metabolize O3 or its metabolites or (2) the plant is
30 able to repair or compensate for the O3 impacts (Tingey and Taylor, 1982; U.S. Environmental
31 Protection Agency, 1996). Therefore, a precondition for O3 to affect plant
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Atmospheric Processe
i
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5 "~ 1 Atmosphere 1 1 Boundary Layer 1
r
Canopy Processes
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i
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1
Ecosystem
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opy J (
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Figure AX9-1. Ozone uptake from the atmosphere. Ozone moves from the atmosphere
above the canopy boundary layer into the canopy primarily by turbulent
air flow. Canopy conductance, controlled by the complexity of the canopy
architecture, is a measure of the ease with which gases move into the
canopy. Within the canopy, O3 is adsorbed onto surfaces as well as being
absorbed into the foliage. Foliage absorption is controlled by two
conductances, leaf boundary layer and stomatal, which together determine
leaf conductance. The solid black arrows denote O3 flow; dotted arrows
indicate processes affecting uptake or response to O3. Boxes at the left
with double borders are those processes described in the figure.
1 function is that it must enter the stomata and be absorbed into the water lining the mesophyll cell
2 walls. The response of each plant is determined by the amount of O3 entering the leaves, which
3 varies from leaf to leaf.
4 Some potentially significant processes have been investigated since the 1996 criteria
5 document, especially detoxification and compensatory processes. The role of detoxification in
6 providing a level of resistance to O3 has been investigated; however, it is still not clear as to what
1 extent detoxification can protect against O3 injury. Data are needed especially on the potential
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1 rates of antioxidant production and on the subcellular localization of the antioxidants. Potential
2 rates of antioxidant production are needed to assess whether they are sufficient to detoxify the O3
3 as it enters the cell. The subcellular location(s) is needed to assess whether the antioxidants are
4 in cell wall or plasmalemma locations that permit contact with the O3 before it has a chance to
5 damage subcellular systems. Although these processes divert resources away from other sinks,
6 detoxification and compensation processes may counteract the reduction in canopy carbon
7 fixation caused by O3. The quantitative importance of these processes requires investigation.
8 As a result of the research since the 1996 criteria document (U.S. Environmental Protection
9 Agency, 1996), the way in which O3 exposure reduces photosynthesis, especially its effects on
10 the central carboxylating enzyme, Rubisco (ribulose-l,6-P2-carboxylase/oxygenasel), is
11 better understood. The rate of leaf senescence has been shown to increase as a function of
12 increasing O3 exposure. The mechanism of the increased senescence is not known, and, hence,
13 it deserves further study.
14 Finally, the role that changes in allocation of resources play in plant response to O3 is now
15 better understood. Most studies have shown that O3 decreases allocation of photosynthate to
16 roots. In some cases, allocation to leaf production has increased. Whether these changes are
17 driven entirely by changes in carbohydrate availability or are controlled by other factors (e.g.,
18 hormones) is not known. Physiological effects within the leaves inhibit photosynthesis; alter the
19 assimilation of photosynthate and shift its allocation patterns; and can lead to reduced biomass
20 production, growth, and yield (U.S. Environmental Protection Agency, 1986, 1996).
21 The major problem facing researchers trying to predict long-term O3 effects on plants is
22 determining how plants integrate the responses to O3 exposures into the overall naturally
23 occurring responses to environmental stressors. Little is now known about how plant responses
24 to O3 exposures change with increasing age and size, but this information is crucial to predicting
25 the long-term consequence of O3 exposure in forested ecosystems.
26 This section focuses on reactions of O3 within cells and cellular tissue, in order to explain
27 known mechanisms that govern plant responses. The processes that occur at cell and tissue
28 levels within the leaf will be divided into several steps beginning with O3 uptake and its initial
29 chemical transformations into a series of currently unknown, but suspected toxic, chemicals
30 (Figure AX9-2). The discussion will then focus sequentially upon various cell regions, their
31 general physiology, and the changes that may occur within a plant after O3 exposure. This is
August 2005 AX9-41 DRAFT-DO NOT QUOTE OR CITE
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Atmospheric Processes
I Canopy Processes |
X
I Leaf Processes | J
x
I Plant Response Processes |
1
| Community Processes
4
I Ecosystem Processes
Plasma Membrane
Second Sites:
Signaling
Second Sites:
Metabolism
General Physiology:
Cell Metabolism
General Physiology:
Whole Plant
Figure AX9-2. Absorption and transformation of O3 within the leaf. The varied
processes are broken down in to smaller mechanistic steps that lead from
uptake of atmospheric O3 into the alterations which may occur within the
individual plant. Each plant responds to the O3 level and therefore
interacts with the total ecological setting to generate an ecosystem
response due to the O3.
1
2
3
4
5
6
7
important because the varying responses of the different plant species in a community ultimately
lead to an ecosystem response. Finally, a general summary is presented that discusses the known
or suspected changes that occur within the whole plant.
AX9.2.2 Mechanisms of Ozone-Induced Plant Alterations
Plants can survive O3 stress through exclusion or tolerance mechanisms (Levitt, 1972)
(Tingey and Taylor, 1982). Ozone may be excluded from tissues or cells via stomatal closure,
by extracellular oxidants, or by membrane impermeability to O3 or its products. Past
investigations of O3 injury have indicated that physiological and metabolic changes occur (Harris
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1 and Bailey-Serres, 1994; Heath, 1988; Heath and Taylor, 1997; Reddy et al., 1993). Many of
2 these changes are likely initiated via gene expression. During the last decade, our understanding
3 of the cellular processes within plants has increased. Although the fundamental hypotheses
4 concerning O3-induced changes in physiology have not changed, a more complete development
5 of the theories is now nearing possibility.
6
7 AX9.2.2.1 Changes in Metabolic Processes: Current Theories
8 The current hypotheses regarding the biochemical response to O3 exposure revolve about
9 injury and its prevention. These are well discussed by Pell et al. (1997) and are listed below in
10 no order of importance. Although they are listed separately, some may be interlinked and related
11 to each other.
12 (1) Membrane Dysfunction. The membrane is altered by O3, principally via protein
changes not involving the lipid portions of the membrane (except at extremely high
levels of O3). These alterations involve increased permeability with perhaps lessened
selectivity, declines in active transport, and changes in the trigger mechanisms of
signal transduction pathways such that the signals are no longer suitable for the state
of the cell. The cellular pools and transport systems of Ca2+/K+/H+ are the primary
suspects.
13 (2) Antioxidant Protectants. Varied antioxidants (both as metabolites and enzyme
systems) can eliminate the oxidant or its products, if present at time of fumigation
and in sufficient abundance. However, oxidant entry that occurs rapidly can
overwhelm the antioxidant response.
14 (3) Stress Ethylene Interactions. Visible injury is caused by the interaction of O3 with
stress-induced ethylene, either by direct chemical transformation to a toxic product or
by alteration of the biochemical relations at the ethylene binding site.
15 (4) Impairment of Photosynthesis. A product of O3 (and less probably, O3 itself) enters
the cell, causing a decline in the mRNA for Rubisco (especially the message RNA
species ofrbcS and rbcL) such that Rubisco levels slowly decline within the
chloroplast, leading to a lowered rate of CO2 fixation and productivity. This process
is very similar to early senescence and may be linked to general senescence.
Alternatively, a false signal is generated at the cell membrane which lowers the
transcription of DNA to mRNA. Ozone alters the normal ionic and water relations of
guard cells and subsidiary cells, causing the stomata to close and limit CO2 fixation.
In any case, the response of the stomata to the current environment does not promote
efficient photosynthesis.
August 2005 AX9-43 DRAFT-DO NOT QUOTE OR CITE
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1 (5) Translocation Disruption. One of the biochemical systems most sensitive to O3
exposure is the translocation of sugars, such that even a mild exposure inhibits the
translocation of carbohydrate (Grantz and Farrar, 1999, 2000).
2 (6) General Impairment/Disruption of Varied Pathways of Metabolism. This is the
oldest and most vague concept of how O3 alters metabolism. It is based upon early
work in which the enzymes and metabolites that could be assayed were. Thus, these
results were based upon what could be done, rather than on a coherent hypothesis.
The best examples are listed in Dugger and Ting (1970).
3 The latter two theories can be restated as a loss of productivity with three possible
4 somewhat-independent causes: (a) a reduced production of the basic building blocks of growth
5 and, hence, a slowing of growth in at least one organ; (b) a reduced ability to reproduce, leading
6 to a decreased production of viable seeds or of fruits and nuts; and (c) a decreased ability to
7 mount a defense against pathogens or insects, leading to weaker plants, which are more liable to
8 be overcome by other stresses. It is important to separate out effects that may be detrimental or
9 disfiguring, such as the production of visible injury, but which have not been shown to lead
10 directly to a loss of productivity due to possible compensation by the remaining tissue.
11
12 AX9.2.2.2 Modifications of Plant Physiological Processes
13 The discussion that follows will focus on physiological processes rather than on species-
14 specific responses; in most cases, the mechanisms of response are similar regardless of the
15 degree of sensitivity of the species. Therefore, Arabidopsis, whose physiology and genome
16 continue to be studied and described by a large number of scientists is an appropriate plant for
17 studying O3 injury. Though the responses of mature trees and understory plants are critical to
18 understanding plant interactions at an ecosystem level, the time required for trees to reach
19 maturity makes using them to study biological mechanisms an inefficient choice.
20 The high levels of O3 used for some investigations do not automatically invalidate the
21 results obtained in those studies. Typically when a new hypothesis is being investigated,
22 extreme levels of the toxicant are used to determine its effects clearly. The older studies that
23 used concentrations as high 1 ppm, an extreme level, helped to define current studies. Later
24 experiments have used concentrations nearer ambient levels. Many of the current studies on
25 physiology use exposures between 0.15 and 0.25 ppm, which though higher than ambient levels
26 in some areas of the country, bypass confounding changes but allow for rapid experiments.
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1 Three forms of air pollutant-induced injury patterns are currently known to exist: (a) acute
2 stress, generated by high atmospheric concentrations of pollutants for short periods of time;
3 (b) chronic stress, generated by lower concentrations of pollutants for long periods of time; and
4 (c) accelerated senescence, generated by very low concentrations of pollutants for very long
5 periods of time. At higher levels, distinct visible injury generally occurs due to cellular and
6 tissue death of regions of leaf mesophyll cells. This leads to a decline in the total area of
7 metabolically active tissue, with consequent loss of membrane integrity, loss of metabolites into
8 the extracellular tissue space, and formation of oxidative products. When no visible injury is
9 observed, lowered rates of photosynthesis or productivity are often used to document injury.
10 Under these conditions, metabolism is altered and the pool sizes of many metabolites are
11 changed. More importantly, the altered biochemical states within the tissue lead to the inability
12 of the plant to respond properly to existing environmental conditions and to other stressors
13 (Heath, 1988, 1994b; Koziol and Whatley, 1984; Manning and Keane, 1988; Schulte-Hosted
14 etal., 1988).
15
16 AX9.2.3 Ozone Uptake by Leaves
17 Plants respond to O3 similarly to other stressors on the levels of exclusion, tolerance, and
18 repair (Levitt, 1972). The response mechanism depends upon the O3 concentration,
19 environmental conditions, and the developmental and metabolic state of the plant (Guzy and
20 Heath, 1993). These responses are detrimental to plant productivity, because they cost the plant
21 metabolic resources. In some cases, the stomata close under the O3 exposure, excluding the
22 pollutant from the leaf interior and preventing injury. However, if this happens too often, CO2
23 fixation is also inhibited and plant productivity suffers.
24 Atmospheric O3 does not cause injury, but rather it is the O3 that enters the plant that
25 causes an effect (Guzy and Heath, 1993; Tingey and Taylor, 1982). Three well-defined,
26 sequential processes control the movement of O3 from the atmosphere into the sites of action
27 within the leaf and must occur to trigger O3 stress (Heath, 1980). The processes are (1) entry of
28 O3 into the leaf, (2) reactions of O3 and its possible reaction product(s) in the water phase at cell
29 surfaces, and (3) movement of an O3 reaction product(s) into the cell with enzymatic or chemical
30 transformation of those products in the cell.
31
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1 Process 1. Entry of O3 into the leaf. Often incorrectly, the external concentration of O3 is
2 used to give an indication of "dose" (Heath, 1994a). Ozone-induced changes on a plant's cuticle
3 are minimal, and O3 does not penetrate the cuticle (Kerstiens and Lendzian, 1989) to cause an
4 effect. As O3 has no easily measured isotope, virtually no measurements have been done on an
5 actual dose of O3, i.e., the amount of O3 which reacts with individual biochemicals in the leaf.
6 Yet the measurement of dose will be the amount of O3 expected to penetrate into the tissue
7 through the stomata. Dose is expressed as a rate of delivery to a surface area (mol/m2 s"1).
8 Whether dose or total accumulation (mole/™2, rate integrated over exposure time) is most critical
9 for the development of injury remains a major question.
10 Ozone uptake includes gaseous diffusion through the leaf boundary layer and stomata into
1 1 the substomatal cavity (Figure AX9-3). Although the movement of pollutants through a
12 boundary layer into the stomata region is known to be important, and even rate limiting in many
13 cases of low wind velocity, its description has been defined from aeronautical concepts and
14 usually relates to smooth surfaces that are not typical of leaf- surf ace morphology; however, it is
15 nearly the only treatment available (Gates, 1968). Once through the boundary layer, the gas
16 must enter the leaf through the stomata. The entry of gases into a leaf is dependent upon the
17 physical and chemical processes of gas phase and surfaces and is a well-defined path that
18 approximately follows a linear flux law of:
19
20
-1 (AX9-1)
21
22
23 where the flux, y, into the internal space of a leaf is related to the conductance, g, through the
24 boundary layer and stomata and the gradient of concentration of gas from the outside, C0,
25 inward, Q. This formulation has been used for years for both water and CO2 (Figure AX9-4),
26 and for regions of varied CO2 concentration that correspond to C0 (CO2 of the atmospheric air,
27 below the leaf proper) and Q (CO2 near the leafs spongy mesophyll cells) (Ball, 1987; Farquhar
28 and Sharkey, 1982).
29 In the past, the internal concentration of O3 has been assumed to be zero (Laisk et al.,
30 1989), due to early studies that found that virtually no O3 could pass through a leaf. That was
3 1 expected because O3 is extremely reactive with cellular biochemicals. If the assumption that the
32 internal concentration zero is correct, then the effective delivery rate for O3 is given as g x C0,
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Bulk Air
Boundary I—K^
Layer \~~^
Stomatal LN.
Aperture V~r
Sub-
stomatal
Cavity
Wind Speed Leaf
Canopy Temperature
Structure
Figure AX9-3.
The uptake of O3 into the leaf. Each of the individual concentration
layers of O3 represents a different process of movement and of
plant/microenvironmental interaction. This figure leads into Table 9-6,
in which the amounts of O3 along the pathway are calculated.
Light
Cuticle
Epidermis rj
Pallisade
Mesophyll
Spongy
Mesophyll
Epidermis
Cuticle
Illlllllllll
Vascular
System
C0 = [CO J
Figure AX9-4.
The microarchitecture of a dicot leaf. While details among species vary,
the general overview remains the same. Light that drives photosynthesis
generally falls upon the upper (adaxial) leaf surface. Carbon dioxide and
O3 enters through the stomata on the lower (abaxial) leaf surface, while
water vapor exits through the stomata (transpiration).
1 with stomatal conductance being the major regulatory control (Amiro et al., 1984; Taylor et al.,
2 1982). However, a recent study by Moldau and Bichele (2002) indicated that the internal O3
3 concentration may not be zero as previous assumed. Moldau and Bichele (2002) permitted
4 leaves ofPhaseolus vulgaris L., which have stomata on both upper and lower leaf surfaces, to
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1 take up O3 at a high rate for 3 to 5 min. Exposure of the lower leaf surface resulted in up to 5%
2 of the O3 that was taken up to be diffused through the leaf, emerging from the stomata on the
3 upper surface. This suggested the presence of above-zero concentrations of O3 in the
4 intercellular leaf air spaces. The descriptive calculations and plots of Moldau and Bichele
5 (2002) indicate that the rise in internal O3 level (for both concentrations of external O3) within
6 the first few minutes of exposure is due to its reaction with an antioxidant, most probably
7 absorbate, within the apoplastic space of the leaf (Figure AX9-5). The rate of rise is probably
8 due to more complete penetration of O3 with a concurrent depletion of the external antioxidant.
9 The rise peaks at about 2 min for 0.88 ppm and 3 min for 0.34 ppm and then falls to a lower
10 level. This may be due to a replenishment of the antioxidant. The authors saw no injury to the
11 plasmalemma (as measured by penetration of a dye) and no change in the stomatal conductance
12 for the lower concentration of O3 (Moldau and Bichele, 2002). The higher level (0.88 ppm)
13 caused the plasmalemma of the mesophyll cells to pass a dye, and a slight decline in stomatal
14 conductance resulted at about 2.5 minutes. These data suggest that the antioxidant hypothesis is
15 correct.
16 Gaseous pollutants flow from the substomatal cavity within the leaf through the cell wall
17 into the cell. It is suspected that the internal concentration of the pollutant is not uniform within
18 the cavity (Taylor, and Hanson, 1992). From within the wall, an equilibrium between the gas
19 and aqueous phase must occur at the interface where the gaseous species dissolve into the water
20 according to Henry's Law (Heath, 1980, 1987; Wellburn, 1990). It is important to understand
21 exactly how much O3 could move into the tissue of the leaves. Calculations in Table AX9-6
22 give an indication of the amount of O3 which may end up near the surface of cells within the
23 leaf. The calculation is done for a standard temperature (25 °C), an ambient concentration of O3
24 (0.10 ppm), and for nonspecific leaves. For example, 0.3 ppm would be the same general
25 numbers but multiplied by 3. Similarly for more closed stomata, the value of 1.0 cm/s
26 (equivalent to about 400 |imole~2-leaf area s"1) for a conductance would be reduced and the
27 smaller values would lead to a smaller amount of O3 moving into the tissue. Nonetheless, these
28 values give some indication of what sort of chemical concentration can be expected. Under
29 these conditions, a delivery rate of O3 into the substomatal cavity near the spongy mesophyll
30 tissue of about 0.42 nmol/(L hr) appears to be reasonable.
31
August 2005 AX9-48 DRAFT-DO NOT QUOTE OR CITE
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160
140
120
o
E
c
0)
o
N
O
100
80
60
03 Exposure
D 0.335
• 0.821
I
I
234
Time (minutes)
40
38
g
36 ^
"
CD
32 §
N
O
30
28
Figure AX9-5. The change in the O3 concentration inside a leaf with time. Data are from
O3 exposures at two different concentrations.
Source: Derived from data in Moldau and Bichele (2002).
1 Process 2. Ozone diffuses into the leaf air spaces and reacts either with varied biochemical
2 compounds that are exposed to the air (path 1) or is solubilized into the water lining the cell wall
3 of the air spaces (path 2). As shown in Figure AX9-6, each reaction has the possibility of
4 transforming O3 into another chemical species (a toxicant) which, in turn, may react with other
5 chemical species and lead to a cascade of reactions.
6 Within the stomata, gases react with the water at the cell's surface and generate new
7 species with the components within the cell wall region. The possible varied pathways are
8 depicted in Figure AX9-7. Although these chemical reactions are poorly understood, some of
9 the fundamentals are known (Heath, 1987, 1988; Wellburn, 1990). Ozone reacts with organic
10 molecules at the double bonds to form carbonyl groups and, under certain circumstances,
11 generates peroxides, such as hydrogen peroxides (H2O2), superoxide (O2 ) and its protonated
12 form (HO2'), hydroxyl radicals (HO'), and peroxy radicals (HO2'). Other chemicals present in
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Table AX9-6. The Flow of Ozone into a Leaf and Possible Reactions
The level of O3 in the atmosphere is chosen to be close to a standard and yet make calculations to other amounts
easy. The same concept will be used for all standard parameters for these calculations.
DESCRIPTION
The atmospheric level of O3 is given as:
For an air temperature of:
The perfect gas law (pV = nRT) is used to convert the O3
level into standard mks. Further, the volume for a mole of
gas (V0 = 22400 m3) will be used, from the perfect gas law
with R= 8.3144
Thus, the concentration of O3 within the atmosphere is:
O3ax 10-6-(Ta + 273.18)
°3 V0 x 273.18
The stomatal conductance of the gas must be chosen to be
standard but adjustable. The number should be as large as
typically measured but allow for easy conversion, if
necessary. For a stomatal conductance of:
The amount of O3 that will penetrate inside the leaf (for a
typical concentration of nearly zero inside the leaf), is:
°3T
18 gsvvv
48 100
In terms of amount of water within the leaf, we can
assume that about 85% of the weight is water and the
density of water is 1 g/mL. A typical leaf has a wet
weight/area:
Thus, the square surface area of the leaf will translate into
water space (for concentration of chemicals), as:
ArT =
FWL x 0.85
100
VALUES
O3a = 0.1ppm
Ta = 25 °C
= 4.873 xlo~12 moles/m3
= 1 cm/s
O3L = 2.984 xlo-14mol/(m2s)
FWL = 30 mg/cm2
Ar, = 0.255 Lr2
The maximum amount of toxic compound that will be
generated, assuming all the O3 is converted, is given
below. Here the units of the leaf area weight are
converted into the mks system and the water space units
are converted into L, such that the concentrations
calculated will be in mol/(L hr). The final units assume
that the O3 is present (and no back reactions occur) for one
hour (short but typical units of exposure).
O3Lc = 4.21 x 10~10 mol/(L hr)
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Table AX9-6 (cont'd). The Flow of Ozone into a Leaf and Possible Reactions
Thus, the maximum amount of toxic chemicals generated
per hour in a leaf would be: O3Lc = 0.42 nmol/(L hr)
Possible errors in these calculations (aside from the input numbers) are (1) the O3 within the leaf does not react
uniformly within the leaf space; (2) the O3 within the leaf does not totally convert to any one species; (3) varied
products of O3 react, leading to innocuous chemicals; and (4) O3 reactions can be catalytic and generate more
reactions by radical reaction cycling.
Plasma
Apoplasm Membrane
Reaction,, p
Solubilization -
4
Reaetion2A
• Reaction2p
»Reaction., p-
Cytoplasm
Reaction1c
• Reaction2c
• Reaction2C-
Figure AX9-6. Possible transformations of O3 within a leaf.
1 the water phase can lead to many other oxygenated moieties (Figure AX9-6). Each of the steps
2 is generally pH dependent (Jans and Hoigne, 2000; Walcek et al., 1997).
3 Sulfhydryls are particularly easy targets, with the formation of disulfide bridges or sulfones
4 (Mudd and Kozlowski, 1975). In water, the reactions become more confusing, but some
5 products have been described by Heath and Castillo (1987), such as H2O2, HO', and O2
6 (Figure AX9-7). Effective detoxification reactions can occur here via antioxidant metabolites
7 and enzymes such as ascorbate, glutathione (GSH), and superoxide dismutase (SOD) if they are
8 present at high enough concentrations (Castillo et al., 1987; Matters and Scandalios, 1987).
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g Superoxide
H+
, Hydrogen
W0**1 Peroxide
Radical
b.
H0_ ^H202
H20
Peroxyl
Radical
Figure AX9-7. Possible reactions of O3 within water, (a) Ozone reacts at the double
bonds to form carbonyl groups, (b) Under certain circumstances,
peroxides are generated.
1 If the levels are low, it is believed that stimulation of their production is a response to O3, albeit a
2 slow one (Harris and Bailey-Serres, 1994). Certainly it is possible that chemical modification of
3 wall-specific biochemicals (Castillo et al., 1987) such as glucan synthase (Ordin et al., 1969) and
4 diamine oxidase (Peters et al., 1988) occurs.
5
6 Process 3. Movement of reaction product(s) into and enzymatic or chemical
1 transformations within the cell. It is believed that the initial site of O3 injury is near or within the
8 plasma membrane. Certainly, membrane functions, such as membrane fluidity (Pauls and
9 Thompson, 1980), permeability (Elkiey and Ormrod, 1979), K+-exchange via ATPase reactions
10 (Dominy and Heath, 1985), and Ca2+ exclusion (Castillo and Heath, 1990), are changed. The
11 similarity of wounding responses (Langebartels et al., 1991) and O3-induced membrane
12 disruption suggests the induction of normal wound-regulated genes (Mehlhorn et al., 1991;
13 Sandermann, 1998). This implies that O3 can react with cell-wall components that are connected
14 to the cytoplasm through the cell wall and membrane by membrane-specific proteins that are not
15 directly involved with transport.
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1 Ozone is soluble in water and once having entered the aqueous phase, it can be rapidly
2 altered to form oxidative products that can diffuse more readily into and through the cell and
3 react with many biochemicals. Again, the presence of an internal antioxidant would be critical
4 to reduce the concentration of most oxidants. A toxic product of O3 may migrate through the
5 cytoplast to react with photo synthetic processes, or a spurious signal generated at the membrane
6 may affect some control process or signal transduction pathway (Schraudner et al., 1998;
7 Overmyer et al., 2000, 2003; DeCaria et al., 2000; Rao et al., 2002; Booker et al., 2004; Leitao
8 et al., 2003; Rao and Davis, 2001; Sandermann, 2000; Vahala et al., 2003).
9
10 AX9.2.3.1 Possible Reactions Within the Leaf
11 Ozone can react with many compounds within the substomatal cavity of the leaf1 to
12 produce a variety of oxidizing and toxic chemicals. Some of the possible reactions that will
13 generate H2O2, HO', and SO2 , as well as charged O3 intermediates, are indicated in Figure
14 AX9-8. Many of these complex reactions have been studied within water solutions through
15 research of O3-induced water purification and are very dependent upon solutes present with the
16 solutions, including FT (see Von Gunten [2003]). An important point is that in alkaline
17 media, O3 forms H2O2, but in acid media, O3 is relatively stable in the absence of free metal ions.
18 The rates of reaction of O3 with several important compounds, including those with a
19 double bond, the so-called Crigee Mechanism shown in Figure AX9-8, can be calculated from
20 the reaction coefficient as given by Atkinson (1990) (Table AX9-7). The double bond of the
21 ascorbate molecule is particularly sensitive to O3 attack. Because of the ring formation of the
22 ascorbate molecule, an unstable ozonide product is formed, which then accelerates the breakage
23 of the double bond, leading to the formation of two products. These products are relatively
24 unstable and can lead to further reactions not shown in Figure AX9-8. The rates of reactions can
25 be calculated (Heath, 1987). At a local concentration of 25 |iM O3, it would take 5000 s
26 (83 min) for all of the O3 to react if there was no further flow of O3. Clearly, O3 does not react
27 rapidly with the compounds in Table AX9-7 and, although some of the products would be
28 formed through the Crigee Mechanism (see Figure AX9-8a), they would be low in
lrThe volume of the substomatal cavity (that are within the leaf immediately below the stomata) must be
regarded as the region in which most O3 reactions occur. That volume, at a relative humidity of near 100%,
possesses many diverse surfaces with varied bonding, which could alter the fate of O3.
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a.
OH-
NO
H2C = CH2
H2C = CH2
H2C = CH2
Crigee _
Mechanism / \
—*~ 9 P
H2C - CH2
OH
•^ H2C-CH2
ON02
H2C —
H
HC=O
0
n
HC-OH
b.
CH(OH)CH 02H
CH(OH)CH O2H
H202
HO OH
\
O=C CH(OH)CHO2H
CHO . CHO
I
Further Oxidation
Figure AX9-8a,b. The Crigee mechanism of O3 attack of a double bond, (a) The typical
Crigee mechanism is shown in which several reactions paths from the
initial product is shown, (b) Typical reaction of ascorbic acid with O3.
Source: Adapted from Mudd (1996).
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Table AX9-7. Some Rates of Reaction of Ozone With Critical Biochemicals
[a] Double bond reactions. The second column is taken from Atkinson (1990) and transformed into Column 3.
Those rate coefficients are used to calculate the rate of reaction at a concentration of 10 ppm for the organic and
0.1 ppm for O3 in the air stream within the leaf (localized concentration of about 25 mM, see Table AX9-6).
Compound x 10~18 cm3/molecules s"1 Rate coefficient (L/mole s"1) Rate of reaction (M/s)
Ethane 1.7 1.02 x 103 4.3 x I0~n
Propene 11.3 6.80 x 103 2.8 x lO"10
1-butene 11 5.91 x 103 2.5 x 10"10
trans-2 Butene 200 1.20 x 105 5.0 x lO"9
a-pinene 85 5.12 x 104 2.1 x 10~9
[b] Possible Oxidative Species. Another possibility is given by the reactions below from Walcek et al. (1997).
Reactions Rate constants
(1) O3 + OH + H2O -> H2O2 + O2 + OH kj = 3.67 x 10 mokT1 L s~'
(2) O3+ O2 -> HO + 2 O2 + OH k2= 1.26 x 109 mole"1 Ls"1
(3) O3+ HO2 -> HO + O2 + O2 k3 = 2.09 x 106 mole"1 L s"1
[c] Possible Concentrations of Other Oxidative Species. Table from Heath (1987). Based upon 100 ppm O3 in
gas stream.
Species
Superoxide Radical (O2')
Ozone Radical
Protonated O3 radical (HO3')
Concentration
pH7
8.75 x 10~15
4.16 x io-15
1.48 x 1Q-16
(M)
pH 9 Molecules within wall
1 x 10~12 5.5 x nr6 6.3 x 10~4
5 x 10~14
1 x 10~18
Number of molecules within apoplastic space of (10~12 L) at 0.1 ppm O3.
1 concentration2. While other radicals, such as the hydroxyl radical (see Figure AX9-8b) can
2 attack double bonds, the products differ. Of particular note for later discussion, is the reaction
3 of O3 with ascorbate (see Mudd (1996) (see Figure AX9-8b), which will cleave the double bond
4 in the ring. Unfortunately, little work has been done to characterize possible products within the
5 leaf (but see next section).
2For example, hydroxylmethyl hydroperoxide would be expected to be formed by the reaction of O3 with
ethylene and its effects have been tested on peroxidases (Polle and Junkermann, 1994). Unfortunately, the
concentration of required for inhibition is much higher than would be expected to be formed within the leaf.
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1 In a paper discussing the stability and reactivity of O3 in the pulmonary air/tissue
2 boundary, Pryor (1992) calculated that O3 has a half-life of about 7 x 1CT8 s in a bilayer.
3 However, the transit time through the lung lining fluid layer is about 2 x 1CT6 s, based upon a
4 reasonable estimate for the diffusion of O3. This means that O3 would suffer nearly 29 half-
5 lives3 in passage through the layer, reducing it to about 3 x 1CT9 of the original concentration —
6 zero for all practical aspects. In the same publication, Pryor points out that any sulfhydryl or
7 ascorbate would interact strongly with O3, further reducing its net concentration. The reactivity
8 of cysteine is 109, while the reactivity of tryptophan, methionine, polyunsaturated fatty acids,
9 and tyrosine is about 2 x 106 and that of phenylalanine is only 103. These numbers are similar to
10 what has been found for O3 reactivity with amino acids and proteins in aqueous solutions.
11 In glycophorin (Banerjee and Mudd, 1992) and cytochrome C (Mudd et al., 1997b; Mudd et al.,
12 1996) in aqueous solutions, only the methionine was oxidized by O3, producing sulfoxide.
13 In other proteins lacking methionine, tryptophans were oxidized only if they were in an exposed
14 position on the surface of the proteins (Mudd et al., 1997b). Treatment of red blood cell ghosts
15 with O3, oxidized peripheral proteins of the plasma membrane before it oxidized lipids (Mudd
16 etal., 1997a).
17
18 AX9.2.3.2 Toxicants Within the Wall Space
19 While Mehlhorn et al. (1990) are often thought to have shown that free radicals were
20 formed in plant leaves under O3 exposure, careful reading of that paper clearly shows that there
21 was no real evidence of free radicals induced by O3. Living tissues have many free radical
22 signals, making it difficult to observe changes in free radicals. Further, the work of Grimes et al.
23 (1983) has also been cited as showing the presence of free radicals in living tissues due to O3
24 exposure; however, no radical signals were found unless certain organic acids (e.g., caffeic acid)
25 were added to the tissue with the O3 exposure. They used the radical trap TMPO
26 (tetramethylphrrolise 1-oxide) which reacts with many types of free radicals to form a stable
27 radical that can be used to "trap" or increase the amount of radical present (see Figure AX9-9a).
28 Ozone would directly react with this trap only if it were bubbled into the solution, not passed
29 over the top of the solution. In the presence of sorbitol or caffeic acid, the trap would indicate
3Here a half-life is the time that it takes the reactive species to travel a distance in which it loses 50% of its
initial concentration. Therefore for a 29 half-life, the concentration has been reduced by 2~29 or about a 10~9 decline.
August 2005 AX9-56 DRAFT-DO NOT QUOTE OR CITE
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a.
ESR spectra from solution treated
with O3 at 0.3 ug/g dissolved for
60 s (1a) control, (1b) with caffeic
acid; spin trapped: DMPO
(Grimes et al., 1983)
ESR spectra from bean
(Phaseolus vulgar!s cv. Pinto)
leaves pretreated with spin
trapped: PEN after a 4-h
fumigation with O3 (0-300
nl/L)(Mehlhornetal., 1990).
C.
EPR signal in white light before
(broken line) and after (dotted
line) exposure of a bluegrass
leaf to 1000 ug rrf3 O3 in the
EPR cavity for 1 h. The solid line
depicts the difference signal.
(Runeckles and Vaartnou, 1997)
Figure AX9-9.
Varied ESR radicals, trapped and not, generated by O3 under somewhat
physiological conditions, (a) The generation of a DMPO-trapped radical
with caffeic acid in water solution (Grimes et al., 1983). (b) The
generation of a DMPO-trapped radical within bean (Phaseolus vulgaris cv.
Pinto) exposed to 70 nL/L O3 for 4 h. The lower trace is the ESR signal
produced with 300 nL/L O3 (Mehlhorn et al., 1990). (c) The EPR signal
produced within a bluegrass leaf exposed to 1000 ug m3 of O3 for 1 h
(Runeckles and Vaartnou, 1997). Although no trapping agent was used in
this experiment, the signal is complex, because of various free radicals
normally present within the illuminated leaf.
1
2
3
4
5
the presence of OH radical, which would mean that O3 ->• HO'. Superoxide dismutase, catalase,
or EDU had no effect upon this signal, suggesting O2 and H2O2 were not involved in the above
sequence. Both O3 and O3 plus caffeic acid had no effect upon the protoplasts' intactness or
viability. Thus, 1CT5 M HO' and/or 0.30 to 0.40 ppm O3 did not react with the cell membrane.
They found no signal in normal cells after subjecting the leaf to O3 and concluded that the
radicals were produced via a concerted mechanism with the acid. This does not fit with the
mechanism postulated by Mehlhorn et al. (1991), which involved a reaction of wound-induced
ethylene and O3 at the wall level to generate some free radicals.
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1 The hypothesis that the production of wound-induced ethylene by O3 exposure and its
2 reaction with O3 would result in the production of radicals was tested by Mehlhorn et al. (1990),
3 using electron paramagnetic resonance spectroscopy. After 4 h of 300 ppb, an EPR signal of a
4 compound was detected which resembled a butonyl radical (Figure AX9-9b [Mehlhorn et al.,
5 1990]). Using 70 ppb, the signal was reduced by about one third that of an ethyl radical4, leading
6 to injury. However, the spraying of the plant with 1-aminoethoxyvinyl-Gly (AVG), which
7 reduces the production of ethylene and visible injury, had no effect upon the EPR signal,
8 suggesting that the radical is not a direct sequela to visible injury.
9 Runeckles and Vaartnou (1997) (Figure AX9-9c) discovered a signal by subtracting other
10 EPR signals of the leaf, which seemed to be due to an O3 reaction with plant material, using
11 0.48 ppm O3. This difference signal looked very much like O2 . At a lower concentration, they
12 observed that this signal still occurred but accumulated more slowly. Both bluegrass and
13 ryegrass leaves seemed to saturate after about 5 h of exposure at 22 to 28 units of signal, while
14 radish leaves reached a maximum of 7 units at 3 h and then declined. The problem, which is
15 typical of any of these methods, was that the detached leaf had to be rolled and placed into the
16 EPR detection cavity. Reichenauer et al. (1998) also detected an undefined free radical signal
17 that seemed to be related to a Mn(II) spectrum. The Nandu and Perlo cultivars of wheat were
18 more sensitive to O3 than Extradur (according to growth rate and closure of stomata under an O3
19 exposure of 80 ppb for 8 h/day, 7 days/week over 100 days), and these more sensitive cultivars
20 had a greater, but insignificant (P = 15%), EPR signal. Thus, data showing any production of a
21 free radical must be approached with some skepticism.
22 With an O3 delivery rate of about 25 |iM/h (Table AX9-6), only 250 jiM would be found
23 after a full day, if all of the O3 were stable. While the use of free radical traps is the best method
24 available to observe any build-up of radicals, the traps are not as specific to individual radicals.
25 Currently, studies should be looking for hydroxyl radicals, superoxide, hyroperoxides, ethylene
26 radicals, and ascorbate radicals.
27
4The reaction would be: O3 + H2C = CH2 -> varied C-l compounds, due to double bond cleavage, at a
rate constant of 1.7 x l(T18 cnf/molecule sec = 1.02 x 103 NT1 s~' (Atkinson, 1990). This should be compared
with a reaction of the hydroxyl radical with ethylene, which has a rate constant of 8.52 x l(T12 cm3 molecule"1 s~',
or 106 x faster.
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1 AX9.2.3.3 Products of Ozone
2 Ozone should reach a certain concentration in the substomatal cavity, which is dependent
3 upon its entry speed and its reactivity with the wall constituents. Once near the apoplastic
4 space, O3 moves in two different pathways (Figure AX9-6). It can react with constituents that
5 are within the wall as a gas in reaction 1A (path 1); or it can solubilize into a water space and
6 travel to another region within the water space and react through reaction 2A (path 2).
7
8 Hydrogen Peroxide
9 Hydrogen peroxide, until recently was thought to be purely a toxic compound for cells.
10 However, it is now clear that it functions as a signaling molecule in plants and mediates
11 responses to abiotic and biotic stressors (Figure AX9-10). Generation of H2O2 is increased in
12 response to various stressors, implicating it as a key factor mediating the phenomena of
13 acclimation and cross tolerance, in which exposure to one stressor can induce tolerance of
14 subsequent exposure to the same or different stresses (Neill et al., 2002). The signaling response
15 to attack by invading pathogens using H2O2 has been described (Mehdy, 1994; Simon-Plas et al.,
16 1997). The reactions leading to hypersensitive cell death are caused by a pathogen recognition
17 step (Figure AX9-10a), probably due to the plant cell wall releasing oligosaccharides in response
18 to the pathogen enzymatically breaking down the cell wall to penetrate it. A feed-forward step
19 in which H2O2 increases the level of benzoic acid leads to the activation of the hydroxylase step
20 in the production of salicylic acid and to a feedback step in which the salicylic acid increases the
21 production of H2O2 (Leon et al., 1995).
22 An elicitor, e.g., a bacterial or fungal pathogen, induces a cascade of reactions within a cell
23 (Figure AX9-10b). Some of the lipid reactions are thought to be due to the opening of the Ca2+
24 channels and the alkalination of the cell wall region. The oxidative burst due to H2O2 production
25 is believed to lead to the transformation of a small population of lipids into jasmonic acid, which
26 is a secondary messenger.
27 Hydrogen peroxide also has an oxidative role in lignification (Schopfer, 1994). In the
28 interaction of lignification and the beginning processes of hypersensitivity, pectinase produced
29 by the pathogen disrupts pectin and dissolves the cell wall. Fragments of the dissolved cell wall
30 trigger an increase in the transcription of peroxidases within the remaining cell wall, leading to
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a.
Pathogen Recognition
I
Free Radical Generation
H2O2 Generation
I
Accumulation of Benzole Acid
^ Benzole Acid
2-Hydroxylase Activation
I
— Salicylic Acid Accumulation
I
Systemic Salicylic Acid Increase
Defense Activation
Hypersensitive Cell Death
Local Resistance
Global Resistance
/ell Membrane C
Ascorbate
Peroxides
OH" \
1 *
\ / „ .(
G Proteins Open Ca2+ Phosphorylation * (
(Rac2/Rap1A) Channels Cascade %
I
Pathogen
/ Kill
/ t i
•( "Oxidative
+ Burst
Cell Wall
->. Structura
Changes
/ NADPH Hydroperoxides
H+ Oxidase ./
Jasmonic
/ Acid
Influx
Signal Transduction
Transcription
Defense Genes
Figure AX9-10. Pathogen-Induced Hypersensitivity. (a) The reactions leading to
hypersensitive cell death and the formation of a global response of
salicylic acid, (b) The cascade of the elicitor-induced reactions within the
cell.
1 lignification, which is a cross-linking of the cell wall that does not use pectin. This prevents
2 further pathogen disruption of the wall and reduces its further entry into the plant cell.
3 It is believed that the first species generated through a one-electron reduction of molecular
4 oxygen is SO2~. That generation is carried out using a cytochrome b6 by the NAD(P)H oxidase
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1 located on the cell membrane (Auh and Murphy, 1995). In the acid region of the cell wall, SO2
2 is converted by a protonation and dismutation to H2O2. The induced oxidative burst is believed
3 to play a role in stimulating the Cl" and K+ efflux, generating an alkalinization of the
4 extracellular space (Cazale et al., 1998). In the wall region, H2O2is not especially toxic, as no
5 necrosis was reported in tobacco when 500 mM peroxide was infiltrated into the leaf tissue.
6 However, the production of salicylic acid and benzoic 2-hydroxylase can be induced with only
7 30 and 0.3 mM H2O2, respectively, indicating some metabolic signaling (Leon et al., 1995).
8 On the other hand, 1M H2O2 infiltrated into soybean will generate lipid peroxidation after 1 h
9 with a peroxidation rate of 15 nmol/g-FW h (Degousee et al., 1994). Cells react to the system5
10 and generate peroxide scavenging compounds within 1.5 to 2 hours, which appear to "mop up"
11 the excess H2O2 (Baker et al., 1995).
12 After O3 exposure in birch, H2O2 has been found in the wall (Pellinen et al., 1999). By
13 using CeCl2 as a cellular stain for H2O2 (as a cerium perhydroxide precipitate), Liu et al. (1995)
14 observed a gradual development of stain after 8 h of O3 exposure (at 150 ppb). After 2 h
15 exposure, H2O2 stain was visible on the surfaces of both sets of mesophyll cells. Accumulation
16 of H2O2 stain continued for 16 h after exposure, suggesting a triggered-reaction rather than O3
17 decomposition itself. H2O2 stain was present in the mitochondria, peroxisomes, and cytoplasm
18 but not in the chloroplast. If methyl viologen (MV) was given to the leaves and then the leaves
19 were exposed to light, H2O2 stain could be observed within the chloroplast. This indicated that
20 the stain worked within the chloroplast if H2O2 were generated by the Mehler reaction
21 (MV + O2~). Thus, apparently, for birch, O3 exposure does not generate excess H2O2 within the
22 chloroplast. Furthermore, these sets of experiments indicate that O3 per se does not generate the
23 H2O2, but rather triggers stress-related H2O2 formation similar to what occurs in a pathogen
24 attack (the Reactive Oxidative Species or ROS reaction).
25 The presence of higher than normal levels of H2O2 within the apoplastic space is a potential
26 trigger for the normal, well-studied pathogen defense pathway. Figure AX9-10b depicts such a
27 pathway and suggests that all the events and activation of pathways/genes caused by pathogen
28 defense could be observed when plants are fumigated with O3. The events shown in Figure
29 AX9-10b will be alluded to in later sections.
Soybean suspension cells were inoculated with Pseudomonas syringae pv syringae, which generate an
active oxygen response. Light emission by luminol, reacting with H2O2, was the assay for the peroxide.
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1 H2O2 has been linked to the hormone ABA-induced closure of the stomata by activating the
2 calcium influx in guard cells (Pel et al., 2000). The addition of H2O2 at a level of only 5 mM to a
3 guard cell preparation will cause a dramatic increase (ca. 9x) in electrical current at the
4 hyperpolarizing potential of-200 mV. Amounts as low 50 mM H2O2 will cause a less, but still
5 sizable, increase. Membrane stability is unaffected by the H2O2 and the activation of the channel
6 requires only about 2 to 3 minutes. Pel et al. (2000) also found that ABA induced the production
7 of H2O2 through ROS accumulation (also see Zhang et al. [2001]).
8 Certain levels of ABA within the leaf lead to stomatal closure. The inactivation of a
9 phospho-tyrosine-specific protein phosphatase (ABI2) is an inhibitor of stomatal opening
10 induced by ABA but that enzyme is inhibited by H2O2 (Meinhard et al., 2002). This means that
11 H2O2 shifts the sensitivity of the stomatal opening to ABA (Figure AX9-11), making the
12 stomatal complex more sensitive to ABA. Thus, for a given level of ABA present in the guard
13 cell complex due to environmental factors (e.g., low humidity, high air temperature, or low soil
14 water potential), the generation of H2O2 would (by inhibiting ABI2) induce a closure of the
15 stomata by increasing the sensitivity of the guard cells to ABA. In the past, it has been difficult
16 to understand why O3 would often decrease conductance in some cases, but not always (Heath,
17 1994b). This interaction between H2O2 and ABA could help understand this complexity.
18
19 Ethylene Reactivity
20 Ethylene (ET) is produced when plants are subjected to biotic stressors, e.g., attacks by
21 insects, fungi, and bacteria or abiotic stressors such as wounding or environmental stressors such
22 as heat, cold, or oxidative stress and O3. If an O3 stress has induced a wounding response with
23 ET release, then ET within the substomatal cavity could react with O3, generating some
24 relatively noxious chemicals (see Figure AX9-6). The relationship between O3 injury and
25 wounding is supported by the observation by Mehlhorn et al. (1991) that an inhibitor of ET
26 formation, AVG (an inhibitor of ACC synthase, a committed step to ET production), would
27 block ET formation and inhibit visible injury. Other studies with polyamine (which is closely
28 linked to ET production), including those of Ormrod and Beckerson (1986) who fed polyamines
29 to the transpirational stream and prevented visible injury, suggested a close involvement of both
30 pathways to the production of visible injury. Both the lack of ET production and an increased
31 level of polyamines slowed or prevented visible injury.
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ABA
Level
H202'
1
ABI2
9s
Control
Stomatal
Closure
[ABA]
Figure AX9-11. The interaction of H2O2 and Ca2+ movements with ABA-induced stomatal
closure. It is well known that certain levels of ABA within the leaf lead to
closure of the stomata within the leaf. That level, however, can be shifted
to make closure more or less sensitive to a given level of ABA. Recently it
has been shown that H2O2 (externally or produced by the plant) within
the cell wall region can shift that sensitivity. Here ABA stimulates the
production of H2O2, which in turn increases the rate of Ca2+ moving from
the wall region into the cytoplasm. That shift in internal Ca2+ level
increases the closure of the stomata. Hydrogen peroxide also blocks the
activation of a polypeptide (ABI2) that inhibits stomatal closure
seemingly induced by ABA.
Source: From Assmann (2003); Assmann and Wang (2001); and Zhang et al. (2001).
1 This concept was taken another step by Langerbartles (1991). The linkage to the pathogen
2 wound responses and visible injury is well established (Sandermann, 1996). Sandermann (1998)
3 used a system of Bell B and W3 tobacco, plants with differential O3 sensitivities, in which the O3
4 exposure level was chosen such that the sensitive cultivar was injured, while the tolerant one was
5 not. This led to a marvelous control that could be used to their advantage. They followed a time
6 sequence to show that the rise of varied systems followed the same order as seen for a pathogen
7 attack (Heath, 1994a).
8 More recent studies, however, indicate that O3 responses resemble components of the
9 hypersensitive response (HR) observed in incompatible plant-pathogen interactions
10 (Sandermann, 1998). The similarity to the HR response may be related to the occurrence of
11 ROS in the apoplast. The O3-derived ROS apparently trigger an oxidative burst in the affected
12 cells by an as yet unknown mechanism. An oxidative burst is similar to one of the earliest
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1 responses of plants to microbial pathogens and is an integral component in HR-related cell death
2 (Overmyer et al., 2000).
3 In plants exposed to O3, ET synthesis is a result of the specific ET induction of the genes
4 encoding 1-aminocyclopropane-l-carboxylase synthase (ACS), one of the fastest and most
5 obvious responses to O3, which has been mechanistically linked to the regulation of O3 lesion
6 formation. Biosynthesis of ET inhibited, with ACS inhibitors significantly reduced, the
7 induction of lesion formation in plant leaves exposed to O3 (Mehlhorn et al., 1991; Mehlhorn and
8 Wellburn, 1987; Vahala et al., 2003). Ethylene biosynthesis correlates best with O3 exposure
9 (Overmyer et al., 2000; Vahala et al., 2003). These data support the concept that elimination of
10 ET formation will prevent visible injury.
11
12 Ethylene-Interaction with Injury and Conductance
13 Increased ET production by plants exposed to O3 stress was identified as a consistent
14 marker for O3 exposure decades ago (Tingey et al., 1976). They exposed more than 20 plant
15 species and cultivars to O3 to determine whether the production of O3-induced stress-ET could be
16 used to determine differences in plant sensitivity to O3. Their studies suggested that increased
17 production of stress-ET correlated well with the degree of foliar injury that developed within
18 hours or days after O3 exposure. The amount of ET released was exponentially related to the O3
19 exposure. Furthermore, the amount of O3-induced ET declined with repeated exposure,
20 indicating an acclimatization to O3. This acclimatization effect associated with repeated
21 wounding has not yet been well described. The release of wound-induced ET is not linear with
22 time, but declines after the initial response (Stan et al., 1981), as is also seen after O3 exposure
23 (Stan and Schicker, 1982). The stress-induced ET production correlates better with O3 exposure
24 level than with exposure duration. In other words, peaks of high O3 (rather than accumulated
25 dose) generate a higher rate of ET release, at least for a single O3 exposure under an acute dose.
26 The production of ET after an O3 exposure is thought to be a typical wounding response
27 (Tingey et al., 1975). Prevention of ET release may prevent the formation of visible injury
28 (Mehlhorn and Wellburn, 1987). However, the question arises as to whether this effect was
29 limited to the prevention of visible injury or if the chemicals used to prevent ET release closed
30 the stomata. Using Glycine max L., Taylor et al. (1988) showed clearly that AVG did not
31 necessarily close stomata nor inhibit carbon assimilation per se.
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1 The correlation of ET release with O3-induced visible injury was likewise shown in pea
2 cultivars (Dijak and Ormrod, 1982). With O3 exposure (generally 6 h at 0.3 ppm), the stomata
3 closed by -50% within 3 h after a dose of 3 x 10~5 mol cm"2 (with an average rate of 2 x 10~9
4 moles cm"2 s"1, as calculated from their data). Both sensitive and insensitive cultivars had a
5 visible-linked-injury ET release, but sensitive cultivars scored higher both in visible injury and
6 in ET release after a given exposure.
7 Gunderson and Taylor (1988, 1991) used exogenous ET to alter the gas exchange of
8 Glycine max and found an exponential, but not simultaneous, decline of both stomatal
9 conductance and carbon assimilation with ET. Interestingly, the exogenous ET caused a slight
10 rise in difference of CO2 within and without the leaf, indicating a lowering of internal CO2,
11 which was not observed in the experiments of Farage et al. (1991) for O3 exposure. Ethylene
12 inhibits both stomatal conductance and carbon assimilation to some extent (Taylor et al., 1988).
13 Thus, one could postulate that O3 generates a wounding response with a production of ET, which
14 would, in turn, generate the change in stomatal conductance and photosynthesis. Clearly, these
15 multiple events may have confounded some earlier studies.
16
17 AX9.2.3.4 Antioxidants Within the Apoplastic Space
18 The first line of defense against O3 is a closure of the stomata to exclude its uptake. This is
19 counterproductive for efficient photosynthesis, but some amount of closure limits the rate of O3
20 deposition into the leaf tissue to allow for a secondary line of defense to detoxify the O3. The
21 secondary line of defense involves a range of antioxidants, which are highly reactive to the types
22 of chemicals that can be generated by O3. Several antioxidant proteins are stimulated by O3 in
23 Plumbagini folia, including glutathione peroxidase (GSH-PX), SOD, and catalase. The
24 timescales for changes in their levels vary: some rise rapidly, while others rise more slowly.
25 The pattern of changes in these particular proteins varies greatly among different species and
26 conditions.
27
28 Ascorbate Within the Cell Wall
29 Most of the recent reports indicate that ascorbate within the cell wall is the real first line of
30 all defense. Ascorbate within the wall declines when the tissue is exposed to O3 (Luwe et al.,
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1 1993; Moldau, 1998). This decline appears to be closely linked to the amount of O3 penetrating
2 the leaf tissue.
3 It has long been suspected that intracellular antioxidants play a role in preventing O3-
4 induced injury to plant cells. Variation in the types of biochemical compounds present in the
5 apoplastic space can give rise to a multiplicity of reactions with O3, but the predominant
6 biochemical species is ascorbate. Ascorbate is water soluble, present in the solution where O3
7 can dissolve, and is highly reactive. Unfortunately, a variety of antioxidants are found
8 throughout the cell and any measurements of one particular type within the total leaf tissue can
9 give misleading results. For example, ascorbate is present within the cell wall, cytoplasm, and
10 chloroplasts (Burkey, 1999; Moldau, 1998); and ascorbate can move between the cytoplasm and
11 the cell wall with relative ease (Figure AX9-12) (Bichele et al., 2000). The total of all ascorbate
12 pools is measured when the tissue is ground and assayed. If the cell wall ascorbate concentration
13 drops by 50% due to O3 exposure but all other tissue concentrations remain the same, the
14 measurement of the total loss is dependent upon the amount of ascorbate within the cell wall.
15 Turcsanyi et al. (2000) showed that, compared to the concentration of apoplastic ascorbate, the
16 rest of the cells contained about 38 times as much. So a 50% loss of apoplastic ascorbate would
17 be converted into only 2 to 3% loss of the total ascorbate.
18 The ascorbate deficient Arabidopsis thaliana mutant has proven to be a powerful tool in
19 furthering the understanding of ascorbate biosynthesis in plants (Smirnoff et al., 2001). Three
20 classes of mutants were formed when A thaliana seed was mutagenized with ethyl
21 methanesulfonate: (a) those deficient in SOD, (b) those that failed to accumulate more
22 antioxidant proteins upon increased O3 exposure, and (c) those that were deficient (but not
23 depleted) in ascorbate. The low-ascorbate mutant type had 50 to 60% less ascorbate than the
24 wild type and displayed more foliar injury. This mutant is involved with the coding of the
25 GDP-D-Mannose pyrophosphorylase enzyme6 in the Smirnoff-Wheeler pathway for ascorbate
26 biosynthesis. Smirnoff et al. (2001) also suggested that other pathways can produce ascorbate
27 without relying upon the pyrophosphorylase step, but most probably at a slower through-put rate,
28 because any fully ascorbate-deficient mutant would be lethal, perhaps because of ascorbate use
EC 2.7.713, Mannose-1 phosphate guanylyltransferase; mannose + GTP —> GDP-mannose + ppi; this
product leads into cell wall polysaccharide synthesis and protein glycosylation through GDP-galactose and
GDP-fucose and, ultimately, through galactose into ascorbate synthesis.
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Cell
Apoplasm Membrane Cytoplasm
a.
Ma late
NAD
NADH
> De hydro-.
ascorbate
Ascorbate
HI Cytb
Dehydro-
ascorbate
-^/AscoTbateJ
II trai sporter(s) \(
De hydro- A
ascorbate
Dehydro-
ascorbate
[ Ascorbate
Dehydro-
ascorbate
b.
Ascorbate
Monodehydro-
ascorbate
Ascorbate
Carrier
Dehydro-
ascorbate
Ascorbate
Cytochrome b
Monodehydro-
ascorbate
NADH
Oxidoreductase
NAD+
Ascorbate
De hydro-
ascorbate
GSH
•GSSG
NADPH
NADP+
Dehydro- Glutathione
ascorbate Reductase
Reductase
Figure AX9-12. The reaction of ascorbate within the apoplasm of the cell wall and its
ultimate reduction/oxidations.
(a) Movements of reducing power (from Dietz [(1997]).
(b) The use of glutathione to maintain the level of ascorbate within
the cell wall region (from Horemans et al. [2000]).
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1 as a cofactor rather than its antioxidant properties. In addition to its lowered antioxidant
2 capacity, the ascorbate-deficient Arabidopsis mutant vtcl (Conklin et al., 1996; Conklin and
3 Barth, 2004) may show suppressed growth due to lower mannose levels that are necessary for
4 cell wall formation. Ozone thus may suppress growth in these mutants through interference with
5 cell wall biosynthesis as well as through lower antioxidant protection.
6 The ascorbate peroxidase (APX, which uses ascorbate to detoxify peroxides) family
7 consists of at least five different isoforms, with isozymes in the apoplastic and cytosolic space.
8 Furthermore, most forms of ascorbate can move through the plasma membrane (Bichele et al.,
9 2000), making the levels of all forms of ascorbate interdependent and able to at least partially
10 influence each other. Dehydroascorbate (DHA) can be broken down into other smaller
11 fragments easily in vivo and represents a continuous loss of ascorbate from varied parts of the
12 cell if ascorbate is allowed to remain in the oxidized form in some regions. In fact, the turnover
13 rate in leaves is estimated to be from 2 to 13% per hour, depending upon species and
14 developmental age (Smirnoff et al., 2001). There are apparently three pathways for ascorbate
15 turnover (Figure AX9-12a). The typical reaction is a reduction of DHA into ascorbate from
16 which an oxidative step generates DHA. Pathway I requires a reductive step using NADH
17 external to the plasma membrane generated from internal malate using a malate/oxaloacetate
18 transporter. Pathway II uses a direct transporter of ascorbate/DHA. Pathway III moves the
19 required electron(s) through a cytochrome b system, maintaining two separate pools of
20 ascorbate/DHA (within the cytoplasm and within the wall). Each of the pathways (Dietz, 1997)
21 represented by Roman Numerals in the Figure AX9-12, require only one NAD(P)H molecule to
22 reduce the DHA molecule back to ascorbate. However, the transport properties and redox
23 potential of the cell differ for each pathway. The efficiency of the reduction of DHA is
24 dependent upon the redox coupling and the region in which the chemical species is located.
25 Turcsanyi et al. (2000) exposed broad bean (Viciafabd) grown under two regimes in
26 duplicate controlled chambers: charcoal/Purafil filtered air (CFA) or (CFA) plus 0.075 ppm O3
27 for 7 h/day for 28 days (chronic exposure) or exposed to 0.150 ppm for 8 h (acute exposure).
28 Responses of the two sets of plants were similar except for stomatal conductance, which was
29 50% lower in the chronically exposed plants. Plants grown under acute exposures developed
30 visible injury, while plants grown under chronic conditions developed no visible injury. Within
31 an hour of the start of the acute exposure, the stomatal conductance was reduced by nearly 40%
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1 and assimilation was reduced by nearly 18% in the clean air plants; the reduction in conductance
2 was only 21% and assimilation 16% in the plants subjected to chronic O3 exposures. The
3 assimilation was affected similarly in both cases, while the conductance showed less of a
4 percentage drop in the chronic O3-exposed plants and began at a lower O3 level. The similarity
5 of the assimilation indicated that the stomata were not limiting assimilation in either case before
6 acute exposure. More to the point, the decline in ascorbate in the apoplastic space due to the O3
7 exposure was ".. .more often than not, on the borderlines of statistical significance." However, a
8 30% decline in ascorbate after 4 h of acute O3 exposure in both cases was observed. This lack of
9 significance may be due to a relatively large standard error of the data, which in turn may be due
10 to the difficulty of extracting and measuring ascorbate from the apoplastic space in quantitative
11 terms.
12 The chemical reaction7 of ascorbate and O3 is given by the molecular rate constant of
13 4.8 x 107 Mr1 s"1. This is some 50,000* the rate constant for ET. Of course, it depends upon the
14 relative concentration of ascorbate and ET, but it is likely that ascorbate is in higher
15 concentration than ET. One would then expect that the rate reaction of ascorbate with O3 would
16 greatly dominate any possible reaction of O3 with ET. For a concentration of ascorbate in the
17 range of 1 mM and for an O3 concentration of about 0.1 ppm or 4.2 x 10~9 M, the detoxification
18 rate would be 4.8 x 107 x i(r3 x 4.2 x 10~9 M s"1 = 2.0 x 10~4 M s"1. Turcsanyi et al. (2000)
19 calculated an O3 flux of about 1.6 x io~9 moles m"2 sec"1. With a wall thickness of 0.12 x io~6 m
20 and all the O3 flux going into the wall region, this would give about 1.3 x 10~2 mol m"3 s"1 or
21 1.3 x 1Q~5 M s"1 flux, which is less than 10% the detoxification rate.
22
23 Glutathione
24 Many of the initial studies of O3 exposure used high concentrations and measured only the
25 total sulfhydryl contents of the tissues. For example, in some of the earlier work, exposures of
26 tobacco to 1 ppm O3 for 30 min induced a 15% loss of the total sulfhydryls (0.74 |imole/g-FW)
27 (Tomlinson and Rich, 1968). These results are similar to other studies at high O3 levels (Dugger
28 and Ting, 1970). It is now suspected that the severe injury in their studies resulted in a massive
29 collapse of the cells and release of most of their internal constituents. Much of the oxidation
7These chemical rate constants are those constants within a bulk solution. In the apoplasm, the possibility
exists for the chemicals to be preferentially oriented near a surface; so the constants may not be the same as for bulk
solutions.
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1 thus observed may have been the result of chemical oxidations of the O3 that subsequently
2 entered the damaged tissue. Even under milder conditions, changes in sulfhydryl components
3 have still been noted and any sulfhydryl on the surface of the cell would be at risk due to its high
4 reactivity with O3 (Mudd et al., 1997b; Mudd et al., 1969). For example, the level of sulfhydryl
5 compounds within the protein of isolated chloroplasts declined about 66% when the chloroplasts
6 were subjected to O3 (about 1 jimole O3) exposure (Mudd et al., 1971).
7 At this stage, it is important to note that there are inherent problems with metabolic studies
8 of full tissues. The first is that most organs have several different types of tissues. For example,
9 leaves have, at the minimum, epidermal and vascular tissues and two types of mesophyll cells.
10 Each type of cell may be metabolizing quite differently and producing very different levels of
11 metabolites and enzymes. Furthermore, most pathways are well regulated and after any small
12 disruption, the pathway tends to return to near its former stability. Changes in the level of
13 enzymes are likewise difficult to measure. Many enzymes function below their maximum
14 activities. Their speeds of reactions are often increased through regulation, rather than through
15 the production of more enzyme.
16 Glutathione is a three-amino acid peptide, which has antioxidant properties due to its free
17 reducing sulfhydryl group (G-SH). Glutathione is generally kept in its reduced form by
18 glutathione reductase (GR) with the reaction:
19
GS - SG(oxidized) + 2e + 2H+ -> 2GSH (9-2)
20
21 GR has six isoforms8 within the chloroplast and six isoforms outside. The optimum activity
22 occurs at pH 7.8, suggesting it is located within the stroma of the chloroplast or in the cytoplasm
23 rather than in the cell wall, which is at pH 4-5 (Madamanchi et al., 1992). Clearly, an increased
An isoform is the same enzyme, with the same structure and perhaps within the same organelle, but its
promoter region has different DNA codes. Thus, each protein segment is induced by different signals, and so its
enzyme can be formed in response to different environments. This is in contrast to isozymes, which classically are
similarly reacting, but structurally different, enzymes in different compartments.
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1 expression of GR (generated through transgenic implants) is important within the chloroplast to
2 prevent some oxidations9 (Aono et al., 1995).
3
4 Catalase
5 Catalase, even though it breaks down H2O2, does not appear to protect plants from O3
6 exposures. Two principal reasons may cause this lack of reactivity: (1) catalase has a high Km
7 for H2O2 and a low rate coefficient, and (2) catalase seems not to occur within the cell wall
8 regions but rather in the cytoplasm and peroxisomes (Buchanan et al., 2000). While a few
9 reports suggest that catalase is increased by exposure to O3 (Azevedo et al., 1998), Booker et al.
10 (1997) found no effect of catalase activity in soybean until late in the growing season, and others
1 1 have found decreased catalase activity in wheat in response to O3 (McKee et al., 1997).
12 Unfortunately H2O2 induced by some forms of wounding in mesophyll cells can lead to
13 induction of an increase in GSH and the transient production of catalase (Vanacker et al., 2000).
14 In general, it seems that catalase is not really involved primarily in the defense of the cell due to
15 O3 attack but rather may be a secondary response. The reaction of catalase (Scandalios, 1993) is
16 as follows:
17
18 2H2O2 -» 2H2O + O2 K= 1.7x lO^M-
,_ .,
(9-3)
19
20
21 Superoxide Dismutase
22 The varied compounds that O3 can produce upon entering an aqueous solution are very
23 similar to those involved in the HR when plants are infected by an avirulent pathogen
24 (Figure AX9-10). The sequence of the plant response to the pathogen is (1) recognition of the
25 gene products of the pathogen by the plant (elicitor), (2) generation of an immediate
26 phytoresponse to attempt to localize the attack and its products, and (3) generation of a systemic
27 acquired resistance (SAR) to subsequent attack by the pathogen. Inducible defense responses are
28 phytoalexin synthesis and production of pathogenesis-related proteins (PR). One aspect of this
29 total response is the production of O2 and H2O2 by the cell (Lamb and Dixon, 1997). The
9Typically this protection is observed in the paraquat sensitivity of plants. In this assay, added paraquat,
the herbicide which intercepts electrons from the reducing end of photosystem I in the chloroplast, caused
oxidations, chlorophyll loss, and death due to the buildup of superoxide and peroxides.
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1 elicitor can generate a transient alkalinization of the apoplast, up to pH 7.2, caused by a lowering
2 of the H+-pump rate and an increase in the H+-influx/K+-efflux exchange. Other effects include a
3 weak accumulation of transcripts for PAL (phenylalanine lyase); a larger and rapid induction of
4 glutathione S-transferase, GSH-PX; oxidative cross linking of cell wall proteins which is blocked
5 by ascorbate acid; generation of localized apoptosis; and rapid influx of Ca2+, which activates
6 apoptosis among other pathways (Lamb and Dixon, 1997). These effects seem to be very similar
7 to those induced by O3 exposure (Sandermann, 1996, 1998).
8 The putative antioxidant enzyme SOD (Equation 9-4 and Table AX9-8) catalyzes the
9 oxidoreductase reaction, which eliminates SO2 by dismutation (Bowler et al., 1992):
10
13
14
2O2- + 2H+ — _» H2O2 + O2 K = 2.4 x 109 M'1 sec'1. (9-4)
12
Table AX9-8. Superoxide Dismutase Isozymes and Isoforms
Reaction: 2H+H
Isozymes
Cu-Zn
Fe
Mn
2 O2 - H2O2 + O2
M.W.
20kDa
23kDa
23kDa
Isoforms
csdl
csd2
csd3
fsdl
sd2
fsd3
msdl
Cytolocation
Plastid
Peroxisomal
Mitochondrial
Plastid
Mitochondrial
1 The number, as well as the activity, of isozymes of each type of SOD in Table AX9-3 can
2 vary with plant species. However, the isozymes that have been tabulated are Cu-Zn SODs, in
3 cytosol and chloroplast; Fe-SOD, active in chloroplast stroma; and Mn-SOD, active in
4 mitochondrial matrix (Karpinski et al., 1993). In the experiment demonstrating the activation of
5 varied SODs, there were three Cu-Zn SODs (csdl, csd2, csd3), three Fe-SODs (fsdl,fsd2,fsd3),
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1 and one Mn-SOD (msdl) (Kliebenstein et al., 1998). Ozone sensitivity was determined by
2 exposure of plants to 8 h of 0.33 ppm of O3. csdl induced by O3 and UV-B was one of the
3 earliest SOD increases and most pronounced responses for mRNA and protein. Also, some
4 increase in csd3 (thought to be peroxisomal) was induced when the plants were exposed to a
5 high-intensity light pulse; msdl was unresponsive to the environmental stressors used here,
6 including O3; and csd2 (thought to be chloroplast) showed little increase. ThefsdJ isozyme
7 (present in the apoplasm) showed a slight decrease. On the other hand, an early report on snap
8 beans in which the experimenters used EDU, N-[2-(2-oxo-l-imidazolidinyl)ethyl]-N-phenylurea
9 (Carnahan et al., 1978; Kostka-Rick and Manning, 1993), to prevent visible injury by O3, 4-h O3
10 exposure at 0.45 ppm was correlated with an increase in general enzyme activity of SOD, i.e.,
11 the level rose nearly 2.5x in 2 weeks at a level of 50 mg EDU per pot (Lee and Bennett, 1982).
12 It is believed that EDU may induce SOD, which then protects the plant. While gross assays of
13 enzyme activity have not proven to be very useful in understanding the mechanism of O3 action,
14 in a well-crafted, long-term study involving ponderosa pine clones. Benes et al. (1995) stated
15 that "changes in antioxidant enzyme activity were not a consistent response to the O3 fumigation,
16 but when observed, they occurred most often in the O3-sensitive clone and in symptomatic,
17 fumigated branches.. .total (intra- and extracellular) activities of the antioxidant enzymes did not
18 appear to be good indicators of O3 tolerance...."
19 Ozone exposure (70 ppb for 7 h/day for 14 to 42 days of exposure)10 caused an increase in
20 POD and a decline in SOD with no change in APX. No GSH was detected, but the
21 concentration of (ASC + DHA) was at 20 to 25 nmol/g-FW of extracellular fluid, compared with
22 2.4 to 3.0 mmol/g-FW of cell fluid. Glutathione within the cell was only 100 to 170 nmol/g-FW
23 of cell fluid. While these results are what one might expect for POD, the decline in SOD and
24 lack of change in APX are not what would be expected if protection was provided by SOD and
25 ascorbate. Yet as noted, because the rate of SOD reaction is many times higher than the rate of
26 O3 entry, there may be no pressure to increase the SOD level.
At a level of 70 ppb, the concentration of O3 in air was about 3.06 x KT6 mol/m3, which with the
conductance of 0.042 mol/m2 s, gives a flux rate of O3 of 1.27 x 10~8moler2 s. Converting the SOD rate of 23
units/g-FW into a SOD rate within the apoplastic space of 6.9 x 10~3 mol/m2 s, or about 500,000 times the entry rate
of03.
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1 Some protection against visible injury (induced by 59 ppb daily mean O3 for 14 h/day for
2 7 days) was observed in genetically modified tobacco plants with excess chloroplast SOD11 (2 to
3 4 times higher), but less protection was observed in plants that had an excess of mitochondrial
4 SOD (8x higher) (Van Camp et al., 1994). In all lines, the conductance of the leaves dropped
5 about 50%, compared with the unmodified plants. There was a correlation with age of leaf (less
6 injury in younger leaves) that corresponded to that found in spruce trees in which the amount of
7 SOD declined in relation to the longer that needles were held on the tree (Polle et al., 1989).
8 A slightly different study, however, found no O3 protection with varied SOD within the needles
9 (Polle and Rennenberg, 1991). Interestingly, in maize, the synthesis of SOD (any form) was not
10 stimulated by O3 exposure (at 0.50 or 0.75 ppm for 8 h, variable times thereafter) but was by
11 exposure to 90% O2 (Matters and Scandalios, 1987). It may be that this high level of O3 does not
12 affect the SOD, or perhaps it stimulates and degrades the enzyme simultaneously.
13 The conclusions to be drawn from these results are not obvious. There seems to be SOD
14 (a Cu-Zn form) present in the apoplastic space of some plants, but it does not necessarily rise
15 with O3 exposure. Thus, either its concentration is sufficient to provide protection or it is not
16 needed. Over expression of any SOD in other organelles may play a role, especially in the
17 chloroplast (Cu-Zn or Fe forms); however, it may be playing a secondary role due to other
18 effects of O3 that generate conditions in which light can overload the chloroplast and generate
19 detrimental circumstances, including the production of SO2 . In addition, SOD is
20 developmentally expressed in varied concentrations, so that long-term exposure to O3 may alter
21 each leafs developmental age and, in turn, alter what level of SOD is observed. In any case,
22 SOD does not seem to be the primary antioxidant system to protect against O3.
23
24 Changes to the Plasmalemma
25 Reports of "peroxidation" generally occur within unicellular organisms subjected to very
26 high levels of O3 (in Chlorella [Frederick and Heath, 1970] and in Euglena [Chevrier et al.,
27 1990]). Heath (1987) determined that by the time biochemical events were altered and MDA
28 was produced in Chlorella., little permeability remained in the cells and most metabolic
29 pathways were greatly disrupted by the subsequent loss of substrates. In fact, MDA production
1 lrThe SOD enzymes were from Nicotiana plumbaginifolia with appropriate transit sequence for targeting
the correct organelle and expressed under control of cauliflower mosaic virus 35S promoter.
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1 was concurrent with a high O3 uptake by the cell, indicating a complete opening of the cell and
2 associated with the concurrent inability to plate the cells on a glucose medium (indicative of cell
3 death). Heath (1987) reached the conclusion that no one had proven that lipid oxidation was in
4 any way a part of the initial reactions of O3 with the cell, a conclusion confirmed by Mudd et al.
5 (1997a). An excellent review regarding the initial action induced by O3 within a plant
6 (Kangasjarvi et al., 1994) should be consulted. There is little data to show that lipids are
7 attacked by O3 in any living system that was not previously severely injured by O3. Most of the
8 data suggesting lipid attack by O3 has been demonstrated in plants subjected to O3 concentrations
9 of 0.5 to 1.0 ppm for several hours, during which gross wilting of the plant tissues usually
10 occurs, suggesting extreme water loss. It is not surprising that lipid and protein injury are
11 observed under these conditions. While those reports were useful in the 1960s and 1970s, they
12 are not especially insightful now when ambient levels of O3 are rarely above 0.2 ppm.
13
14 AX9.2.4 Wounding and Pathogen Attack
15 The decline of an enzyme is more difficult to measure than the rise of a new enzyme; an
16 increase from 0 to 2% may be within the precision of any assay, but a decrease from 100 to 98%
17 is often masked by simple variation of the assay. Thus, measuring enzymes, which are in great
18 abundance in prefumigated tissue, is a risky operation. On the other hand, if O3 induces a
19 general physiological change that has characteristics similar to other well-studied, stress-induced
20 changes, then O3 studies could "piggyback" onto those studies to gain insight into the full scope
21 of metabolic alterations. It is now becoming clear that wounding and pathogen attack of plants
22 are similar to O3-induced changes in plants, and a reasonable hypothesis is that O3 must induce
23 one or more of the first steps seen in the wounding/pathogen-attack response.
24 Systemic acquired resistance (SAR) has been heavily investigated, and DNA probes have
25 existed for some time for a series of expressed genes (see Table AX9-9). Several enzyme
26 classes are associated with O3 injury, including glucanases and peroxidases and others, such as
27 the PR proteins and chitinases. Thus, strong evidence exists from enzyme function and genetic
28 material that O3 induces an activation of a S AR-like response.
29 Mehdy (1994) described a model of how an elicitor produced by the pathogen attack
30 activates a G-protein, which opens the inward-flowing Ca2+ channel. The flow of Ca2+ into the
31 cytoplasm raises the internal level (at the jiM level) and activates a protein kinase that increases
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Table AX9-9. Gene Families and cDNA Clones Used as Probes for SAR
(Ward et al., 1991)
Probe
Relevant Properties of Encoded Protein
Reference
PR-1
PR-2
PR-3
PR-4
PR-5
PR-1 basic
PR-O'
Basic, glucanase
Basic chitinase
Acidic peroxidase
Acidic, extracellular; function unknown most
abundant PR protein in tobacco; >90% identical to
PR-lbandPR-lc
Acidic, extracellular b-1,3-glucanase, >90% identical
to PR-N and PR-O
Acidic, extracellular chitinase; also known as PR-O;
>90% identical to PR-P
Acidic, extracellular; unknown function; homologous
to C-terminal domain of Winl and Win2 of potato
Acidic, extracellular; homologous to thaumatin and
bifunctional amylase/proteinase inhibitor of maize;
also known as PR-R or PR-S
Basic isoform of acidic PR-1
Acidic, extracellularb-l,3-glucanase; approximately
55% identical to PR-2 group
Vacuolar; approximately 55% identical to PR-2 group
and PR-O'
Vacuolar; approximately 65% identical to PR-3 group
Extracellular; lignin-forming
(Payne etal., 1989)
(Wardetal., 1991)
(Payne etal., 1990)
(Friedrichetal., 1991)
(Payne etal., 1989)
(Payne etal., 1989)
(Payne etal., 1990)
(Shinshi et al., 1988)
(Shinshietal., 1987)
(Lagrimini and Rothstein, 1987)
1
2
3
4
5
9
10
11
12
the activity of the plasma membrane NAD(P)H oxidase and generates O2 . Superoxide
dismutase converts O2 into H2O2. Both O2 and H2O2 are responsible for the active oxygen
species response, which is believed to be a defense mechanism to kill the pathogen. In this
normal defensive reaction, a subsequent system induces either localized lipid peroxidation per se
or a membrane lipase to produce j asmonic acid or inositol triphosphate, which act as secondary
messages to activate the defense gene products.
Booker et al. (2004) found that G-proteins might be involved in the perception of O3 in the
extracellular region using A. thaliana G-protein null mutants. The activation of a passive inward
flow of Ca2+, e.g., by an O3-induced response, would serve the same function as activation of the
G-protein. Once the level of cytoplasmic Ca2+ rises, all else follows. It is suspected that
exposure of plants to O3 does just that, as Castillo and Heath (1990) demonstrated — the in vivo
fumigation of bean plants both inhibits the outward-directed ATP-requiring Ca2+ pump and
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1 increases the passive permeability of Ca2+. It was thought that the calcium transporter system
2 has a sensitive sulfhydryl group which, if oxidized, would alter normal Ca2+ movements.
3 In addition, Dominy and Heath (1985) observed that the Reactivated ATPase (believed to be
4 involved in K+transport) was inactivated by in vivo exposure to O3 and that inactivation was
5 traced to a sensitivity sulfhydryl. Mudd et al. (1996) argued that several amino acids are very
6 sensitive to O3, including any with an exposed sulfhydryl. Thus, the O3-induced change in Ca2+
7 permeability may be the trigger to most, if not all, the wounding responses. However, the
8 difficult problem of proving that the cytoplasmic Ca2+ change is the first event in O3 injury
9 remains.
10 Some wound- and pathogen-induced genes that are activated or repressed in Arabidopsis
11 thalania are found with DNA arrays (Cheong et al., 2002). While these responses may not be
12 uniform for all plants, they suggest the possibility of wide-ranging gene changes that may occur
13 with a simple wound and that those changes are wide-ranging and diverse. As an example, these
14 responses are related to hormonal responses that are related to jasmonic acid, ET, and auxin
15 pathways; signal transduction responses; and transcription factors for a variety of pathways.
16 The involvement of ET in wounding and pathogen attacks is discussed in Section 9.2.3.3.
17
18 AX9.2.4.1 Peroxidases
19 Increases in cytosolic and apoplastic peroxidase activity in response to O3 are often
20 observed, but the reasons and outcomes of these changes have yet to be fully explained.
21 Increased activity is frequently correlated with O3 injury. Dass and Weaver (1972) observed that
22 increases in peroxidase after O3 injury was similar to that observed for plant infection by a virus.
23 Tingey et al. (1975) observed a 35% decrease in peroxidase activity immediately following O3
24 exposure; however, within 24 to 48 h, activity had increased significantly and was above control
25 level and remained there throughout the remainder of the study. Dijak and Ormrod (1982) also
26 observed increases in peroxidase activity when two O3-sensitive and two O3-resistant varieties of
27 garden peas (Pimm sativum) were exposed to O3. Peroxidase activity was not related to cultivar
28 sensitivity nor to visible injury. Unfortunately, there are many peroxidases (Birecka et al.,
29 1976); therefore, any general increase is not specific. In ET-treated leaves, peroxidase reaction
30 products were found between the plasma membrane and the cell wall, suggesting that ET itself
31 could induce peroxidase activity (Abeles et al., 1989a,b; Birecka et al., 1976).
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1 At the same time, others examined peroxidase reactions in general and found two types of
2 peroxidases (designated as acidic or anionic and basic or cationic, EC 1.11.1.7, but also listed as
3 EC 1.14.18.1). Many types of peroxidases are located in diverse organelles, and each seems to
4 be activated by different conditions (e.g., pH for anionic and cation types and substrates such as
5 guaiacol, syringaldazine, and ascorbate). Peroxidases belong to at least two groups, which
6 catalyze two separate reactions: (1) the reaction of H2O2 with ascorbate to form DHA, discussed
7 earlier (Thorn and Maretzki, 1985), which is regenerated by plasma membrane electron transport
8 using a dehydrogenase (Gross and Janse, 1977) now believed to be a malate/oxaloacetate shuttle
9 through the membrane coupled to a NAD(P)H-cytochrome-b-reductoxidase; and (2) the reaction
10 with coniferyl alcohol (from phenylalanine through phenylalanine ammonia lyase) to form lignin
11 within the wall. The anionic peroxidase thought to be involved with lignification is within the
12 cell wall (Buchanan et al., 2000; Taiz and Zeiger, 2002). Some basic peroxidases are maintained
13 within the cell, while some are external to the cell. After wounding (Gasper et al., 1985;
14 Lagrimini and Rothstein, 1987), some basic peroxidases can be activated by processes leading to
15 the synthesis of stress ET (Yang and Hoffman, 1984) and/or by excess Ca2+ (Gasper et al., 1985).
16 Elicitor treatment of plants change a series of peroxidases, some of which are similar to those
17 seen in O3-induced changes (see Table AX9-9).
18 The formation of lignin is due to the phenylpropanoid metabolism (Buchanan et al., 2000).
19 Tyrosine and phenylalanine are converted to cinnamic and/>-coumaric acid, which are, in turn,
20 converted to/>-coumaryl, coniferyl, and sinapyl alcohols, and then into lignins. Hence, the
21 peroxidase activity is often measured by one of these substrates (Espelie et al., 1986; Gasper
22 et al., 1985). However, it is questionable whether apoplastic peroxidase activity is limiting for
23 lignification; laccases have a prominent role as well. Also, availability of monolignols is critical
24 for core lignin formation, and it is unclear whether levels of these metabolites change in
25 response to O3. Studies by Booker and others (Booker et al., 1991, 1996; Booker, 2000; Booker
26 and Miller, 1998) indicated that O3 did not increase core lignin concentrations in foliage of
27 loblolly pine, soybean, or cotton; although levels of phenolic polymers and cell wall-bound
28 phenolics were elevated in soybean. Increased phenolic polymers appear to be lignin in acid-
29 insoluble lignin assays and may well be responsible, along with polyphenol oxidase, for the
30 stippling injury observed in O3-treated plants. Cell wall function implies the transport of
31 peroxidase molecules out of the cell and, most likely, the regulation of their activities within the
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1 wall space. These extracellular peroxidases may be observed by vacuum infiltration of buffer
2 into leaf air spaces and subsequent centrifugation of the tissues to remove the buffer with the
3 apoplastic enzymes that wash out (Castillo and Greppin, 1986). However, exposure to O3
4 induces important changes in the plant. For example, extracellular peroxidase activity in Sedum
5 album leaves increased nearly 3-fold over that in the control plants after 2-h exposure to 0.40
6 ppm O3 (Castillo et al., 1984). This O3-induced increase in extracellular peroxidase appears to
7 be under the control of Ca2+ (Castillo et al., 1984; Heath and Castillo, 1987). Initially, no effect
8 on the anionic activity as measured with syringaldazine (specific electron donor for lignifying
9 peroxidases) was observed, yet 21 h later, the anionic peroxidase activity was increased, whereas
10 the cationic (ascorbate measured) peroxidase activity was decreased, in O3-treated plants. This
11 suggests an immediate response (ascorbate peroxidase activation) and a secondary response that
12 activates the lignifying peroxidase via gene activation.
13 The rapid response of cationic peroxidase after O3 exposure may not result from de novo
14 protein synthesis but from the secretion and direct activation by Ca2+ ions of enzyme molecules
15 already present in the tissue. Cationic peroxidases might attack the peroxides and, in this
16 manner, act as a detoxifying agent with ascorbate as the substrate in the apoplasm. The effect of
17 Ca2+ upon peroxidase activity is stronger at low H2O2 concentrations (Penel, 1986). Thus, one
18 can imagine that, when the H2O2 concentration is low, this peroxidase activation would have a
19 greater in vivo importance. Furthermore, the secretion of cationic peroxidases into the free
20 spaces as a result of O3 treatment is accompanied by a simultaneous release of at least one of its
21 natural substrates (ascorbic acid); this cationic peroxidase exhibits a much higher affinity toward
22 ascorbate (up to 6-fold) than the anionic isozyme (Castillo and Greppin, 1986).
23
24 AX9.2.4.2 Jasmonic Acid and Salicylic Acid
25 Jasmonic acid (JA) and salicylic acid (SA) are considered to be regulators of the plant
26 defense response (Figure AX9-13) (Buchanan et al., 2000). They tend to respond more slowly
27 than ET, causing widespread effects in the plant tissues. Both seem to be heavily involved in
28 responses of the plant to O3, once again linking the pathogen/wounding defense to O3-induced
29 injury; however, their roles are far from clear.
30
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Phospholipid-
Ethylene—
Salicylic Acid-
-> /AO Synthas^
0='
JasmonicAcid
(Howe ef a/., 2000)
COOH
Allene Oxide
if
Reductase
HOOC
13-Hydroperoxy-
. A • i
Imolenic Acid
(HPOT)
Traumatic Acid
(Croft etai, 1993)
COOH
Systemin
Figure AX9-13. The pathway leading from phospholipids to jasmonic and traumatic acid.
The role of lipoxygenase and the production of a hydroperoxyl moiety
from the unsaturated fatty acid is clearly demonstrated. More
importantly, several of the enzymes within this pathway have been shown
to be activated by oxidative conditions including O3 exposure. The
production of both of these acid species could lead to a general global
response of a whole plant to the O3 exposure of a single leaf.
Sources: Howe et al. (2000); Croft et al. (1993); Buchanan et al. (2000).
1
2
3
4
5
9
10
11
One of the lipoxidase isoforms is activated by pathogen infection (POTLX-3) within 6 h
and accumulates for a week (Kolomiets et al., 2000). This enzyme is the first stage of the JA
pathway which leads to 13-hydroperoxide linolcenic acid (HPOT) which is converted either to
allene oxide through AOS or to C6 aldehydes through hydroperoxide lyase. These aldehydes act
as signaling agents via systemic (Sivasankar et al., 2000) or volatile odiferous compounds
(oxylipins) that have been implicated as antimicrobial toxins (Froehlich et al., 2001).
Interestingly, these compounds seem to target the chloroplast envelop where they interact with
its metabolism. As HPOT and AOs are both implicated in plant defense and are activated by O3,
these interactions may be related to how chloroplast enzymes and their mRNAs are involved
in O3-induced injury.
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1 AX9.2.4.3 Stress-Induced Alterations in Gene Expression
2 Early studies addressed the qualitative and quantitative effects of O3 on protein metabolism
3 (Harris and Bailey-Serres, 1994). Subsequent reports suggested that the physiologic and
4 metabolic consequences of exposure to O3 were, in part, mediated by increased gene expression.
5 A summary of the gene-linked changes in proteins induced by SAR may be seen in
6 Table AX9-9. Of particular note are the productions of PR proteins, chitinase, glucanase, and
7 acidic peroxidases that appear to be common markers used in many O3 studies. A summary of
8 varied proteins as measured by changes in the mRNA in A. thalania induced by O3 exposure is
9 shown in Table AX9-10. While studies on Arabidopsis thaliana required high concentrations of
10 O3 to produce a response, the levels reported in most of the studies did not induce visible injury.
11 The types of messages induced included glutathione S-transferase, PAL, ACC synthase, SOD,
12 and some PRs. Slower increases in messages are seen for other PR and SAR-senescence
13 proteins. Declines in messages were observed for varied chloroplast enzymes, including those
14 for Rubisco and chlorophyll binding proteins. A few new proteins were found — a casein kinase
15 and three plasma membrane proteins. It is interesting to note that few messages for "new"
16 proteins were generated by O3 exposure.
17 The working hypothesis is that O3, which is not eliminated by antioxidants in the cell wall,
18 alters the properties of the plasma membrane. Specific polypeptides, indicative of these
19 antioxidants, are induced. If specific receptor molecules or channels on the membrane are
20 affected, the ionic balance within the cytoplasm is changed, leading to altered transcription or
21 translation of the genes controlling those and other types of polypeptides. Once this membrane
22 disruption occurs, the cell must mobilize repair systems to overcome the injury. Thus, carbon
23 and energy sources once destined for productivity, must be used in repair processes. Some of
24 these repairs are thought to result from the induction of specific genes. Photosynthesis is
25 inhibited by direct inhibition of some of the enzymes, through byproducts of O3 attack or
26 by altered ionic balance. At the very least, the decrease in photosynthesis is a result of
27 an O3-induced decrease in rbcS mRNA.
28
29
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OQ
C
S-
to
o
o
VO
oo
to
o
p>
H
6
o
2;
o
H
O
o
H
W
O
O
H
W
Table AX9-10. Proteins Altered by Ozone as Measured by Molecular Biological Techniques as mRNA Level or
Exposure
150/300 ppb for
6 h daily
300 ppb for 6 h daily
200/500/1 000 ppb
for2h
350 ppb for 1-6 h
300 ppb for 6 h
1 50 ppm for 6
h/8 and 14 days
160 ppb for 3-72 h
(a) 250 ppb for 8 h;
(b) 250 ppb for 2 h;
(c) 175 ppb for
8 h/4 days
Other Gene Activity Rather than Enzyme Activity
Identified
Proteins Fast
Increase Slow Increase Examined,
Physiological Events Response Response Decline But No Change
Leaf curling; reduced GST, PAL Pxase, SOD CAT,LOX1
growth
lObandsoflORNA
Wilting (8 h); premature
senescence
Ethylene production; ACS-6 ACS-1, -2, -4, -5
downward curvature; water
logging
Necrosis in NahG and Cvi-0 Chi SOD, Chi GPX Cab mRNA,
(accumulating SA), not in cytAPX, GST1 cyto SOD,
Col-0 chl GR
Downward rolling of leaf; BCB,ERD1, Cab, rbcS Atgsr2,MTl,
early senescence SAG21 SAG 12, SAG 13,
SAG 19, SAG 20
Early senescence GST1,VSP2 MT1 CCH
(a) little chlorosis or lesions; GSTApx, PAT1 Fe-SODl GR,
(c) growth retardation CuZn-SOD cab, rbs
Unknown
Proteins Reference
Grosjean
etal. (1994)
AtOZIl » casein Sharma and
kinasell Davis (1995)
3 plasma Tokarska-
membrane Schlattner
proteins: 75-, 45-, etal. (1997)
35-kDapeptides
Vahala et al.
(1998)
Rao and
Davis (1999)
Miller et al.
(1999)
Mira et al.
(2002)
Conklin and
Last (1995)
-------
to
o
o
VO
oo
H
6
o
o
H
O
O
H
W
O
O
HH
H
W
Table AX9-10 (cont'd). Proteins Altered by Ozone as Measured by Molecular Biological Techniques as mRNA Level or
Other Gene Activity Rather than Enzyme Activity
Exposure
200 ppb for 24 h
250 ppb for 6 h
Identified
Proteins Fast
Increase Slow Increase
Physiological Events Response Response Decline
PR-l,PR-2a,
PR-5,AtEDSl, PR-3b,PR-4
AtGSTl,AtGST2
Lesion initiated on margin
and spread inward
Examined,
But No Change
LOX2,AtOZIl,
PAL,Lhcb,PATl,
HSP
Unknown
Proteins
rcdl, on
chromosome 1,
single Mendelian
trait
Reference
Matsuyama
et al. (2002)
Overmyer
et al. (2000)
Abbreviations used in Tables 9-9 and 9-10.
GST = Glutathionine synthase
PAL = Phenylalanine ligase
PR-1 = Promoter region 1
Pxase = Peroxidase
CAT = Catalase
LOX1 = Lipoxygenase
ACS-6 = ACC synthatase
SOD = Superoxide dismutase
CuZn-SOD = cyto SOD
Fe-SODl = Chi SOD
cyt APX = Ascorbate peroxidase
Chi GPX = Glutathione peroxidase
Cab mRNA = Chlorophyll a/b binding protein
chl GR = Gluthatione reductase
BCB = Blue copper binding protein
ERD1 = Ethylene response
SAG21 = Senescence
rbcS = Rubisco small subunit
MT1 = Mitochondria
-------
1 AX9.2.5 Primary Assimilation by Photosynthesis
2 AX9.2.5.1 Photooxidation: Light Reactions
3 Photooxidation refers to the oxidation of chlorophyll within the light reaction due to an
4 imbalance between light absorption and the CO2 use to produce carbohydrates. It was
5 discovered in the 1920s and studied under the concept of chlorophyll bleaching and photo-
6 autooxidation (Asada, 1999; Rabinowitch, 1945). What generally occurs is that electron transfer
7 from H2O to NADPH declines, and a light reaction overload occurs. The slowdown of electron
8 transfer may also be due to inhibition of the dark reactions, through the poor use of small
9 molecular weight carbohydrates or a lowered amount of the fundamental substrate CO2.
10 To counteract these detrimental reactions, a series of "antioxidant" reactions exist, which
11 eliminate the buildup of oxidative intermediates.
12 A lowered CO2 level, which can be caused by stomatal closure (Heath, 1996), blocks the
13 use of reduced plastoquinone (PQH2) in Photosystem II through NADP reduction in
14 Photosystem I (Hankamer et al., 1997). The buildup of PQH2 reduces the amount of QA,
15 resulting in a buildup of P680+|Pheo~ species (the primary photoact). The inability to reduce this
16 radical leads to injury to the Dx protein (32 kDa) and its fragmentation into 23-, 16-, and 10-kDa
17 fragments (Hankamer et al., 1997). Ozone exposure of bean plants leads directly to the loss of
18 this Dj protein (Pino et al., 1995). The loss of Dj stimulates the production of new Dx (and its
19 mRNA). Also, the production of the oxidized form of P680 (P680+) is harmful to the plant, because
20 electron flow from water to P680+ is limited, generating a P680T (the triplet form of P680), which is
21 highly oxidizing and can lead to dangerous reactions. One form of protection is the use
22 of p-carotene to convert the triplet form back to its normal state; however, that reaction can lead
23 to the loss of P-carotene. Without the protection of P-carotene, oxygen reacts with oxidized
24 products to produce singlet state of O2. This, in turn, can react with chlorophyll, leading to ring
25 breakage that, in essence, leads to chlorosis. These types of reactions do not seem to occur
26 often, but chlorosis is one form of visible injury, and loss of P-carotene has been reported.
27 Using a FACE system to expose soybean to elevated O3, Morgan et al. (2004)found that,
28 in leaves at the top of the canopy, there was no effect on the maximum light-adapted apparent
29 quantum efficiency of PSII ((|)PSII,max), electron transport at growth [CO2], and saturating
30 light (Jsat) nor on the probability of an absorbed photon reaching an open PSII reaction
31 center (Fv'/Fm'; the quantum yield). There was a small, but significant, decrease in
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1 photochemcial quenching (qP) at the top of the canopy. As leaves aged, the decrease in qP
2 was significant as leaves began to senescence, likely due to losses of chlorophyll. The results
3 of these studies and others suggest that alterations to the dark reactions are much more common
4 than to light reactions (Farage et al., 1991; Farage and Long, 1999).
5
6 AX9.2.6 Alteration of Rubisco by Ozone: Dark Reactions
7 A large body of literature published since 1996 shows that O3 exposure affects Rubisco
8 concentrations (Pell et al., 1997). Treatment of a variety of plants with O3 at near-ambient levels
9 results in a loss of Rubisco and of the mRNA coding for both subunits of Rubisco (rbcS, small
10 and rbcL, large). In addition, oxidation of Rubisco by O3-generated ROS may be an important
11 factor in suppressing photosynthesis (Pell et al., 1997). Increased carbonyl concentrations of
12 Rubisco are correlated with O3 injury (Kanoun et al., 2002; Leitao et al., 2003). Because
13 Rubisco plays such an important role in the production of carbohydrates (Figure AX9-14), any
14 loss may have severe consequences for the plant's productivity.
15 Chronic O3 exposure, both with and without elevated CO2, significantly lowered
16 assimilation and leaf conductance of soybean in aging mature leaves (Fiscus et al., 1997; Reid
17 and Fiscus, 1998), which was associated with significant decreases in Rubisco content in aging
18 leaves. Noormets et al. (2001) also found that O3 had the greatest effect on older leaves of aspen
19 clones using a FACE exposure facility in which areas of ambient CO2 (daytime 360 ppm) and
20 ambient with added CO2 (560 ppm), with added O3 (97.8 ppb), and with added CO2 and O3, were
21 used. They found an O3- induced decline in assimilation and in conductance, and subsequently
22 confirmed that the internal CO2 (calculated for within the leaf) is not affected by O3 exposure.
23 Higher levels of CO2 increased the assimilation and lowered the conductance, maintaining the
24 internal to external CO2 ratio identical to that found with the ambient CO2 level, corresponding
25 to the theory of Farquhar et al. (1980). More to the point was that the stomatal limitation12 was
26 not altered by O3 exposure, with or without excess CO2. It is critical to point out that mesophyll
27
12The limitation was defined as the ratio of stomatal resistance to the total resistance, which included the
operating point of the assimilation (A) verses internal CO2 concentration (Q) curve and the resistance of the
boundary layer. The operating point of the curve was defined as the internal CO2 level, which is calculated by the
conductance and assimilation. The resistance of this operating point was calculated as the cotangent of the slope to
the operating point. Unfortunately, the slope is not a dimensionless parameter but is rather moles of air per area of
leaf ~s of time and, thus, it is unclear whether the slope changes with added CO2 and O3.
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{precursors}
{precursors}r
DNA —*> {rbcJi
{products}r
{products}
{Triose-P}
Figure AX9-14. The production of Rubisco and its Calvin Cycle pathway reactions. Two
peptides are used to build Rubisco: rbcS, the small subunit produced by
DNA within the nucleus; and rbcL, the large subunit produced by DNA
within the chloroplast itself. Clearly both polypeptides must be closely
regulated to produce the enzyme in a coherent manner. Furthermore, at
least five isoforms of DNA can produce rbcS, each of which is regulated
by a different promotor region.
1 conductance is directly linked to internal CO2 level13. So if Q/C0 (C0 = CO2 outside) is constant
2 and gs declines, then gm must likewise decline. If, as it is argued, Rubisco levels are constant or
3 at least increasing, then a regeneration of RuBP must be the cause of the decline in gm. Farquhar
4 et al. (1980) were more concerned with high levels of CO2 and had little to say about O3
5 exposure.
6 In a similar study, Morgan et al. (2004) examined elevated O3 using a FACE system to
7 increase O3 by 20% over the entire growing season. They examined O3 effects from emergence
8 through the entire life cycle to senescence. Leaf photosynthetic performance was measured
9 using a LI-COR 6400 with integrated chlorophyll fluorescence capabilities to examine both dark
10 and light reactions. This study found no effect of elevated O3 on newly expanded leaves over the
13T
Respiration is generally small at saturating A and often is ignored. By transforming the term {A = gs (C0
- Q)} into {A = gs C0 - gm C0 = (gs - gm) Co} where gm= gs (Q/C0) or the mesophyll conductance in earlier
literature.
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1 growing season. There were little O3-induced changes in the light reactions; however, as leaves
2 aged, there were significant changes in the dark reactions. For example, there were significant
3 losses in the maximal photosynthetic assimilation in saturating light (Asat) and the maximum rate
4 of carboxylation (an in vivo measure of Rubisco efficiency), and maximum rate of electron
5 transport for the regeneration of RuBP (Jmax). The findings showed the greatest impact of O3 on
6 the oldest leaves and demonstrated the significant impact on seed production.
7 The level of carbohydrate within the cell has an effect upon the amount of mRNA for
8 Rubisco (rbcS). Experiments by Krapp et al. (1993) indicated that a decline in carbohydrate
9 levels is probably due to the increased production of control metabolites, such as fructose
10 2,6-bisphosphate, which can shut down important sugar production pathways. This report also
11 leads to a measure of half-time for the decline in rbcS of about 2 days14 when 50 mM glucose is
12 added to a cell suspension of Chenopodium. Also, the carbohydrate level was increased by cold
13 girdling the petioles of intact tobacco and potato plants. The levels of carbohydrate nearly
14 doubled in 5 days and the level ofrbcS declined rapidly (reaching 25% after 12 h). A decline in
15 Rubisco followed, but more slowly (with an estimated half-time of about 108 h after a lag of at
16 least 12 hours). This, of course, is expected; the level of the enzyme would decline slowly with
17 a lag after a loss of the message.
18 A better estimation of the half-life ofrbcS can be found in the Jiang et al. (1993) study of
19 the destabilization of the message by an antisense message. The wild-type rbcS in tobacco had a
20 half-life of about 5 h compared to that in the mutant with the antisense. It was argued that the
21 antisense message increased the degradation of the normal rbcS. The estimated half-life ofrbcS
22 under O3 fumigation is about 1 h (Pell et al., 1994). Although comparisons of these diverse
23 systems cannot be easily made, the normal half-life ofrbcS may be closer to 5 to 10 h; and O3
24 fumigation does not simply stop the transcription of DNA, but rather it alters the rate of
25 degradation, either independently of, or simultaneously with, transcription.
26 Williams et al. (1994) developed a correlation between the levels of ABA after water stress
27 in Arabidosis thaliana leaves and the loss ofrbcS. Although their data were not quantitative, the
28 level of ABA had a half-time rise of about 1 to 2 h and the level ofrbcS had a half-life decline of
29 about 2 to 4 h. Their work suggests that drought stress may alter the CO2 metabolism by
30 changing enzyme relationships much more than by merely closing the stomata. If an ABA rise
14The amount of Rubisco drops from an initial 0.12 to a final amount of 0.04 umole/g-FW s in 6 days.
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1 is lowering rbcS, rbcS may not be a good marker of O3 fumigation except under highly
2 controlled conditions.
3
4 AX9.2.7 Carbohydrate Transformations and Translocation
5 The question of whether translocation of the sugars out of the leaf is inhibited by O3
6 exposure arises, because productivity is often dramatically inhibited by O3 fumigation. Though
7 nearly 35 years have passed since Dugger and Ting (1970) investigated the question of sugar
8 transport within the leaf, the question has since been little studied. Translocation (Cooley and
9 Manning, 1987) appears to be inhibited, because root functions are impaired by O3 exposure.
10 Many observed events suggest that while carbon assimilation within the leaf declines,
11 translocation of carbon is inhibited even more so, because plant growth points are inhibited and
12 root/shoot ratios are altered (Dugger and Ting, 1970; Gerant et al., 1996; Tjoelker et al., 1995).
13 Many of the experiments with O3 fumigation indicate that O3 exposure decreases the net
14 growth or dry mass of the plant, but the mechanism is poorly understood. Generally the
15 decrease in assimilation is much less than the decrease in growth, but not always. Under many
16 conditions, the stomata will close partially, decreasing assimilation by a smaller factor. Only a
17 long exposure, or high levels of exposure for a short time, generate enough decline in Rubisco to
18 make the growth of the plant problematical. No convincing argument has linked the decrease in
19 growth with a small decline in assimilation, either by a conductance- or Rubisco-limitation.
20 Measures of assimilation with crops are frequently done on upper canopy leaves, which are the
21 last leaves to exhibit O3 injury, while leaves deeper in the canopy exhibit injury and early
22 senescence. Crop root growth must be sensitive to these and other O3 effects, because root
23 biomass is often suppressed early by elevated O3.
24 Volin et al. (1998) found O3 exposures statistically decreased leaf area ratio, specific leaf
25 area, leaf weight ratio, and root weight ratio in Populus tremuloides and two C3 grasses
26 (Agropyron smithii and Koeleria cristatd) but not in Quercus rubra and in the C4 grasses
27 Bouteloua curtipendula and Schizachyrium scoparium. There was no statistically significant
28 change in any species in leaf conductance (4% level decline in K. cristatd) nor in assimilation
29 (although there was a decline in assimilation at the 6% level for P. tremuloides and a decline at
30 the 1% level in B. curtipendula). They also reported a correlation between growth decline and
31 decreased stomatal conductance among all species.
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1 Tradeoffs are made by plants. Birch grown in highly fertilized conditions exhibited a
2 greater leaf turnover when exposed to O3, in that leaves not only formed faster but abscised
3 faster, presumably due to early senescence; whereas birch grown under poorer fertilized
4 conditions retained their leaves longer and had a greater respiration rate within those leaves
5 (Maurer and Matyssek, 1997). Again, one must be careful in comparing short-term versus long-
6 term exposures. Grulke et al. (2001) observed that maximum concentrations of carbohydrates in
7 1-year-old needles that had not abscissed due to early senescence declined when subjected to
8 year-long exposures along an increasing pollution gradient. Furthermore, the monosaccharide
9 concentrations (along with starch) in fine roots were largely decreased, suggesting that needle
10 sugars were limiting, leading to root-sugar limitations. However, determination of the total
11 productivity and detailed balance of carbohydrate was impossible, because these were older,
12 larger trees and the data were taken over a full growing season. For a shorter-term exposure of 9
13 days, Smeulders et al. (1995) observed that O3 appeared to increase the retention of labeled
14 photosynthates within the needle, and, at higher exposures (400 versus 200 or 0 mg/m3), the total
15 starch within the needle decreased, suggesting that less carbohydrate was produced within the
16 cell or perhaps that it was in compounds not measured.
17 Studies with Pima cotton (Gossypium barbadense), aspen (Populus spp.) and bean
18 seedlings (Phaseolus vulgaris) indicate that acute O3 exposures inhibit export of the current
19 assimilate that provides carbohydrates to the roots from source leaves of cotton as well as recent
20 assimilate from the older leaves of aspen and bean (Grantz and Yang, 2000). Grantz and Yang
21 (2000) attempted to distinguish between potential mechanisms of O3 phytotoxicity operating at
22 the level of the whole plant. Four hypotheses were tested by fumigating cotton: (1) O3 exposure
23 reduces leaf pools of soluble sugars; (2) pruning leaf area and reducing source strength to match
24 that of O3-treated plants reproduces O3 effects; (3) pruning lower leaf area more closely
25 reproduces O3 effects than pruning the upper leaf area; and (4) manipulating plant age and,
26 thereby, plant size to match O3-treated plants reproduces O3 effects. All were shown to be
27 incorrect. Under each of the above conditions, Grantz and Yang (2000) reduced the amount of
28 foliage to match that caused by O3 injury. While the treatments reduced the biomass and leaf
29 area, they did not alter biomass allocation nor root function. They concluded that a simple loss
30 of foliage does not induce the changes in translocation to the roots to the same extent as does O3
31 injury.
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1 This finding by Grantz and Yang (2000) is important in that it suggests that O3 can trigger
2 a plant-wide response that may be linked to alterations in signal transduction and the generation
3 of whole plant signals. Stitt (1996) suggested that"... allocation is regulated by long-distance
4 signals that act to influence growth of selected sinks and to modify the delivery of resources to
5 these sinks in parallel." Cooley and Manning (1987), citing McLaughlin and McConathy
6 (1983), suggested three possible ways that O3 fumigation might alter translocation:
7 (1) malfunction of the phloem loading process, (2) increased translocation to leaf injury repair,
8 and (3) an altered balance between the leaf and sinks caused by reduced carbon fixation and a
9 greater demand for assimilate in the leaf.
10 Ethylene has been shown to reverse this sugar inhibition of development and to be
11 antagonistic to the ABA effect (Finkelstein and Gibson, 2002). However, these effects depend
12 greatly upon the developmental stage of the plant. Thus, the balance of the effectors (sugars,
13 ABA, and ET) may interact to generate the variation observed in the O3-induced productivity
14 decline. For example, O3 fumigation can induce a shift in the carbon transfer between roots and
15 shoot, and this shift can be amplified by mild drought (Gerant et al., 1996). Furthermore a
16 regulation of source-sink relations with the defense responses induced by elicitors was observed
17 by wounding the leaves of Chenopodium rubrum (Roitsch, 1999). Ethylene appears to be able to
18 repress the expression of extracellular invertase, which is critical for control and downloading of
19 sucrose derived from the translocational stream (Roitsch, 1999). In addition, the development of
20 Arabidopsis at high concentrations of glucose or sucrose is arrested by increasing the ABA level
21 (Coruzzi and Zhou, 2001).
22 Clearly more work is needed on the interactions between assimilation, translocation, and
23 source/sink relations with O3 exposure. In these interactions, one must be aware of the
24 developmental age of the plants and their phytohormonal status.
25
26 AX9.2.7.1 Lipid Synthesis
27 Heath (1984) summarized several early reports of O3-exposure induced lipid alterations.
28 Most concerned the production of MDA as a measure of lipid oxidation as well as the loss of
29 unsaturated fatty acids. However, a series of experiments by Sakaki and coworkers (Sakaki
30 et al., 1983, 1985) concentrated on one type of fumigation system and one metabolic pathway.
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1 This literature provides the best, most complete story with regard to lipid metabolism and O3
2 fumigation and suggests that O3 injures cellular membrane systems via lipid destruction.
3 Sakaki and coworkers (Sakaki et al., 1983, 1985) used spinach, which is a sensitive plant
4 but which has not been much evaluated with respect to O3 fumigation. While the O3 level was
5 high (0.5 ppm), enough work has been done to be able to discern what is happening. The first
6 paper showed that chlorophyll bleaching does not begin until the plants have been exposed to O3
7 for more than 10 h, whereas some MDA production begins with as little as 6 h exposure (Sakaki
8 et al., 1983). Consistent production of MDA, indicative of gross disruptions, occurred only after
9 8 h exposure (Sakaki et al., 1985), within the timescale when chlorophyll and carotenoid levels
10 begin to decline. Concurrently, the total fatty acid (FA) level decreased from -481 to 358
11 nmol/cm2 as the MDA level increased from 0.6 to 2.4 nmol/cm2, indicating FA peroxidation
12 (Sakaki etal., 1985).
13 Sakaki et al. (1983) also studied the development of changes by cutting disks from exposed
14 leaves and floating them on water solutions for varied time periods (up to 24 h). This permitted
15 feeding experiments to be done easily, whereas the cutting gives rise to an additional wound
16 response and eliminates metabolite movement to and from other portions of the plant. The
17 floating experiments indicated that, after exposure, scavengers of singlet oxygen (1O2), such
18 as D2O, and of hydroxyl radicals, such as benzoate and formate, have no effect on development
19 of the MDA response after 8 h of in vivo fumigation, while scavengers of (O2 ), such as tiron
20 and ascorbate, lowered the amount of MDA formed. By measuring metabolites immediately
21 after cessation of fumigation, they were able to show that ascorbate loss began with the onset of
22 fumigation, as did SOD loss. A lag time associated with the production of DHA suggested that
23 the reaction of ascorbate with fumigation did not immediately produce the oxidation product.
24 The first 4 h of exposure yielded 30 nmole/cm2 of ascorbate loss with 5 nmole/cm2 of DHA
25 production, whereas the second 4 h of exposure yielded 20 nmole/cm2 of ascorbate loss with 20
26 nmole/cm2 of DHA production.
27 Nouchi and Toyama (1988) exposed Japanese morning glory (Ipomea nil) and kidney bean
28 (Phaseolus vulgaris) to 0.15 ppm O3 for 8 h. Under these conditions, little visible injury was
29 found with up to 4 h of exposure, while injury increased by -50% after 8 h of exposure.
30 Morning glory produced more MDA than kidney bean, which produced the same as the zero-
31 time control. Morning glory also demonstrated a slight (5%) drop in MDGD
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1 (monogalactosyldiacylcerol), with increases in PC (phosphatidylcholine), PG
2 (phosohatidylglycerol), PI (phosphatidylinositol), and PE (phosphatidylethanolamine) after 4 h.
3 Twenty-four hours later, the drop in MGDG (mongalactosyldiacyglyerol) was much larger and
4 was thought to be related to an inhibition of UDP-galactose galactotransferase due to a rise in
5 free fatty acids (FFAs) in the chloroplast. Note that the two distinct timescales involved in O3
6 fumigation, immediately postfumigation and a day or so later, allows for comparison after the
7 plant metabolism responds to the fumigation event.
8 The pathway for the formation of MGDG and DGDG (digalactosyldiacylglycerol) is
9 located on the chloroplast envelope. Diacylglycerol (DG) arrives from either the endomembrane
10 system or the stroma and the enzyme UDP galactose-l,2-diacylglycerol galactosyltransferase
11 (UDGT) forms MGDG with galactose from UDP-galactose. Sakaki et al. (1990) suggested that
12 the O3-induced inhibition of UDGT was due to a release of FFAs from within the chloroplast.
13 These FFAs are inhibitory to UDGT, but not to GGGT, which is stimulated by high
14 concentrations of Mg2+ (Sakaki et al., 1990). The Sakaki et al. (1990) data indicate that the in
15 vivo measured activities of both enzymes isolated after fumigation are not affected by O3
16 fumigation. Both enzymes have sensitivity sulfhydryls, and both are located on the envelope.
17 Ozone, if it reaches those sulfhydryls, should inhibit these enzymes; yet inhibition was not seen.
18 It has been thought for years that tocopherols functioned as antioxidants in biological
19 systems (Tappel, 1972). Hausladen et al. (1990) examined the role of antioxidants in red spruce
20 (Picea rubens Sarg.) by following seasonal changes. They fit the level of tocopherol within the
21 needles (/g-FW) to the time of the year and found little change (fit as level = A + Bt + Ct2).
22 From this empirical fit, they found that the constant A was lower with higher levels of O3. The
23 seasonal variation coefficients, B and C, were also lower, suggesting year-long low tocopherol
24 levels. Variation with the season is not particularly surprising, given that phytochrome action
25 may be linked to tocopherol biosynthesis (Lichtenthaler, 1977). Hausladen et al. (1990) reported
26 a significant (p < 0.05) trend in the difference between the high and low level of treatment;
27 although there was no discussion of why it occurred or what it meant in relation to metabolism.
28 Their major conclusion was that the antioxidant changes due to O3 exposure may decrease cold
29 hardiness.
30 Sterols, believed to act as membrane stabilizers, have been investigated by several groups
31 with mixed results. Tomlinson and Rich (1971), who exposed common bean at 0.25 ppm for
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1 3 h, and Grunwald and Endress (1985), who exposed soybean at 0.07 ppm for 6 h for 48 days,
2 reported an increase in free sterols and a decline in esterified sterols. However, Trevathen et al.
3 (1979) exposed tobacco at 0.3 ppm for 6 h and reported opposite results. None of these
4 investigators believed that O3 had attacked the sterols directly, instead, they believed that these
5 changes involved metabolism and membrane stability. If O3 induced a metabolic shift that
6 disturbed the polar lipid to sterol balance, membrane reactions to other stressors, such as cold
7 tolerance, would certainly also be affected, perhaps detrimentally.
9
10
11
12
13
14
15
16
17
18
AX9.2.8 Role of Age and Size Influencing Response to Ozone
Clearly many changes occur with O3 exposure can be observed within hours, or perhaps
days, of the exposure. This document has argued that many of those events are connected with
wounding and elicitor-induced changes in gene expression, but those are relatively fast acting
changes (a timescale of tens of hours). Two other effects due to O3 take longer to occur and tend
to become most obvious under long periods of low-O3 concentrations. These have been linked
to senescence or some other physiological response very closely linked to senescence. These
two responses, separated by a time sequence, are shown diagrammatically in Figure AX9-15.
03
]•<—Ascorbate-<—
"First Event"
{Membrane Alteration} U-
ROS
I
Hypersensitivity Reactions —'
Accelerated
Senescence
Figure AX9-15. Linkage of senescence with hypersensitivity reactions and the first event
of O3 attack of plants.
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1 The understanding of how O3 affects long-term growth and resistance to other biotic and
2 abiotic insults in long-lived trees is unclear. Often, the conditions to which a tree is subjected to
3 in one year will affect the response of that tree in the next year. This has been called "memory
4 effect", although the term "carry-over" is preferred. In other words, a condition in an earlier
5 year sets the stage for a reaction in the next year; thereby giving a "cause-effect" scenario.
6 In perennial plant species, growth affected by a reduction in storage carbohydrates may
7 result in the limitation of growth the following year (carry-over effects) (Andersen et al., 1997).
8 Carry-over effects have been documented in the growth of tree seedlings (Hogsett et al., 1989;
9 Sasek et al., 1991; Temple et al., 1993; U.S. Environmental Protection Agency, 1996) and in
10 roots (Andersen et al., 1991; U.S. Environmental Protection Agency, 1996). Accumulation of
11 the carry-over effects over time will affect survival and reproduction. Data on the cumulative
12 effects of multiple years of O3 exposures have been, for the most part, the result of 2- to 3-year
13 seedling studies. The difficulty of experimentally exposing large trees to O3 has lead to the tacit
14 assumption that seedling response to O3 is a good predictor of large-tree response to O3 (U.S.
15 Environmental Protection Agency, 1996).
16 The carry-over effects of O3 exposures as observed in tree seedlings cited above by Hogsett
17 et al. (1989) have been termed "memory effects" by Langebartels et al. (1997) and proposed by
18 Schmieden and Wild (1995) to explain the sensitivity of spruce seedlings to frost in the winter
19 after having been exposed to O3 during the previous summer. Norway spruce (Picea abies L.)
20 exposed to 80 ppb O3 for a whole growing season, demonstrated visible injury symptoms the
21 following year when the new needle flush appeared (Langebartels et al., 1997). Additional
22 studies using Norway spruce and Scots pine (Pinus sylvestris L.) seedlings have shown similarly
23 delayed responses following O3 exposures. Carry-over symptoms were noted to develop at
24 different times of the year, depending on the species of seedling exposed: in early spring for
25 Norway spruce, and in early autumn for Scots pine (Lange et al., 1989). Visible effects of O3
26 exposures on spruce and pine may develop after a substantial delay during the "sensitive"
27 periods of the year when chlorophyll and needle loss normally occur. Norway spruce and Scots
28 pine differ in their sensitive periods because of the different needle classes normally remaining
29 on the tree (Langebartels et al., 1997).
30 Nutrient status of the tree during the overwintering phase of its life (Schmieden and Wild,
31 1995) and chronic exposure to ambient O3 (less severe with fewer peaks of very high levels)
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1 induce (1) mineral nutrient deficiency; (2) alterations of normal metabolism, including allocation
2 of carbohydrates and probably nitrogen; and (3) disturbance of normal transpiration and diurnal
3 cycling, leading to water stress. This condition, termed "Montane yellowing", appears to be
4 related to nutrient deficiencies rather than senescence (although early loss of leaves and needles
5 occurs). While generalized low nutrient concentrations may not occur within the foliage,
6 localized deficiencies may. However, they are hard to observe or prove without a great deal of
7 work involving all portions of a tree and without a general hypothesis of what is occurring.
8
9 AX9.2.9 Summary
10 As the understanding of wounding responses of plants and more genome details and varied
11 plant mutants become available, the cellular and physiological responses of plants to O3
12 exposures are slowly becoming clearer. However, more studies are needed on a larger variety of
13 species. Nevertheless, several key findings and conclusions can be highlighted:
14 (1) The entrance of O3 into the leaf through the stomata remains the critical step in O3
sensitivity. Not only does O3 modify the opening of the stomata, usually closing it
partially, but O3 also appears to alter the response of stomata to other stressful
situations, including a lowering of water potential and ABA responses. The
concentration of O3 within the leaf is not the same as the external concentration due
to reactions within the leaf, but it is not "zero".
15 (2) The initial reactions of O3 within the leaf are still unclear, but the involvement of
H2O2 is clearly indicated. The detection of possible products by EPR spectroscopy
has progressed, but has not reached the point where any products can be identified.
Nonetheless, reaction of O3 (or its product) with ascorbate and possibly other
antioxidants present in the apoplastic space of the mesophyll cells is clear and
serves to lower the amount of O3 or product available to alter the plasma membrane
of the cells.
16 (3) The initial sites of membrane reactions seem to involve transport properties and,
possibly, the external signal transducer molecules. The alteration and mechanism of
the alteration of the varied carriers of K+and Ca2+ is far from clear, but it would
seem that one of the primary triggers of O3-induced cell responses is a change in
internal Ca2+ levels.
17 (4) The primary set of metabolic reactions that O3 triggers now clearly includes those
typical of "wounding" responses generated by cutting of the leaf or by
pathogen/insect attack. Again, this seems to be due to a rise in cytoplasmic Ca2+
levels. Ethylene release and alteration of peroxidases and PAL activities, as well as
activation of many wound-derived genes, seem to be linked to some of the primary
reactions.
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1 (5) The alteration of normal metabolism due to wounding has effects outside of the
cytoplasm. What effects are due to the "spreading of the problem" to other cellular
organelles is less clear. One of the secondary reactions is linked to an activation of
a senescence response. The loss of Rubisco and its messenger RNA is linked to an
early senescence or a speeding up of normal development leading to senescence.
The loss of photosynthetic capacity is linked to the lowered productivity of plants,
and problems with efficient translocation are indicated, although photosynthesis and
translocation still occur at a reasonable rate. The loss of productivity is not yet
clearly explained.
2 It is important to note that the dramatic strides in understanding the genetic makeup of
3 plants, gene control, and signal transduction/control over the last few years will likely accelerate
4 in the future. That understanding will translate into better models of the hypotheses listed above
5 and into more detailed schemes of how O3 alters much of basic plant metabolism. Thus, while
6 understanding of how O3 interacts with the plant at a cellular level has dramatically improved,
7 the translation of those mechanisms into how O3 is involved with altered cell metabolism, with
8 whole plant productivity, and with other physiological facts remain to be more fully elucidated.
9
10
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1 AX9.3 MODIFICATION OF FUNCTIONAL AND GROWTH RESPONSES
2 AX9.3.1 Introduction
3 The responses of plants to any air pollutant may be significantly influenced by a wide
4 range of biological, chemical, and physical factors. A plant's genetic makeup is an important
5 inherent biological determinant of its response, but response can also be modified by other
6 biological agents such as disease-causing organisms, insects and other pests, and by other higher
7 plant species with which it may be competing for resources. Chemical factors that may
8 influence response to an air pollutant range from mineral nutrients obtained from the soil to other
9 air pollutants and agricultural chemicals. Physical factors that may influence response include
10 light, temperature and the availability of moisture, which are components of climate and climate
11 change.
12 Some environmental factors can be controlled, to some degree, by man, while others
13 cannot. The biological factors (e.g., pests, diseases, symbioses, competition) are partly
14 controllable in agriculture but much less so (if at all) in natural ecosystems. It is possible to
15 control agricultural soil fertility and the use of agricultural chemicals, as well as to exercise some
16 control over the supply of water and airborne chemical factors. In contrast, the physical factors
17 (e.g., light and temperature) are uncontrolled in the field even though they may be controllable in
18 specialized situations such as greenhouses or shade houses. Although light and temperature are
19 components of climate, they are initially reviewed as individual physical factors, even though
20 temperature effects are revisited to some extent in the discussion of interactions with climate
21 change.
22 The impacts of these various factors on plant response to O3 and other oxidants were
23 extensively reviewed in the 1996 O3 AQCD (U.S. Environmental Protection Agency, 1996).
24 It was noted in that document that, since any combination of these factors may come into play at
25 some time in a plant's life history, "response will be dictated by the plant's present and past
26 environmental milieu, which also includes the temporal pattern of exposure and the plant's stage
27 of development." That document also stressed that both the impact of environmental factors on
28 response to oxidants and the corollary effects of oxidants on responses to environmental factors
29 have to be considered in determining the impact of oxidants on vegetation in the field. The
30 variability observed in plant responses to defined exposures to O3, particularly under field
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1 conditions, is a consequence of the influences of genetics and the range of environmental
2 variables.
3 In view of the large number of factors to be considered, this section focuses mainly on
4 situations in which there is clear evidence that environmental factors truly interact with oxidant
5 effects, i.e., they magnify or diminish the impact of O3 and are not merely additive to it.
6 Conversely, it will cover situations where O3 acts synergistically or antagonistically, but not
7 additively, with effects induced by other factors. It will also emphasize those interactions as a
8 result of which overall plant growth, development, and yield are adversely affected, rather than
9 the details of interactions at the mechanistic level, unless the latter are deemed to be essential to
10 an understanding of larger-scale effects.
11 Few studies reported since the 1996 document have systematically investigated
12 quantitative responses to O3 exposure concurrent with other variables. Although the 1996
13 document cited almost 300 references pertaining to environmental interactions, and the present
14 review cites more than 350 new references, the bulk of the recently published work has
15 continued to be determined by the specific, and frequently narrowly focused, interests of
16 individual researchers or groups. Hence, the new findings are scattered and far from uniformly
17 distributed among the various subtopics. In some instances, little or no new research has been
18 published that adds to our understanding since the 1996 document. In such cases, the present
19 review is, therefore, restricted to summarizing the understanding that was current in 1996.
20 A few reviews have appeared since the early 1990s dealing with various environmental
21 interactions, and these are cited in relevant sections below. More general recent reviews are
22 those of Wellburn (1994); multi-authored volumes edited by Alscher and Wellburn, 1994; Yunus
23 and Iqbal, 1996; De Kok and Stulen, 1998; and Bell and Treshow, 2002); and reports by the
24 United Nations Environment Programme (UNEP, 1999) and the Intergovernmental Panel on
25 Climate Change (IPCC, 2001). Several biotic and abiotic interactions involving forest trees are
26 discussed in the review by Johnson et al. (1996b).
27 Although many reports have provided quantitative information on interactive effects,
28 in most cases the information simply describes a specific situation involving only two or three
29 levels of a variable. While this may be adequate to provide statistical information about the
30 existence of interactions with environmental factors, it does not permit the development of
31 response surfaces or models to show the form that any influence of such factors might take
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1 on O3 exposure-response relationships or how O3 might quantitatively influence responses to the
2 factors in question. This, together with the fragmented information available on the effects of
3 most factors, has contributed to the relative lack of development of simulation models of
4 oxidant-environmental factor interactions. Yet, as noted by Taylor et al. (1994), the large
5 number of variables constrains the assessment of pollution effects by experimentation alone.
6 The only alternative is to use mathematical models to attempt to predict the outcome of
7 different O3 and environmental factor scenarios, building up their complexity in stages.
8 The few models thus far used to investigate O3 stress have been adapted from existing process
9 models of crop or tree growth that include limited numbers of physical or chemical variables
10 such as temperature, soil water stress, or nutrient deficiency. Taylor et al. (1994) provided a
11 listing of several simulation models developed for trees at the individual, stand, and regional
12 levels; these and many other models have been critically reviewed by Kickert and Krupa (1991)
13 and Kickert et al. (1999). However, regardless of whether such models are descriptive/empirical
14 or process/mechanistic, their outputs will always be associated with varying degrees of
15 uncertainty and require validation against observable responses wherever possible. Kickert et al.
16 (1999) also noted that very few of the models that have been described provide risk assessments
17 that address likelihood, in contrast to consequence assessments that address the magnitudes of
18 effects. Thus, even though capable simulation models of plant response to O3 involving complex
19 mixes of many biological, physical, and chemical factors may be out of reach at the present time,
20 the use of newer mathematical approaches such as artificial neural networks (ANNs) has
21 enabled insightful analyses to be performed in several field studies involving numerous
22 micrometeorological and other environmental variables (e.g., Balls et al., 1996; Mills et al.,
23 2000).
24 Because the ensuing subsections deal with studies of O3 interactions involving an
25 extremely wide array of biological, physical, and chemical factors in the plant's environment, it
26 is inevitable that many different exposure facilities and regimes have been used in these studies.
27 To provide specific information regarding the O3 exposure concentrations, profiles, hours and
28 days of exposure (as well as the types of systems and facilities used for the exposures) would
29 add a wealth of detail that would do little to assist our understanding of the roles of environment
30 factors in modifying the impact of O3 on vegetation or to facilitate our ability to estimate the
31 magnitudes of any such modifications. Thus, only experiments in which the exposure levels and
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1 regimes were within the bounds of ambient experience in North America are discussed in the
2 ensuing subsections, regardless of the type of exposure profile used. The cutoffs used have
3 been -200 ppb for peak hourly concentrations or for short-term exposures, -100 ppb for daytime
4 means involving prolonged exposures for several hours, or a doubling of ambient levels in cases
5 in which enriched exposure levels were a function of ambient levels. Actual details of the
6 exposure regimes and conditions can, of course, be obtained from the original references but are
7 only stated here when any distinction is required between the effects of different exposure levels.
8 Hence, it should be understood that ensuing statements such as ". . .it was found that O3
9 caused. . ." should always be read as ". . .it was found that exposures to O3 [within the range
10 of those that have been measured in ambient air] caused."
11
12 AX9.3.2 Genetics
13 The response of individual plants to O3is affected by several factors, including the
14 environment in which it is growing, competition with neighboring plants, ontogeny, and
15 genetics. This section examines the role of genetics in plant response to O3. In addition, major
16 knowledge gaps in the understanding of genetic aspects of O3 response are pointed out.
17 It is well known that species vary greatly in their responsiveness to O3 (U.S. Environmental
18 Protection Agency, 1996). This again has been recently demonstrated for grassland species
19 (Bungener et al., 1999b; Bungener et al., 1999a; Franzaring et al., 2002; Nussbaum et al., 2000a;
20 Pleijel and Danielsson, 1997; Warwick and Taylor, 1995), wild herbaceous plants (Bergmann
21 et al., 1999; Danielsson et al., 1999), agricultural crops (Benton et al., 2000; Elagoz and
22 Manning, 2002; Fumagalli et al., 2001; Heagle and Stefanski, 2000; Kollner and Krause, 2003;
23 Nali et al., 2002; Nussbaum et al., 2000b; Ollerenshaw et al., 1999; Postiglione et al., 2000;
24 Renaud et al., 1997), horticultural shrubs and trees (Findley et al., 1997; Hormaza et al., 1996),
25 and forest trees (Bortier et al., 2000a; Guidi et al., 2001; Landolt et al., 2000; Matsumura, 2001;
26 Momen et al., 2002; Nali et al., 2002; Oksanen and Rousi, 2001; Paakkonen et al., 1997; Pell
27 et al., 1999; Saitanis and Karandinos, 2001; Volin et al., 1998; Zhang et al., 2001). These
28 studies have shown a wide range of responses to O3, from growth stimulation by a few species
29 such as Festuca ovina L. (Pleijel and Danielsson, 1997) and Silene dioica and Chrysanthemum
30 leucanthemum (Bungener et al., 1999b; Bungener et al., 1999a) to significant growth reduction,
31 depending on environmental conditions and exposure dose.
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1 While determining the explanation for differences in species sensitivity to O3 remains one
2 of the challenges facing plant biologists (Pell et al., 1999), a number of hypotheses have been
3 suggested. Reich (1987) proposed that variation in O3 sensitivity could be explained by variation
4 in total uptake of the gas. Others have suggested that (1) fast-growing species are more sensitive
5 than slower-growing ones (Bortier et al., 2000b), (2) overall O3 sensitivity may be closely linked
6 to root responses to O3 (Warwick and Taylor, 1995), or (3) the relative ability of species to
7 detoxify O3-generated reactive oxygen free radicals may determine O3 sensitivity (Alscher et al.,
8 1997; Pell et al., 1999). Volin et al. (1998) suggested that the relative rate of stomatal
9 conductance and the photosynthesis rate at a given conductance both contribute strongly to
10 determining a species sensitivity to O3. Likely, there is more than one mechanism determining
11 sensitivity, even in a single species.
12 Within a given species, individual genotypes or populations can also vary significantly
13 in O3 sensitivity (U.S. Environmental Protection Agency, 1996). For example, the intraspecific
14 variation in O3 sensitivity was a factor of two for Phleum pratense (Danielsson et al., 1999) and
15 Trifolium repens L. (Postiglione et al., 2000). A similar range of intraspecific variations in O3
16 responses was demonstrated for clonal differences in Betulapendula by Paakkonen et al. (1997)
17 and Prunus serotina (Lee et al., 2002). These examples of wide ranges within species responses
18 suffice to show that caution should be taken when ranking species categorically as having an
19 absolute degree of O3 sensitivity (Davison and Barnes, 1998).
20
21 AX9.3.2.1 Genetic Basis of Ozone Sensitivity
22 Plant response to O3 is determined by genes that are directly related to oxidant stress and to
23 an unknown number of genes that are not specifically related to oxidants. The latter includes
24 genes that control leaf and cell wall thickness, stomatal conductance, and the internal
25 architecture of the air spaces. Although there is currently a great emphasis on individual
26 antioxidants that can be manipulated by molecular methods, the challenge is to determine the
27 relative contributions of all of the components to plant response and to understand the interplay
28 between them. Recent studies using molecular biological tools are beginning to increase the
29 understanding of O3 toxicity and differences in O3 sensitivity.
30 While much of the research in developing the understanding of O3 responses has been
31 correlative in nature, recent studies with transgenic plants have begun to positively verify the
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1 role of various genes and gene products in O3 tolerance. The finding that the overexpression of
2 MnSOD in transgenic tobacco plant chloroplasts increased O3 tolerance (Van Camp et al., 1994)
3 provided the first definitive proof of antioxidants key role in O3 tolerance. Subsequently,
4 Broadbent et al. (1995) showed that the simultaneous overexpression of pea glutathione
5 reductase in both chloroplasts and mitochondria enhanced O3 tolerance in transgenic tobacco.
6 Similarly, increased O3 tolerance to O3-induced foliar necrosis was shown for transgenic tobacco
7 plants overexpressing the cytosolic Cu/Zn-SOD gene (Pitcher and Zilinskas, 1996). Transgenic
8 tobacco plants expressing antisense RNA for cytosolic ascorbate peroxidase, which reduces
9 ascorbate peroxidase production, showed increased susceptibility to O3 injury, suggesting a key
10 role in O3 tolerance for the antioxidant ascorbate peroxidase (Orvar and Ellis, 1997).
11 The consensus among molecular studies of O3 sensitivity is pointing to O3 as triggering
12 salicylic acid, ethylene, and jasmonic acid production and that the signaling of these molecules
13 determines, in some cases, the O3 susceptibility of plants (DeCaria et al., 2000; Moeder et al.,
14 2002; Nunn et al., 2002; Overmyer et al., 2000; Rao and Davis, 1999; Tamaoki et al., 2003;
15 Vahala et al., 2003a,b). Increased levels of jasmonic acid production in O3-tolerant compared to
16 O3-sensitive plants has been shown for Arabidopsis (Overmyer et al., 2000) and Populus (Koch
17 et al., 1998, 2000). Blockage of ethylene production by using antisense methods with
18 1-aminocyclopropane-l-carboxylate (ACC) synthase and ACC oxidase suggest strongly that
19 ethylene synthesis and perception are required for H2O2 production and cell death following O3
20 exposure of Lycopersicon esculentum (Moeder et al., 2002). Ethylene signaling may have
21 multiple roles in O3 tolerance determination as was demonstrated recently by Vahala et al.
22 (2003a,b) who found that, in Populus tremula x P. tremuloides hybrid clones differing in O3
23 sensitivity, ethylene accelerated leaf senescence in sensitive plants under low O3, but under acute
24 O3, ethylene seemed to be required for protection from cell death.
25 While changing the expression of single antioxidant genes has proven very useful in
26 identifying possible mechanisms of O3 sensitivity and tolerance (Kuzniak, 2002), it should be
27 noted that increased O3 tolerance has not been shown in some studies of transgenic plants with
28 enhanced antioxidant production (Strohm et al., 1999; Strohm et al., 2002; Torsethaugen et al.,
29 1997). Clearly, ethylene production plays a role in O3 sensitivity, but the roles of various
30 antioxidants in O3 tolerance regulation are yet to be fully elucidated (Wellburn and Wellburn,
31 1996). It is unlikely that single genes are responsible for O3 tolerance responses, except in rare
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1 exceptions (Engle and Gabelman, 1966). Regulation of stomatal opening and leaf structure
2 (Elagoz and Manning, 2002; Fujita et al., 1992) are likely to play key roles in O3 tolerance in
3 plants. Newly developing opportunities to examine simultaneous regulation of larger numbers
4 of genes are also likely to yield more clarification of the genes controlling O3 tolerance (Desikan
5 et al., 2001; Matsuyama et al., 2002).
6 Attempts to demonstrate conclusive changes in antioxidant and protective pigments for O3
7 sensitive and tolerant mature trees growing in the field have largely been unsuccessful (Tausz
8 et al., 1999a,b). However, evidence for antioxidant expression differences contributing to
9 differences in O3 sensitivity of 4-year-old Populus tremuloides trees has been found (Wustman
10 etal., 2001).
11
12 AX9.3.3 Environmental Biological Factors
13 The biological factors within the plant's environment that may directly or indirectly
14 influence its response to O3 in a positive or negative manner encompass insects and other animal
15 pests, diseases, weeds, and other competing plant species. Although such interactions are
16 ecological in nature, those involving individual pests, plant pathogens, or weeds, or agricultural
17 crop or forest tree species are considered in this section. More complex ecological interspecies
18 interactions are dealt with in Section AX9.5.
19 The different types of biological factors are dealt with separately, as in the 1996 O3 AQCD
20 (U.S. Environmental Protection Agency, 1996). Still, it is important to recognize certain general
21 features of relationships of plants with the biological components in their environments:
22 • Successful infestation or infection involves complex interactions among the target or host
species, the causal organism and environmental factors.
23 • Infestations and infections may co-occur.
24 • The successful development and spread of a pest, pathogen, or weed require favorable
environmental factors.
25 • Significant losses to crops and forest trees result from pests and pathogens.
26 • Significant losses to crops and seedling trees result from weed competition.
27 Ozone and other photochemical oxidants may influence the severity of a disease or
28 infestation by a pest or weed, either by direct effects on the causal species, or indirectly by
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1 affecting the host, or both. In addition, the interaction between O3, a plant, and a pest, pathogen,
2 or weed may influence the response of the target host species to O3. A perceptive overview of
3 the possible interactions of O3-exposure with insect pests and fungal diseases has been provided
4 by Jones et al. (1994), based on a model system involving two insects and two pathogens
5 affecting cottonwood (Populus deltoides). Their study also included effects on the
6 decomposition of leaf litter.
7 In contrast to detrimental biological interactions, there are mutually beneficial relationships
8 or symbioses involving higher plants and bacteria or fungi. These include (1) the nitrogen-fixing
9 species Rhizobium and Frankia that nodulate the roots of legumes and alder and (2) the
10 mycorrhizae that infect the roots of many crop and tree species, all of which may be affected by
11 exposure of the host plants to O3.
12 In addition to the interactions involving animal pests, O3 may also have indirect effects on
13 higher herbivorous animals, e.g., livestock, due to O3-induced changes in feed quality.
14
15 AX9.3.3.1 Oxidant-Plant-Insect Interactions
16 The 1996 O3 AQCD (U.S. Environmental Protection Agency, 1996) stressed the variability
17 in the reported effects of O3 on host plant-insect interactions. Since relatively few plant-insect
18 systems have been studied, few consistent patterns of response have emerged, as noted in other
19 reviews such as those of Colls and Unsworth (1992), Heliovaara and Vaisanen (1993), Whittaker
20 (1994), Docherty et al. (1997), and, most recently, Fluckiger et al. (2002).
21 None of the studies reported in the past decade have clarified the situation in terms of
22 clearly consistent effects. A 1997 review by Docherty et al. (1997), for example, examined
23 17 reports of studies of aphid species on a range of hosts and classed the O3 effects on aphid
24 performance as 35% positive, 41% negative, and 24% showing no significant effect.
25 A tabulation of 19 studies by Fluckiger et al. (2002) gave the corresponding figures: 42%, 21%,
26 and 37%.
27 Other recent studies with the aphids Schizolachnuspineti and Cinarapinea on Scots pine
28 (Pinus sylvestris) and Cinarapilicornis on Norway spruce (Picea abies) have also yielded
29 variable results, but suggested that O3 enhances aphid density on pine and aphid performance on
30 spruce (Holopainen et al., 1997; Kainulainen et al., 2000a). In an earlier study with
31 Schizolachnus pineti on Scots pine, Kainulainen et al. (1994) had observed no significant effects
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1 of O3-treatment on aphid performance. However, more recent observations of long-term effects
2 on aphid populations on aspen (Populus tremuloides) exposed to O3 in a FACE system revealed
3 that O3 significantly increased aphid populations while decreasing the populations of predatory
4 insects (Percy et al., 2002).
5 The observations of Brown et al. (1993) and Jackson (1995) led Whittaker (1994) and
6 Brown (1995) to suggest that aphid response was dependent on ambient temperature as well as
7 the dynamics of O3 exposure and that growth tended to be stimulated with maximum
8 temperatures below -20 °C but was reduced at higher temperatures. The present situation with
9 plant-aphid responses, therefore, remains confused and, although numerous suggestions have
10 been offered to explain specific findings, they are difficult to assemble into a coherent picture.
11 Variability has also been found with the interactions involving chewing insects. For
12 example, Lindroth et al. (1993) reported a small negative O3 effect (8% reduction) on the growth
13 of gypsy moth larvae (Limantra dispar) on hybrid poplar (Populus tristis x P. balsamifera) but
14 no effect when growing on sugar maple (Acer saccharum). Ozone exposure reduced the growth
15 rate of the larvae of the bug Lygus rugulipennis on Scots pine, but enhanced the growth of larvae
16 of the sawfly Gilpiniapallida (Manninen et al., 2000). Costa et al. (2001) observed no
17 significant O3 effects on the growth and fecundity of the Colorado potato beetle (Leptinotarsa
18 decemlincatd) on potato (Solanum tuberosum L.) in greenhouse and field experiments.
19 Fortin et al. (1997), in a 2-year study of the forest tent caterpillar (Malacosoma disstrid) on
20 sugar maple, observed that O3 exposure increased the growth rate of female larvae in only one
21 year; fourth- and fifth-instar larvae also showed a feeding preference for treated foliage in that
22 year. However, studies based on open-air exposures of aspen indicated O3-enhanced growth of
23 M. disstria in terms of pupal weight (Percy et al., 2002) and larval performance (Kopper and
24 Lindroth, 2003). Jackson et al. (2000) observed inconsistency in studies on the larva of the
25 tobacco hornworm (Manduca sexto) on tobacco (Nicotiana tabacum). In one year, feeding
26 on O3-treated foliage resulted in significantly greater larval weight, whereas the increase was not
27 statistically significant in a second year although survival was increased. Also, oviposition by
28 hornworm moths was increased if ambient O3 levels were increased by 70% and returned to
29 normal in ambient O3 levels (Jackson et al., 1999).
30 Studies of the two-spotted spider mite (Tetranychus urticae) on white clover (Trifolium
31 repens) and peanut (Arachis hypogeae) by Heagle et al. (1994) and Hummel et al. (1998)
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1 showed that O3-exposure stimulated mite populations on an O3-sensitive clover clone and on
2 peanut. The lack of significant effects on mites on the O3-resistant clover clone suggests that the
3 responses were host-mediated.
4 There, therefore, appears to be a clearer indication of the likelihood that increased chewing
5 insect and mite performance will result from O3-induced changes in the host plant. However but
6 negative effects continue to be reported, indicating that the response is probably also being
7 determined, in part, by other environmental, genetic, or temporal variables.
8 Reported O3-induced enhancement of attack by bark beetles (Dendroctonus brevicomis) on
9 Ponderosa pine (Pinusponderosd) has been suggested by Dahlsten et al. (1997) to be due to
10 greater brood development on injured trees, possibly related to decreased numbers of predators
11 and parasitoids. This view gains some support from the observation that O3 exposure adversely
12 affected the searching behavior of the parasitoid Asobara tabida for larvae ofDrosophila
13 subobscura which led to fewer parasitized fly larvae (Gate et al., 1995). Such observations
14 reveal another level of complexity in the O3-plant-insect interrelationship: O3 may reduce the
15 effectiveness of the natural control of insect pests. The phenomenon is probably related to
16 effects on olfactory cues, as it was shown by Arndt (1995) that O3 can affect fly behavior by
17 modifying the pheromones involved in fly aggregation.
18 These reports focus on the direct or indirect effects on the insect or mite feeding on foliage
19 previously or currently exposed to O3. They provide little, if any, information on the host plant
20 effects other than qualitative references to the injury caused by the O3 exposure. Enhanced pest
21 development will ultimately lead to increased adverse effects on the hosts in the long term, but
22 the only report of an O3-plant-insect interaction directly affecting the host plant in the short term
23 still appears to be that of Rosen and Runeckles (1976). They found that infestation by the
24 greenhouse whitefly (Trialeurodes vaporariorum) sensitized bean plants (Phaseolus vulgaris) to
25 injury by otherwise noninjurious low levels of O3, leading to premature senescence of the leaves.
26 The overall picture regarding possible O3 effects on plant-insect relations, therefore,
27 continues to be far from clear. Only a few of the very large number of such interactions that
28 may affect crops, forest trees, and other natural vegetation have been studied. The trend
29 suggested in the 1996 O3 AQCD (U.S. Environmental Protection Agency, 1996) that O3 may
30 enhance insect attack has received some support from a few recent studies. However, the
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1 variability noted in most of the studies makes it clear that we are still far from being able to
2 predict the nature of any particular O3-plant-insect interaction or its magnitude or severity.
3
4 AX9.3.3.2 Oxidant-Plant-Pathogen Interactions
5 Plant diseases are caused by pathogenic organisms, e.g., fungi, bacteria, mycoplasmas,
6 viruses, and nematodes. Ozone impacts on disease are briefly discussed in earlier reviews by
7 Ayres (1991) and Colls and Unsworth (1992) and, more recently, by Fluckiger et al. (2002).
8 Biotic interactions with forest trees have been reviewed by Chappelka and Samuelson (1998);
9 Sandermann (1996) and Schraudner et al. (1996) have summarized molecular similarities and
10 interrelationships between necrotic O3 injury to leaves and pathogen attack. A few recent
11 publications have added to our fragmented knowledge of O3-plant-disease interactions and the
12 mechanisms involved, but there appear to have been no reports to date of studies involving
13 mycoplasmal diseases.
14 The 1996 O3 AQCD (U.S. Environmental Protection Agency, 1996) noted the concept put
15 forward by Dowding, (1988) "that pathogens and pests which can benefit from damaged host
16 cells and from disordered transport mechanisms are enhanced by pollution insult to their hosts,
17 whereas those pathogens and other symbionts which require a healthy mature host for successful
18 invasion are depressed by pollutant stress to their host." The pathogens of the first type are
19 mostly facultative necrotrophic fungal parasites, whereas the second type are largely obligate
20 biotrophic fungi, bacteria, and viruses. Based on this distinction, the majority of the cases cited
21 in the 1996 document supported Dowding's (1988) view, as have several more recent studies
22 summarized in Table AX9-11. However, there are also some contradictions.
23 Most investigations have focused on the incidence and development of disease on plants
24 previously or concurrently exposed to O3, rather than on the corollary effect of disease on the
25 response to O3. In all of the studies of facultative pathogens and the nematode studies, exposure
26 to O3 tended to result in increased disease severity through increased spore germination or
27 increased fungal growth and development; although in the case of grey mold (Botrytis cinered)
28 on kidney bean (Phaseolus vulgaris), this was only observed on an O3-sensitive cultivar
29 inoculated with conidia (Tonneijck, 1994). After mycelial inoculation, O3 exposure reduced
30 disease development in the O3-sensitive cultivar, but no satisfactory explanation was offered to
31 account for the difference in response. With poplar leaf spot, Marssonina tremulae., on hybrid
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Table AX9-11. Interactions Involving O3 and Plant Pathogens
_-
C/3
to
o
\D
i
^J
u
l>
H
1
O
o
o
H
/O
O
1
w
0
n
H
w
Host Plant
Obligate Biotrophs
Bottle gourd (Lagenaria siceraria)
Cucumber (Cucumis sativa)
Pea (Pisum sativum)
Aspen (Populus tremuloides)
Hybrid poplar (Populus trichocarpa
x balsamifera)
Broad bean (Viciafaba)
Facultative Necrotrophs
Kidney bean (Phaseolus vulgaris)
Scots pine (Pinus sylvestris)
Pathogen
Powdery mildew
(Sphaerotheca fulginea)
Powdery mildew
(Sphaerotheca fulginea;
Powdery mildew
(Erysiphe polygoni)
Leaf rust (Melampsora
medusae f. sp.
tremuloidae)
Leaf rust (Melampsora
larici-populina orM
allii-populina)
Bean rust (Uromyces
viciae-fabae)
Grey mold (Botrytis
cinerea)
Grey mold (Botrytis
cinerea)
White mold (Sclerotinia
sclerotiorum)
Annosus root and butt rot
(Heterobasidion annosum)
Effect of O3 on Disease
Increased in 50ppb O3;
decreased in 100+ppb
Increased in 50ppb O3;
decreased in 100+ppb
Decreased infection
Increased severity
Increased infection and
severity
Not reported
Increased from conidia on O3-
sensitive cultivar; decreased
from mycelium
Increased infection
Increased infection
Increased development*
Effect of Disease on O3 Response
Decreased; partial protection
Synergistic increase in 50ppb O3;
antagonistic decrease in 100+ppb;
partial protection
Decreased; partial protection
Not reported
Increased sensitivity (synergistic)
Decreased; partial protection
Not reported
Not reported
Not reported
Not reported
Reference
Khan and
Khan(1998a)
Khan and
Khan (1999)
Rusch and
Laurence
(1993)
Karnosky
et al. (2002)
Beare et al.
(1999a)
Lorenzini
et al. (1994)
Tonneijck
(1994)
Tonneijck and
Leone (1993)
Tonneijck and
Leone (1993)
Bonello et al.
(1993)
-------
Table AX9-11 (cont'd). Interactions Involving O3 and Plant Pathogens
1—
to
o
o
VO
1
oo
o
H
6
O
0
H
O
O
0
o
H
W
Host Plant
Facultative Necrotrophs (cont'd)
Loblolly pine (Pinus taeda)
Hybrid poplar (Populus deltoides x
nigra)
Hybrid poplar (Populus trichocarpa
x balsamifera)
Wheat (Triticum aestivum)
Nematodes
Tomato (Lycopersicon esculentum)
* Increase completely countered by
Pathogen
Pitch canker (Fusarium
subglutinans)
Canker (Septoria musiva
[=Mycosphaerella
populinum})
Leaf spot (Marssonina
tremulae)
Blotch (Septoria
nodorum)
Tan spot (Pyrenophora
tritici-repentis)
Root-knot nematode
(Meloidogyne incognita)
Effect of O3 on Disease Effect of Disease on O3 Response
Increased development Increased sensitivity
Increased incidence Not reported
Increased spore germination Not reported
and lesion growth after
lOOppb O3 (30 days);
decreased germination after
200ppb (15 days)
Increased infection Not reported
Increased infection of Not reported
disease-susceptible genotypes
Increased development Increased foliar injury; reduced plant
growth (synergistic)
Reference
Carey and
Kelley (1994)
Woodbury
et al. (1994)
Beare et al.
(1999b)
Tiedemann
and Firsching
(1993)
Sah et al.
(1993)
Khan and
Khan (1997);
Khan and
Khan(1998a)
mycorrhizae (Hebeloma crustuliniforme).
-------
1 poplar (Populus trichocarpa x balsamifera), low level exposures to O3 increased disease (in
2 agreement with theory) but higher levels (200 ppb, 8 h per day for 15 days) reduced conidial
3 germination (Beare et al., 1999b).
4 The situation with obligate biotrophic pathogens is less consistent. The effects on powdery
5 mildew (Sphaerotheca fulginea) on both bottle gourd (Lagenaria siceraria) and cucumber
6 (Cucumis sativa) resembled the situation with the necrotrophic poplar leaf spot disease, since
7 low O3 exposures increased disease severity (in disagreement with theory), although higher
8 levels decreased it. The decreased infection in the pea-powdery mildew (Erysiphe polygoni)
9 situation agrees with theory, but the situations with leaf rust (Melampsora sp.) on hybrid poplar
10 or aspen do not. However, these reports are in contrast to earlier reports included in the 1996 O3
11 AQCD (U.S. Environmental Protection Agency, 1996) of observations with other species of
12 Erysiphe (Tiedemann et al., 1991) and Melampsora (Coleman et al., 1987). In contrast to the
13 recent report of a synergism with Melampsora on poplar, infections caused by the other
14 biotrophs (Sphaerotheca, Erysiphe, Uromyces spp.) reduced the severity of injury caused by O3
15 (in agreement with numerous earlier reports), but only at high O3 exposures in the case of
16 Sphaerotheca on cucumber. At low exposure levels, the disease and O3 exposure acted
17 synergistically. The only other recent observations of such disease-related synergisms are the
18 tomato-nematode reports of Khan and Khan (Khan and Khan, 1997, 1998b).
19 It is, therefore, clear that the type and magnitude of exposure to O3 plays an important role
20 in determining both the responses of both the disease organism and the host.
21 No recent studies involving interactions between O3 and bacterial diseases appear to have
22 been reported since 1996. With regard to viruses, a laboratory study by Yalpani et al. (1994)
23 added to several reports of O3 decreasing the severity of tobacco mosaic virus infection of
24 tobacco; and Jimenez et al. (2001) reported that previous O3 exposure resulted in increased
25 adverse effects on tomato yield attributed to several viral diseases.
26 Similarities between the sensitivities of different cultivars or clones to O3 and to specific
27 diseases have been noted. For example, Sah et al. (1993) found that the severity of injury caused
28 by tar spot and standard O3 exposures of 12 wheat (Triticum aestivum L.) cultivars were closely
29 correlated (R2 = 0.986). Such similarities appear to have a mechanistic basis, as several studies
30 have noted similarities in the molecular and biochemical changes that occur in plants infected
31 with pathogens and in O3 exposed plants. Schraudner et al. (1992), Ernst et al. (1992), Eckey-
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1 Kaltenbach et al. (1994a,b), Yalpani et al. (1994), and Bahl et al. (1995) have presented evidence
2 that O3 exposures result in responses such as increased levels of salicylic acid, the signaling
3 agent for increased induced resistance to pathogens. This, in turn, leads to the activation of the
4 genes that encode defense proteins, including the so-called pathogenesis-related proteins. The
5 induction of such proteins might account for the decreased infection with Sphaerotheca and
6 Melampsora at higher O3 exposures but does not account for increased infections seen at lower
7 exposure levels. The issue is discussed more fully by Sandermann (1996) and Schraudner et al.
8 (1996). More recently Sandermann (2000) has extended the theory relating O3 exposure and
9 disease by suggesting that, because of O3 "memory effects" in affected host plants that may
10 persist over weeks or months, analysis for various induced biomarkers of gene activation may
11 provide a useful tool for improving our ability to predict the outcome of O3-plant-pathogen
12 interactions.
13 There have been no reports of O3 studies with mixed infections by pathogens, but the
14 complete suppression of Heterobasidion butt and root rot of Scots pine by the mycorrhizal
15 symbiont Hebeloma crustuliniforme indicates the possibility of interactions involving more than
16 one fungus (see Section AX9.3.4.3.3 below).
17 In summary, our understanding of oxidant-plant-disease interactions is far from complete.
18 However, a combined tabulation of the evidence presented in the 1996 O3 AQCD (U.S.
19 Environmental Protection Agency, 1996) and that noted in Table AX9-11 leads to the following
20 summary of O3 effects on plant diseases and corollary effects of infection on plant response
21 to O3, as indicated by the number of studies showing increases or decreases in disease or
22 susceptibility.
23 For obligate biotrophic fungi, bacteria, nematodes:
24 O3 increased disease: 9 Increased susceptibility to O3: 3
25 O3 decreased disease: 15 Decreased susceptibility to O3: 9
26 For facultative necrotrophic fungi:
27 O3 increased disease: 25 Increased susceptibility to O3: 2
28 O3 decreased disease: 3 Decreased susceptibility to O3: 4
29 Thus, although O3 may reduce the severity, but not the incidence, of some of the diseases caused
30 by the obligate pathogens, the evidence overall indicates that with most diseases, their severity is
31 more likely to be increased by O3 than not. However, the actual consequences will be specific to
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1 the disease and level of exposure, and, most importantly, will be determined by environmental
2 suitability and epidemiological requirements for disease to develop. Conversely, some evidence
3 suggests that infection by obligate pathogens may confer some degree of "protection" against O3,
4 a dubious benefit from the plant's point of view.
5
6 AX9.3.3.3 Oxidant-Plant-Symbiont Interactions
7 No further studies have appeared regarding O3 effects on the important bacterial symbiont
8 of legumes, Rhizobium, since those summarized in the 1996 O3 AQCD (U.S. Environmental
9 Protection Agency, 1996). Hence, our present understanding is that, although relatively high
10 levels of exposure (>200 ppb) can result in severe (>40%) reductions in nodulation (and
11 therefore nitrogen-fixation) on soybean roots, lower O3 exposures may cause lesser reductions in
12 nitrogen fixation. However, the data are inadequate to attempt to define any quantitative
13 exposure-response relationships.
14 There have been a few recent reports on O3-plant-mycorrhizae interrelationships. These
15 have mostly involved seedlings of coniferous tree species. A transient O3-induced stimulation of
16 mycorrhiza on Scots pine roots reported by Kasurinen et al. (1999) was not observed in a later
17 study by Kainulainen et al. (2000b). Studies of the mycorrhiza Paxillus involutus on birch
18 (Betulapenduld) seedlings showed that, although O3 reduced mycorrhizal growth rate, it led to
19 greater extension growth which in turn resulted in greater mycorrhizal infection of neighboring
20 Aleppo pine (Pinus halepensis) seedlings (Kytoviita et al., 1999). However, O3 reduced nitrogen
21 acquisition by P. halepensis from its mycorrhizal symbiont (Kytoviita et al., 2001). The
22 complex interrelationships that may occur in the rhizosphere were revealed by the observation
23 by Bonello et al. (1993) that the mycorrhiza Hebeloma crustuliniforme could overcome
24 the O3-stimulated severity of root rot on Scots pine caused by the fungus Heterobasidion
25 annosum (noted in Section AX9.3.4.3.2).
26 In summary, the available evidence is far too fragmented and contradictory to permit
27 drawing any general conclusions about mycorrhizal impacts. The negative effects of O3 on
28 mycorrhizae and their functioning that have been reported have not necessarily been found to
29 lead to deleterious effects on the growth of host plants. Thus, little has changed from 1991 when
30 Dighton and Jansen (1991) asked: "Atmospheric Pollutants and Ectomycorrhizae: More
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1 Questions than Answers?". Because of their important roles in ecosystems, mycorrhizae are
2 further discussed in Section AX9.6.
3
4 AX9.3.3.4 Oxidant-Plant-Plant Interactions: Competition
5 Plant competition involves the ability of individual plants to acquire the environmental
6 resources needed for growth and development: light, water, nutrients, and space. Intraspecific
7 competition involves individuals of the same species, typically in monocultural crop situations,
8 while interspecific competition refers to the interference exerted by individuals of different
9 species on each other when they are in a mixed culture.
10 In cropping situations, optimal cultural practices for row spacing and plant density/row
11 tend to balance the negative effects of intraspecific competition with the goal of maximum yield.
12 Although interspecific competition is agriculturally undesirable when it involves weak
13 infestations, the use of mixed plantings may be agriculturally deliberate, e.g., grass-legume
14 mixtures used for pasture or forage. In natural plant communities, monocultures are rare, and
15 complex interspecific competition is the norm.
16 Although weak competition is the largest global cause of crop losses, little is known about
17 the impact of O3 on crop-weed interactions. The topic does not appear to have been investigated
18 in recent years. We can only speculate as to the possible consequences of O3 exposure on weed
19 competition based on our limited understanding of the effects on a few, mostly two-component
20 mixtures of cultivated species.
21 The tendency for O3-exposure to shift the biomass of grass-legume mixtures in favor of
22 grass species, reported in the 1996 O3 AQCD (U.S. Environmental Protection Agency, 1996) has
23 been confirmed by recent studies. In a ryegrass (Lolium perenne) + clover (Trifolium repens)
24 mixture grown in an open-air fumigation system, clover growth was impaired by extended
25 exposures to above-ambient O3, leaving patches for weed invasion (Wilbourn et al., 1995). An
26 open-top chamber study (OTC) by Nussbaum et al. (1995b) using the same species confirmed
27 the greater effect on clover but observed that the magnitude of the effect depended highly on the
28 pattern of O3-exposures over extended growing periods. Low-level exposures shifted species
29 composition in favor of Lolium, but exposures to higher peak O3 levels depressed total mixture
30 yield. With an alfalfa (Medicago sativa) + timothy (Phleumpratense) mixture, Johnson et al.
31 (1996a) noted that O3 decreased alfalfa root growth and increased timothy shoot growth and
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1 height. Nussbaum et al. (2000a) reported that, with increased exposure to O3, well-watered red
2 clover (Trifolium prateme) plants suffered from increased competition from the grass Trisetum
3 flavescens, but the O3 exposure also negatively affected grass growth, depressing overall total
4 yield. However, a greater adverse effect on Trisetum resulted from O3-induced increased
5 competition when grown with brown knapweed (Centaureajacea\ a weed species.
6 Andersen et al. (2001) demonstrated the potential for competition and O3 exposure to work
7 together to affect the growth of tree seedlings. Ozone had no direct adverse effect on pine
8 growth in a 3-year study of ponderosa pine (Andersen et al., 2001) seedlings grown in
9 mesocosms with three densities of blue wild-rye grass (Elymus glaucus), but O3 exposure
10 increased the competitive pressure of the grass which caused a major reduction in pine growth.
11 Three studies have been reported on more complex plant associations. Ashmore and
12 Ainsworth (1995) studied mixed plantings of two grasses, Agrostis capilaris and Festuca rubra,
13 with two forbs15, Trifolium repens (a legume) and Veronica chamaedrys, exposed to O3 in OTCs.
14 The proportion of forbs, Trifolium in particular, declined, especially when cut at biweekly
15 intervals. In a related study, Ashmore et al. (1995) used artificial mixtures of grasses and forbs
16 and transplanted swards of native calcareous grassland species and found that, regardless of
17 whether total biomass was adversely affected by exposures to O3, higher exposures progressively
18 shifted species composition, usually at the expense of the forb species. The observed shifts in
19 competitive balance in favor of grasses is consistent with observations that many grass species
20 are less sensitive to O3 than forbs. However, as previously shown by Evans and Ashmore
21 (1992), knowledge of the relative sensitivities to O3 of the component species grown in isolation
22 or in monoculture does not always predict the impact of O3 on the components in a mixed
23 culture.
24 Barbo et al. (1998) exposed an early successional plant community to O3 in OTCs for two
25 growing seasons. Ozone decreased community structure features such as height of canopy,
26 vertical canopy density (layers of foliage), and species diversity and evenness. Surprisingly,
27 blackberry (Rubus cuneifolius), a species considered to be O3-sensitive, replaced sumac (Rhus
28 copallind) canopy dominance. Barbo et al. (2002) also demonstrated the role of competition in
29 determining the impact of O3 on loblolly pine (Pinus taeda). They reported that the increased
30 growth of natural competitors in OTCs using charcoal-filtered air to reduce the ambient O3
15 Forb: any non-grassy herbaceous species on which animals feed.
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1 concentrations resulted in decreased pine growth. They noted that this is contrary to the
2 frequently reported increased growth observed in reduced O3 levels in the absence of
3 interspecific competition.
4 McDonald et al. (2002) classified four clones of aspen (Populus tremuloides) as either
5 competitively advantaged or disadvantaged, based on their height relative to the height of
6 neighboring trees, and exposed them to 1.5 x ambient O3 in a FACE facility over a 4-year period.
7 Competitively disadvantaged trees were proportionately more adversely affected by O3 than
8 competitively advantaged or neutral trees (McDonald et al., 2002). However, one clone of the
9 disadvantaged trees demonstrated enhanced growth.
10 In summary, our present knowledge of how O3 may affect the competitive interspecific
11 plant-plant relationships typifying the agricultural and natural worlds is very limited. However,
12 as noted in the 1996 O3 AQCD (U.S. Environmental Protection Agency, 1996), "the
13 development and use of field exposure systems have permitted many recent studies of crop
14 species to be conducted at normal planting densities and hence have incorporated intraspecific
15 competition as an environmental factor." Such facilities were used in most of the studies of
16 interspecific competition discussed above. But we are still far from being able to use small
17 model competing systems to extrapolate to the realities of natural ecosystem complexity.
18
19 AX9.3.4 Physical Factors
20 The physical features of a plant's aerial and edaphic environments exercise numerous
21 controls over its growth and development. Thus, many of their effects may be modified by
22 exposure to atmospheric oxidants and, alternatively, plants may modify responses to such
23 exposures. As in the 1996 O3 AQCD (U.S. Environmental Protection Agency, 1996), this
24 section focuses on the defining features of plant microclimate: light, temperature, relative
25 humidity (HR, or saturation vapor pressure deficit), and the presence and availability of water,
26 especially in the soil. Monteith and Elston (1993) suggested that light energy and mass of water
27 should be viewed as climatic resources and that the other two elements (temperature and
28 saturation vapor pressure deficit) be viewed as rate modifiers that determine how fast the
29 resources are used. The modifications of plant response by physical environmental factors has
30 recently been reviewed by Mills (2002).
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1 Another physical feature of the microclimate, wind and air turbulence, which affects the
2 thicknesses of the boundary layers over leaves and canopies and, hence, affects gas exchange
3 rates (including the fluxes of O3 and other oxidants into the leaves) is discussed elsewhere
4 (Section AX9.4).
5 Physical features of the environment are also important components of larger-scale
6 regional and global climates. However, the following discussions are confined to issues related
7 to individual factors at the plant level; meso-scale effects are reviewed in Section AX9.3.4.8,
8 which addresses the issues of climate change interactions.
9
10 AX9.3.4.1 Light
11 Plants are the primary producers of biomass on the planet through their ability to capture
12 light energy (by the process of photosynthesis) and convert it to the many forms of chemical
13 energy that sustain their own growth and that of secondary consumers and decomposers. Light
14 intensity is critical because the availability of light energy (a resource, sensu Monteith and Elston
15 (1993) governs the rate at which photosynthesis can occur, while light duration (i.e.,
16 photoperiod) profoundly effects development in many species. Although light quality (i.e., the
17 distribution of incident wavelengths) may also affect some physiological plant processes, there is
18 no evidence to indicate that such effects are of relevance to concerns over oxidant pollution,
19 except at the short wavelengths of UV-B. This topic is discussed in the context of climate
20 change in Section AX9.3.8.2, and as a stress factor per se affected by atmospheric O3 in
21 Chapter 10. However, as noted above and in the 1996 O3 AQCD (U.S. Environmental
22 Protection Agency, 1996), none of these features is controllable in natural field situations.
23 A brief discussion of light intensity-O3 interactions is included in the review by Chappelka and
24 Samuelson (1998).
25 The conclusion in the 1996 O3 AQCD (U.S. Environmental Protection Agency, 1996) that
26 low light intensities and short photoperiods tended to increase susceptibility to foliar O3-injury
27 may still be valid, but this may or may not translate into adverse effects on growth. For
28 example, Tjoelker et al. (1993) found that, when seedlings of sugar maple, a shade-tolerant
29 species, were grown in 7% full sunlight, O3 reduced shoot and root growth, but had no
30 significant effect in 45% sunlight (a 6-fold increase). In contrast, the reverse was observed with
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1 a shade-intolerant hybrid poplar, with the greater impact of O3 occurring in the higher light
2 intensity treatment.
3 The greater sensitivity of maple in low light has also been confirmed in other studies.
4 Tjoelker et al. (1995) noted a greater O3-induced inhibition of photosynthetic CO2 assimilation in
5 shaded leaves than in leaves in full sunlight. However, in the absence of differences in stomatal
6 conductance, the effect was considered to be independent of O3 flux; it appeared to be a
7 consequence of reduced chlorophyll contents and quantum efficiencies induced by O3. In
8 contrast, Back et al. (1999), who also observed a greater inhibition of net photosynthesis by O3 in
9 shaded leaves, reported decreased stomatal conductance. Although reduced conductance might
10 suggest reduced O3 flux and, therefore, decreased adverse effects, the authors concluded that the
11 effects of reduced conductance were offset by long-term changes in leaf structure, leading to less
12 densely packed mesophyll cells and greater internal air space within the leaves. Morphological
13 differences between lower and upper crown leaves of black cherry (Primus serotina) have been
14 suggested as the basis for the greater O3-susceptibility of the lower crown leaves (Fredericksen
15 et al., 1995). Back et al. (1999) also observed accelerated foliar senescence induced by O3 on
16 shaded leaves, a response also noted by Topa et al. (2001). Sensitivity to O3 was found to be
17 increased in shade- but not sun-leaves of shade-tolerant red oak (Quercus rubrd) (Samuelson and
18 Edwards, 1993). Similarly, Mortensen (1999) observed that seedlings of mountain birch (Betula
19 pubescens) grown in 50% shade suffered greater foliar injury from O3 than those grown in full
20 sunlight.
21 Not all shade-intolerant species exhibit greater reductions in photosynthesis and growth
22 due to O3 when grown in full sunlight. Higher than ambient levels of O3 failed to inhibit
23 photosynthesis in leaves of shade-intolerant yellow poplar (Liriodendron tulipiferd) grown in
24 nearly full sunlight (Tjoelker and Luxmoore, 1991). Greater foliar injury in the lower, shaded
25 leaves of shade-intolerant black cherry trees and saplings, was attributed to higher stomatal
26 conductance and greater O3 uptake relative to net photosynthetic rate (Fredericksen et al.,
27 1996a). However, in a 3-year study of Norway spruce seedlings in OTCs, Wallin et al. (1992)
28 observed that photosynthetic efficiency was more adversely affected by O3 in high than in
29 low light.
30 The suggestion of greater sensitivity to O3 of shade-tolerant species in low-light conditions
31 and the greater sensitivity of shade-intolerant species in high light is somewhat of an
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1 oversimplification when dealing with mature trees, for which light intensity varies considerably
2 within the canopy because of shading. Chappelka and Samuelson (1998) noted that the
3 interaction between sensitivity to O3 and the light environment in forest trees is further
4 complicated by developmental stage, with seedlings, saplings, and mature trees frequently giving
5 different results. Topa et al. (2001) also cautioned that O3 effects on leaf-level photosynthesis
6 may be poor predictors of the growth responses of sugar maple in different light environments.
7 In high-light intensities, many species exhibit some degree of photoinhibition of the
8 photosynthetic process through the overloading of the mechanisms that protect the
9 photosynthetic reaction centers in the chloroplasts. Guidi et al. (2000) reported complex
10 interactions between high-light intensities (inducing photoinhibition) and O3 exposures in kidney
11 bean with high intensities tending to enhance the detrimental effect of O3 on photosynthesis.
12 One of the studies in the extensive European Stress Physiology and Climate Experiment-wheat
13 (ESPACE-wheat) program (Bender et al., 1999), conducted in 1994 and 1995, included an
14 investigation of the effects of climatic variables on yield response to O3 using two simulation
15 models, AFRCWHEAT2-O3 and LINTULCC (Ewart et al., 1999; Van Oijen and Ewart, 1999).
16 Among the observed trends, it was noted that relative yield loss of wheat due to elevated O3
17 tended to increase with light intensity. In contrast, Balls et al. (1996) used ANNS to investigate
18 microclimatic influences on injury caused by O3 to clover (Trifolium subterraneum) and found
19 that, especially at mid-range cumulative O3 exposures (350 to 500 ppb-h), injury tended to
20 decrease with increasing light intensity. Similar observations by Davison et al. (2003) of foliar
21 injury to wild populations of cutleaf cone flower (Rudbeckia laciniata) exhibiting a range of
22 PAR levels within their canopies led the authors to conclude that the variation in injury
23 symptoms observed was "unlikely to be due to differences in ozone flux and more likely to be
24 due to variation in light." Antonielli et al. (1997) found evidence indicating that the high
25 sensitivity of the bioindicator tobacco cultivar Nicotiana tabacum cv. Bel-W3 is partly
26 determined by its high photosynthetic electron transport rates at high-light intensities, which
27 exceed the capabilities of the plant to dissipate energy and oxyradicals.
28 The 1996 O3 AQCD referred to the important role of light in controlling stomatal opening
29 and suggested that light duration (i.e., photoperiod) might dictate the actual uptake of O3 to some
30 degree. However, it should also be noted that Sild et al. (1999) found that clover plants could
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1 suffer foliar injury even if they were exposed to O3 during the dark period of the day-night cycle,
2 when stomatal conductance is at its lowest.
3 A possible indirect effect of light intensity was noted by Reiling and Davison (1992) in
4 their study of the O3-tolerance of common plantain (Plantago major L.) plants grown from seeds
5 collected from populations at 28 different sites in Britain. Ozone-tolerance, defined in terms of
6 plant growth, was found to be a function of both previous O3-exposure history and hours of
7 bright sunshine during the year before the seeds were collected. However, the authors cautioned
8 that, since tropospheric O3-formation is itself dependent upon irradiation, the observation does
9 not necessarily imply a direct effect of light intensity on the plants' response to O3.
10 The only recent studies concerning interactions with light quality appear to be those
11 involving O3 and UV-B as a component of climate change. These are dealt with in Section
12 AX9.3.4.8.2. The effects of photoperiod on response to O3 or the converse do not appear to have
13 received any recent attention.
14 Although the intensity, quality, and duration of light are not controllable in the natural
15 world, the interactions of O3 with light intensity, in particular, clearly have relevance to the
16 growth of shade-tolerant and shade-intolerant species in mixed forest stands. It appears that the
17 nature of light intensity-O3 interactions may depend upon the type of light environment to which
18 the species are best adapted, with increased light intensity increasing the sensitivity of light-
19 tolerant and decreasing the sensitivity of shade-tolerant species to O3. Although there is
20 certainly some evidence to the contrary, this hypothesis is a reasonable summation of current
21 understanding with regard to O3-light intensity interactions.
22
23 AX9.3.4.2 Temperature
24 "Temperature determines the start and finish and rate and duration of organ growth and
25 development" (Lawlor, 1998). Such processes depend on fundamental physiological activities
26 that are mostly enzyme-mediated and whose kinetics are directly affected by temperature. Since
27 the processes of enzyme deactivation and protein denaturation also increase as temperatures rise,
28 each enzymatic process has a unique optimum temperature range for maximal function.
29 However, the optima for different processes within the plant vary appreciably and, hence, the
30 optimum temperature range for overall plant growth is one within which all of the individual
31 reactions and vital processes are collectively functioning optimally, not necessarily maximally.
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1 Furthermore, individual features of plant development (e.g., shoot and root growth, flowering,
2 pollen tube growth, fruit set, seed development) have different specific optima, so that
3 differential responses to temperature occur, leading to temperature-induced developmental
4 changes. For example, despite increased assimilation, increased temperatures may result in
5 decreased grain yields of crops such as wheat, because the growing season is effectively
6 shortened by a more rapid onset of senescence (Van Oijen and Ewart, 1999).
7 Rowland-Bamford (2000) noted that a plant's response to temperature changes will depend
8 upon whether it is growing at its near optimum temperature for growth or its near maximum
9 temperature and whether any increase in mean temperature results in temperatures rising above
10 the threshold for beneficial responses. Impairment by O3 of any process may be thought of as
11 being analogous to a downward shift below and away from the temperature optimum or an
12 upward shift above and away from the optimum. Since a temperature rise toward the optimum
13 would result in a rate increase, the combined effects of O3 and such an increase might neutralize
14 each other, while the effects of O3 and a decrease in temperature would likely be additively
15 negative. Above the optimum temperature, the situations would be reversed with the effects of
16 increased temperatures and O3 being additively negative, and decreasing temperatures
17 counteracting any negative effect of O3. Thus, it is difficult to generalize about the interactions
18 of temperature and O3 on overall plant responses such as growth in which the different
19 temperature-rate relationships of different growth components are merged, because they depend
20 upon the relationship of any temperature changes to the optimum for a species.
21 Studies of the effects of temperature on the impact of O3 have increased recently because
22 of an increased need to understand the consequences of global warming as a component of
23 climate change. Direct interactions of temperature with O3 are reviewed here, but the issues are
24 addressed again in Section AX9.3.8.1 in relation to changes in atmospheric CO2 levels.
25 The 1996 O3 AQCD (U.S. Environmental Protection Agency, 1996) stressed the
26 interdependence of the temperature within the tissues of the leaf (where the various temperature-
27 sensitive processes occur) on three distinct components: the ambient air temperature, the heating
28 effect of incident infrared radiation during the photoperiod, and the evaporative cooling effect
29 caused by transpirational loss of water. It also cautioned that, especially in experiments using
30 controlled environment chambers, the effects of temperature could well be confounded with
31 those of humidity /vapor pressure deficit (VPD). Temperature and VPD are strongly interrelated,
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1 and VPD plays an important role in regulating stomatal transpiration. Because of the role that
2 evaporative cooling plays in determining internal leaf temperatures, any factor that causes
3 stomatal closure and reduced conductance inevitably leads to increased leaf temperatures. Such
4 interactions add to the difficulties in distinguishing the effects of temperature from those of other
5 factors, as actual leaf temperatures are rarely measured and reported.
6 Despite these caveats, there is some evidence that temperature per se influences plant
7 response to O3. For example, in rapid-cycling Brassica (Brassica rapd) and radish (Raphanus
8 sativus), marked O3-inhibitions of growth were observed at low root temperatures (13 °C) but
9 not at 18 °C (Kleier et al., 1998, 2001). With regard to air temperature, this was included in the
10 range of micrometeorological variables studied in several recent extensive field studies and was
11 found to have a significant effect on response to O3 in most cases. Balls et al. (1996) used ANNs
12 in an analysis of the growth of clover (Trifolium subterraneuni) and concluded that light and
13 VPD had greater influences than temperature on the visible injury response to O3. However, in
14 three studies with different cultivars of white clover {Trifolium repens), temperature was found
15 to be important to the growth response. Ball et al. (1998) exposed T. repens cv. Menna to
16 ambient O3 in OTCs at 12 European sites at a range of latitudes and altitudes from 1994 to 1996.
17 The impact of O3 on growth was determined as the ratio of growth with and without treatment
18 with the O3-protectant, EDU (see Section AX9.2). Artificial neural network analysis showed
19 that O3 exposure (measured as the AOT40 index, see Section AX9.3.6), VPD, and temperature
20 were consistently the three most important variables governing response to O3 over a range of
21 different ANN models. However, the authors did not describe the form of the O3-response
22 relationship with temperature. Similar observations were reported by Ball et al. (2000) and Mills
23 et al. (2000) for O3-sensitive and -tolerant clones of T. repens cv. Regal, grown at 14 to 18
24 European locations from 1995 to 1998. In both studies, the impact of O3 was measured as the
25 sensitive/tolerant growth (biomass) ratio. Although Ball et al. (2000) found temperature to be
26 less important than O3 exposure and VPD, Mills et al. (2000) found temperature to be the most
27 important input variable after O3 exposure (AOT40). In both cases, the adverse effect of O3
28 increased with increasing temperature.
29 A study of black cherry seedlings and mature trees in Pennsylvania, using
30 micrometeorological variables aimed to predict O3 uptake, found temperature to be unimportant
31 (Fredericksen et al., 1996b), but in the study of populations of common plantain referred to in
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1 Section AX9.3.4.1, Reiling and Davison (1992) noted a weak, positive correlation between mean
2 temperature at the collection site and O3 tolerance (based on growth rate) of the different
3 populations. In contrast, Danielsson et al. (1999) collected genotypes oiPhleum arvense from a
4 wide range of Nordic locations and found a positive effect of temperature on the growth of
5 genotypes from locations with higher summer temperatures, but sensitivity to O3 did not vary
6 systematically with geographic location.
7 Van Oijen and Ewart (1999) studied the effects of climatic variables on the response to O3
8 in the ESPACE-wheat program, based on two distinctive simulation models (AFRCWHEAT2-
9 O3 and LINTULCC [Ewart et al., 1999]) and noted that although the relative yield loss of wheat
10 due to elevated O3 tended to increase with temperature, the effect was of minor significance.
11 In contrast to the variable results obtained in studies of the effects of temperature on
12 response to O3, the corollary effect of O3 exposure on subsequent sensitivity to low temperature
13 stress, noted in the 1996 criteria document, is well recognized. In reviewing low temperature-O3
14 interactions, Colls and Unsworth (1992) noted that winter conditions produce three kinds of
15 stress: desiccation, chilling or freezing temperatures, and photooxidation of pigments. Of these,
16 they suggested that while the first two were important, the last may play a particularly significant
17 role because the "combination of high irradiance and low temperatures permits a buildup of free
18 radicals in leaf tissue, and these free radicals then attack chlorophyll." Chappelka and Freer-
19 Smith (1995) suggested that the injury and losses to trees caused by this delayed impact of O3
20 may be equally or more important than the direct impacts of O3 on foliage of visible injury and
21 necrosis, or the disruption of key physiological processes such as photosynthesis. In this
22 context, the 1996 O3 AQCD (U.S. Environmental Protection Agency, 1996) referred to the
23 conceptual framework of Eamus and Murray (1991), which is still valid: brief periods of mild
24 temperatures in the severest winters result in dehardening; O3 decreases frost hardiness per se,
25 but also increases the predisposition to dehardening; dehardening places O3-exposed trees at
26 greater risk from subsequent low temperatures. However, no quantified models of these effects
27 have yet appeared.
28 The 1996 O3 AQCD also noted that O3 adversely affects cold hardiness of herbaceous
29 species. More recently, Foot et al. (1996, 1997) observed winter injury and decreased growth in
30 low-growing perennial heather Calluna vulgaris exposed to O3 (70 ppb, 8 h/day, 5 days/week for
31 6 months) during the winter (6.8 °C mean), but found no significant effects from the same
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1 exposures during the summer (12.3 °C mean). Although Potter et al. (1996) observed a similar
2 situation with the moss Polytrichum commune, the reverse was found with the moss Sphagnum
3 recurvum.
4 In summary, unequivocal evidence exists that O3 causes sensitization to the adverse effects
5 of low temperatures, but there is no clear pattern in the evidence regarding the effects of
6 temperature on O3 response. The many contradictory responses to temperature and O3 probably
7 reflect our lack of detailed knowledge of the temperature optima for the different growth
8 components of the studied species. The topic of temperature-oxidant interactions is revisited
9 later in Section AX9.3.4.8 in the context of global warming as a feature of climate change.
10
11 AX9.3.4.3 Humidity and Surface Wetness
12 The moisture content of the ambient air (or its VPD) is a rate modifier (sensu Monteith and
13 Elston (1993)) and an environmental regulator of stomatal conductance. Both of the previous O3
14 AQCDs (U.S. Environmental Protection Agency, 1986, 1996) concluded that the weight of
15 evidence indicated that high RH (=low VPD) tended to increase the adverse effects of O3,
16 principally because the stomatal closure induced in most situations by O3 is inhibited by high
17 RH, leading to increased O3 flux into the leaves.
18 Recent reports have confirmed this role of RH. The studies by Balls et al. (1995, 1996)
19 and Ball et al. (1998) showed that VPD was an important determinant of O3-induced injury
20 and reduced growth in two species of clover, Trifolium repens cv. Menna and T. subterraneum.
21 However, Mills et al. (2000) found it to be unimportant in the case of T. repens cv. Regal. Such
22 difference between cultivars is not unexpected , because considerable differences also occur
23 among species and genera. For example, Bungener et al. (1999a) studied 26 Swiss grassland
24 species and found clear evidence that O3 injury increased with decreased VPD (i.e., increased
25 RH) in only eight species. However, the 1995 data from the European cooperative study of O3
26 injury, which involved 28 sites in 15 countries and six crop species, led to the development of
27 two 5-day critical-level scenarios involving O3-exposure (calculated as the AOT40 index) and
28 mean VPD (0930-1630h): 200 ppb-h at >1.5 kPa, and 500 ppb-h at < 0.6 kPa (Benton et al.,
29 2000).
30 With forest tree species, Fredericksen et al. (1996b) found significant correlations between
31 stomatal conductance of black cherry leaves and RH (+ve) and VPD (-ve), and studies on free-
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1 standing Norway spruce and larch (Larix decidud) showed that although ambient VPD was
2 highly positively correlated with ambient O3 concentration, increased VPD caused stomatal
3 closure, reducing O3 uptake and impact (Wieser and Havranek, 1993, 1995).
4 Surface wetness may affect O3 response through its direct effects on deposition to the
5 surface and through changes in RH. Effects on the deposition of O3 have been reviewed by Cape
6 (1996). A surface film of water on leaves was found to increase O3 deposition in four studies
7 involving field-grown grape (Vitis vinifera) (Grantz et al., 1995), red maple {Acer rubrum)
8 (Fuentes and Gillespie, 1992), deciduous forest dominated by largetooth aspen (Populus
9 grandidentatd) and red maple (Fuentes et al., 1992), and clover-grass mixed pasture (Trifolium
10 pratense, Phleumpratense, and Festucapratensis) (Pleijel et al., 1995). In each case, the
11 increased deposition could be attributed partly to an increased stomatal conductance through the
12 abaxial (lower) surface and partly to uptake into the aqueous film on the adaxial (upper) surface.
13 In contrast, decreased deposition was noted by Grantz et al. (1997) with field-grown cotton
14 (Gossypium hirsutum). Since cotton is amphistomatous, with functional stomata on both leaf
15 surfaces, it was suggested that, in this case, the water layer effectively sealed the adaxial surface
16 stomata, more than offsetting any increase in conductivity of the stomata in the abaxial surface.
17 However, none of the studies investigated the consequences of the differences in deposition.
18 Although it could be inferred that, with part of any increased deposition being the result of
19 increased O3 flux into the leaves, there would be the likelihood of increased O3 adverse effects,
20 as suggested by earlier studies (Elkiey and Ormrod, 1981) that, by misting bluegrass (Poa
21 pratensis) during exposure to O3, injury was significantly increased.
22 To conclude, the effects of high RH (low VPD) and surface wetness have much in
23 common, as they both tend to enhance the uptake of O3, largely through effects on stomata
24 leading to increased impact.
25
26 AX9.3.4.4 Drought and Salinity
27 The 1996 O3 AQCD (U.S. Environmental Protection Agency, 1996) concluded that the
28 available evidence clearly indicated that exposure to drought conditions could reduce the adverse
29 effects of O3 on the growth of herbaceous and woody plants, but it also noted that no quantitative
30 models of the O3-soil moisture deficit (SMD) interaction had yet appeared in print.
31 Nevertheless, the "protective" effect was inconsistent, and only appeared when SMD was
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1 accompanied by high evaporative demand. Since that time, further studies have confirmed the
2 interaction, and simulation models have begun to appear. Mills (2002) has recently provided a
3 brief review of the topic.
4 With regard to herbaceous species, Vozzo et al. (1995) observed less O3-induced injury and
5 suppression of net photosynthesis and growth in water-deficient soybean (Glycine max) than in
6 well-watered plants. In several studies with wheat (Triticum aestivum), on the other hand,
7 although adverse effects of both O3 and SMD were noted, they were consistently additive
8 (Bender et al., 1999; Fangmeier et al., 1994a,b; Ommen et al., 1999).
9 In attempting to model the stomatal conductance of wheat in relation to O3 and soil
10 moisture, Griiters et al. (1995) found that although O3-induced stomatal closure was enhanced by
11 SMD, reducing O3 uptake, the R2 of the overall model was only 0.40, indicating that other
12 significant factors or relationships were involved.
13 With regard to native vegetation, Bungener et al. (1999a) used mixed plantings of 24 Swiss
14 grasses, herbs, and legumes and observed that, although O3-drought interactions were species-
15 specific, they tended to reflect stomatal functioning. They found that SMD reduced O3 injury in
16 two clovers (Trifolium repens and T. pratensis) and two grasses (Trisetum flavescens and
17 Bromus erectus), but noted no interactions in the other 20 species. With relative growth rate as
18 the measure of response to O3, interactions with SMD were noted in only three species:
19 Trifolium repens and two weedy herbs, Knautia arvense and Plantago lanceolata (Bungener
20 et al., 1999b). Although this variability in response among species was noted in the review by
21 Davison and Barnes (1998), they also pointed out that in severely draughted regions of Europe,
22 notably in Greece and Spain, O3-induced injury and growth reductions were common on many
23 (usually irrigated) crops, but there were virtually no records of injury symptoms in wild species.
24 Thus, the situation with herbaceous species is essentially unchanged from 1988 when
25 Heagle et al. (1988) summarized the extensive NCLAN experiments that incorporated water
26 stress as a variable: "SMD can reduce the response of crops to O3 under some conditions but not
27 under other conditions. Probably the occurrence of O3 by SMD interactions was dependent on
28 the degree of SMD-induced plant moisture stress."
29 With regard to trees, O3 interactions with soil water availability have been discussed in
30 several recent reviews: Chappelka and Freer-Smith (1995), who focused on O3-induced
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1 predisposition to drought stress (Johnson et al., 1996b; Chappelka and Samuelson, 1998; and
2 Skarbyetal., 1998).
3 Several recent studies with conifers have yielded mixed results. No interactions with
4 drought were observed by Broadmeadow and Jackson (2000) on Scots pine, by Karlsson et al.
5 (2002) on Norway spruce, or by Pelloux et al. (2001) on Aleppo pine. More recently, Le Thiec
6 and Manninen (2003) reported that drought reduced O3-induced growth suppression of Aleppo
7 pine seedlings. Panek and Goldstein (2001) inferred less impact of O3 on draughted Ponderosa
8 pine, and Van Den Driessche and Langebartels (1994) reported that drought reduced injury and
9 O3-induced ethylene release by Norway spruce. But Karlsson et al. (1997), in a comparative
10 study of fast- and slow-growing clones of P. abies, only observed a drought-induced reduction of
11 O3-inhibited root growth in the fast-growing clone. In contrast, Grulke et al. (2002) noted a
12 synergistic interaction between O3 and drought stress on gross photosynthesis ofPinus
13 ponderosa, and Wallin et al. (2002) reported a synergistic growth response of Norway spruce in
14 the third year of a 4-year study. A similar response was noted by Dixon et al. (1998) with the
15 Istebna strain of Norway spruce.
16 With broad-leaved trees, studies of Durmast oak (Quercuspetraea) (Broadmeadow et al.,
17 1999; Broadmeadow and Jackson, 2000) and European ash (Fraxinus excelsior) (Broadmeadow
18 and Jackson, 2000; Reiner et al., 1996) showed that drought provided partial protection
19 against O3-induced growth reduction. Although European beech (Fagus sylvaticd) is reportedly
20 an O3- and drought-sensitive species, neither Pearson and Mansfield (1994) nor Broadmeadow
21 et al. (1999) observed any interactions between these stresses, while Dixon et al. (1998)
22 observed partial protection. Paakonen et al. (1998) observed only additive effects in a sensitive
23 clone of birch (Betulapendula). However, the experiments of Schaub et al. (2003) and the
24 survey by Vollenweider et al. (2003) on black cherry clearly indicate antagonism between
25 drought and O3 stresses on this species.
26 With regard to the converse effect, in a critical review of the evidence for predisposition to
27 drought stress being caused by O3, Maier-Maercker (1998) supported the hypothesis and
28 suggested that the effects were caused by the direct effects of O3 on the walls of the stomatal
29 guard and subsidiary cells in the leaf epidermis, leading to stomatal dysfunction.
30 The Plant Growth Stress Model (PGSW) developed by Chen et al. (1994) is a physiology-
31 based process model which includes drought among several environmental variables.
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1 Simulations for Ponderosa pine incorporated antagonistic effects between O3 and drought
2 stresses, i.e., partial protection, although Karlsson et al. (2000) have since emphasized that
3 drought-induced "memory effects" should be considered when developing simulation models
4 incorporating stomatal conductance.
5 Retzlaff et al. (2000) used the single-tree model, TREEGRO, to simulate the combined
6 effects of O3 and drought on white fir {Abies concolor). Although simulated reductions in
7 precipitation >25% reduced growth, they also reduced O3 uptake (and impact). But lesser
8 reductions in precipitation combined synergistically with O3 stress to reduce growth, leading the
9 authors to conclude that moderate drought may not ameliorate the response of white fir to O3.
10 On a much larger scale with a modified forest ecosystem model (PnEt-II) incorporating
11 O3-response relationships for hardwood species, Ollinger et al. (1997) showed how predicted
12 changes in net primary production and mean wood production in the northeastern U.S. hardwood
13 forests due to O3 would be reduced (but not countered or reversed) by drought stress, particularly
14 in the southern part of the region. This geographic distribution of the effect was substantiated by
15 the work of Lefohn et al. (1997) on the risk to forest trees in the southern Appalachian
16 Mountains, based on localized estimates of O3 levels and SMD. The TREEGRO and ZELIG
17 models were combined by Laurence et al. (2001) to predict the impacts of O3 and moisture (as
18 precipitation) on the growth of loblolly pine and yellow poplar. Based on O3 and precipitation
19 data from three sites in the eastern United States, the six model regressions developed for the
20 two species included both positive and negative coefficients for O3 exposure and precipitation as
21 determinants of growth.
22 As noted in the 1996 O3 AQCD (U.S. Environmental Protection Agency, 1996), the effects
23 of soil salinity are similar to those of SMD. In a study of rice (Oryza sativci) cultivars of
24 differing sensitivity to salinity, Welfare et al. (1996) noted that although both O3 and salinity
25 reduced many features of growth additively, antagonistic interactions were only seen for leaf
26 length and potassium accumulation. Similarly, a recent study on chickpea (Cicer arietimmi)
27 found no interactions with regard to most components of biomass accumulation (the effects
28 of O3 and salinity were additive), but with root growth, salinity suppressed the adverse effects
29 ofO3.
30 In summary, the recently described interactions of O3 and drought/salinity stresses are
31 consistent with the view that, in many species, drought/salinity reduces the impact of O3, but O3
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1 increases sensitivity to drought stress, i.e., the type of response is determined by the sequence of
2 stresses. However, synergisms have also been observed and any antagonisms are species-
3 specific and unpredictable in the absence of experimental evidence. In no case has an
4 antagonism been found to provide complete protection.
5
6 AX9.3.5 Nutritional Factors
7 The 1996 O3 AQCD (U.S. Environmental Protection Agency, 1996) noted that the large
8 number of macro- and micronutrients and the wide range of species had limited the number of
9 experimental investigation to all but a few cases of nutrient-O3 interactions and most of these
10 concerned nitrogen (N) and crops or forest tree species. The document also provided a
11 comprehensive tabulation of the results of the relevant studies up to 1992.
12 The suboptimal supply of mineral nutrients to plants leads to various types of growth
13 reductions. The consequences of suboptimal nutrition might, therefore, be expected to have
14 some similarities to those of O3 exposure. One might expect nutritional levels below the
15 optimum either to amplify any effects of O3 or at least lead to additive responses. The difficulty
16 with this suggestion is that the available information has mostly been obtained from
17 experimentation conducted using two or more arbitrarily selected levels of fertility with little or
18 no regard to optima. Hence, it is not surprising that there have been contradictory reports, even
19 among studies with the same species or cultivars conducted by different workers at different
20 locations using different soils or soil mixes.
21 There appear to have been no recent studies on O3 interactions with specific mineral
22 nutrients other than N. Hence, the previous conclusions are still valid, viz. that increasing levels
23 of the major elements potassium (K) and sulfur (S) usually reduce the impact of O3, or,
24 deficiency increases susceptibility, whereas increased phosphorus (P) usually increases injury,
25 or, deficiency decreases susceptibility.
26 However, with N, a relationship to the optimum is usually demonstrable. Several earlier
27 studies of O3 x N interactions reported that the adverse effects of O3 on growth were greatest at
28 the optimum and decreased with increasing N-deficiency, a finding supported by the work on
29 aspen of Pell et al. (1995), who also confirmed that excess N decreased O3 impact on growth.
30 Similarly, the adverse effects of O3 on growth rate in wheat diminished with decreased N supply
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1 (Cardoso-Vilhena and Barnes, 2001). However, the effects of N are far from consistent. For
2 example, Greitner et al. (1994) reported that O3 and N-deficiency acted additively in aspen in
3 reducing leaf surface area and rate of photosynthesis, Bielenberg et al. (2001) reported that the
4 rate of O3-induced senescence was increased by N-deficiency in hybrid poplar (Populus
5 trichocarpa x P. maximovizii), and Paakkonen and Holopainen (1995) observed the least adverse
6 effects of O3 on European white birch (Betulapenduld) at optimum N-fertility levels. With
7 cotton, increased N-levels more than overcame the adverse effect of O3 on growth and boll yield
8 (Heagle et al., 1999b). In view of these contradictions, one may conclude that other, unrecorded
9 factors may have contributed to the various findings. Thus, much remains unclear about O3 x N-
10 fertility interactions.
11 There have been two recent studies on the effects of overall soil fertility. Whitfield et al.
12 (1998) observed that low general fertility increased O3 sensitivity in selections of common
13 plantain. At the biochemical level, well-fertilized European white birch saplings were found to
14 be less adversely affected by O3 than nutrient-stressed plants (Landolt et al., 1997).
15 TREEGRO model simulations of the growth of red spruce (Picea rubra) in conditions of
16 nutrient deficiency and O3 stress showed that, in combination, the two stresses acted less than
17 additively (Weinstein and Yanai, 1994). Minimal amelioration by nutrient deficiency was
18 predicted with Ponderosa pine.
19 Plants may also obtain N and S from airborne sources such as NOX, HNO3, NO3 , SO2,
20 and SO42 , although, depending upon their concentration, these may also be phytotoxic.
21 In various parts of the world, the deposition of N and S in these forms contributes significantly
22 to the levels of nutritionally available N and S in soils. Such depositions may, in turn, influence
23 the impact of O3 on sensitive species through their roles as nutrients independent of any
24 interactions that may occur because of their acidic properties (see Section AX9.3.6.5). For
25 example, Takemoto et al. (2001) recently reviewed the situation in southern California's mixed
26 conifer forests and noted that, where N-deposition is appreciable, its combination with O3 is
27 causing a shift in Ponderosa pine biomass allocation toward that of deciduous trees, with
28 increased needle drop so that only 1- and 2-year needle classes overwinter. Such changes are
29 having significant consequences on the balance of the forest ecosystem and are discussed more
30 fully in Section AX9.5.
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1 Of the micronutrient elements, only manganese (Mn) appears to have been studied
2 recently. In beans (Phaseolus vulgaris) Mn-deficiency increased O3 toxicity, despite causing
3 reduced O3 uptake (through decreased stomatal conductance) and inducing increased levels of
4 Mn-SOD (Mehlhorn and Wenzel, 1996).
5 In view of the foregoing, it is impossible to generalize about the interactions of soil fertility
6 with O3. While this is especially true of the interactions involving soil nitrogen, for which there
7 is much conflicting evidence, the interactions with other nutrients need much more thorough
8 investigation than has occurred to date, before any clear patterns become apparent.
9
10 AX9.3.6 Interactions with Other Pollutants
11 The ambient air may have pollutant gases other than O3 and its photochemical oxidant
12 relatives. In particular, industrial, domestic, and automobile emissions and accidents can lead to
13 significant atmospheric concentrations of gases such as sulfur dioxide (SO2) and nitric oxide
14 (NO) and nitrogen dioxide (NO2), collectively referred to as NOX, both locally and regionally.
15 Local releases of gases such as hydrogen fluoride (HF), hydrogen chloride (HC1), and
16 chlorine (C12) may result from industrial emissions and accidents. Agricultural fertilizer and
17 manure usage can lead to significant increases in ambient ammonia (NH3) and ammonium
18 sulfate ((NH4)2SO4). The sulfur and nitrogen oxides may undergo reactions in the atmosphere
19 leading to the formation of sulphate (SO42 ) and nitrate (NO3 ) ions and resultant acid deposition.
20 The 1996 O3 AQCD (U.S. Environmental Protection Agency, 1996) discounted much of
21 the early research on pollutant combinations, because of its lack of resemblance to the ambient
22 experience: the concentrations used were unrealistically high or the exposure regimes employed
23 almost invariably used gas mixtures, whereas Lefohn et al. (1987) showed that the co-occurrence
24 patterns of significant levels of O3 with SO2 or NO2 in the United States were most frequently
25 sequential or partially sequential with overlap; only rarely were they entirely concurrent. On the
26 other hand, O3 and peroxyacetylnitrate (PAN) frequently co-occur, as both form photochemically
27 under similar conditions.
28 To the list of reviews mentioned in the 1996 O3 AQCD should be added the more recent
29 ones by Barnes and Wellburn (1998), Robinson et al. (1998), and Fangmeier et al. (2002), which
30 also explore some of the potential mechanisms underlying pollutant-pollutant interactions.
31
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1 AX9.3.6.1 Oxidant Mixtures
2 In 1998, Barnes and Wellburn noted that virtually no information existed on the effects on
3 plants of concurrent exposures to O3 and other components of photochemical oxidant other than
4 PAN. The situation has not changed since their review appeared, and the topic appears to have
5 attracted no research interest since before the 1996 O3 AQCD (U.S. Environmental Protection
6 Agency, 1996). The continuing conclusion must, therefore, be that, from the limited information
7 available, the two gases appear to act antagonistically, with O3 raising the threshold for the
8 visible injury response to PAN and PAN reducing the harmful effects of O3.
9
10 AX9.3.6.2 Sulfur Dioxide
11 In reviewing O3 x SO2 interactions, Barnes and Wellburn (1998) remarked: "The outcome
12 of exposure to this combination of pollutants has probably been the most studied, yet is one of
13 the least understood." More recent studies have only added to the conflicts referred to in the
14 1996 criteria document (U.S. Environmental Protection Agency, 1996), rather than resolve them.
15 For example, Diaz et al. (1996) reported that, after a year of daily exposures of Aleppo pine
16 seedlings to 50 ppb O3 and/or 40 ppb SO2, the combination of pollutants synergistically reduced
17 shoot and root growth and impaired mycorrhizal colonization of the roots. With tomato
18 (Lycopersicon esculentum), on the other hand, effects on growth ranged from synergistic at low
19 exposures (50 ppb) to antagonistic at exposures of 200 ppb of each gas (Khan and Khan, 1994).
20 Although various physiological measurements were made in these and earlier studies, it has not
21 been possible to determine any consistent mechanism or mechanisms that might account for the
22 conflicting results.
23 Since the information available about O3 x SO2 interactions appears to be highly dependent
24 upon species, the type of response measured, and the experimental protocol used, it would still
25 appear prudent to heed the statement of Heagle et al. (1988) in their summary of the studies
26 undertaken in 12 field experiments over several years within the NCLAN program: "There were
27 no cases where O3 and SO2 interactions significantly affected yield." (emphasis added.)
28
29 AX9.3.6.3 Nitrogen Oxides, Nitric Acid Vapor, and Ammonia
30 The major oxides of nitrogen that occur in ambient air are nitrous oxide (N2O), NO,
31 and NO2, of which the latter two (conveniently symbolized as NOX) are particularly important in
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1 connection with O3, because they are components of the reaction mix that leads to photochemical
2 O3 formation and because they can interact with O3-responses. Their reactions in the atmosphere
3 can also lead to the occurrence of nitric acid vapor (HNO3) in ambient air. The other major
4 N-containing contaminant of ambient air in many parts of the world is NH3, largely released
5 through agricultural practices.
6 Despite various combinations of O3 and NOX being probably the most common air
7 pollutant combinations found in the field, Barnes and Wellburn (1998) noted that they have been
8 little studied. Much early work with O3 and NOX focused on O3 x NO2 interactions and can be
9 discounted, because of the unrealistic concentrations employed and their use as mixtures rather
10 than in types of sequences. The 1996 O3 AQCD (U.S. Environmental Protection Agency, 1996)
11 concluded that evidence from studies involving concurrent exposures to both O3 and NOX at
12 realistic concentrations was so fragmented and varied that no firm conclusions could be drawn as
13 to the likelihood and nature such interactions. However, the few recent investigations taken
14 together with the earlier data are now beginning to reveal a pattern of response.
15 With regard to NO, Nussbaum et al. (1995a, 2000b) reported their findings with concurrent
16 exposures to NO and O3 and observed that, at low O3 levels, NO tended to act similarly to O3 by
17 increasing the scale of responses such as growth reductions. However, in ambient air in
18 which O3 is a dominant factor, the effects of NO were usually found to be negligible due to
19 low levels, although the authors admitted that the effects observed were confounded by the
20 inevitable O3-induced oxidation of NO to NO2.
21 Two possible mechanisms whereby NO may influence plant response to O3 are suggested
22 by recent biochemical studies. First, there is growing evidence for the role of NO as a signaling
23 agent in plants that can induce defense responses to a range of biotic and abiotic stressors
24 (Beligni and Lamattina, 2001; Neill et al., 2002). Second, a role for NO as an antioxidant
25 scavenger of reactive oxygen species has been demonstrated by Beligni and Lamattina (2002) in
26 potato leaves and chloroplasts. However, both of these cases concern endogenously synthesized
27 NO, and it must be noted that in none of these or other reports of studies of NO signaling have
28 the authors considered the potential significance of exogenous NO in ambient air.
29 An independent case for O3 x NO interactions comes from Mills et al. (2000). The ANN
30 model developed to predict the O3 effects on white clover biomass based on experiments at
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1 18 locations throughout Europe suggested that the minimum daily NO concentration (at 5 p.m.)
2 may have been a contributor to adverse effects.
3 Turning to NO2, Maggs and Ashmore (1998) found that, although concurrent but
4 intermittent exposures of Bismati rice (Oryza saliva) revealed no significant growth
5 interactions, NO2 reduced the rate of O3-induced senescence, an antagonistic response possibly
6 related to enhanced N-metabolism.
7 With regard to sequential exposures, two studies on gene activation in tobacco revealed
8 that NO2 counteracted the effect of O3 in reducing mRNA levels for three genes encoding
9 photosynthetic proteins (Bahl and Kahl, 1995) and tended to counteract the O3-induced
10 enhancement of defense-protein gene activation (Bahl et al., 1995). However, despite
11 compelling evidence for significant interactive effects provided by earlier studies (Bender et al.,
12 1991; Goodyear and Ormrod, 1988; Runeckles and Palmer, 1987), the only recent investigation
13 of growth effects seems to have been that of Mazarura (1997) using sine-wave exposure profiles.
14 He found that although 4 weeks of twice daily 3-h exposures to NO2 (120 ppb peak
15 concentrations) slightly stimulated growth of radish (Raphanus saliva) and while daily 6-h
16 exposures to O3 (120 ppb peak concentration) did not significantly reduce growth, the daily
17 sequence, NO2 - O3 - NO2, led to a 13% drop in dry matter production.
18 The combined evidence to date, therefore, suggests that, in leguminous species, the effects
19 of these sequences are antagonistic with NO2 tending to reduce (or reverse) the negative effects
20 of O3 on growth, while the effects are increased in other species. These conclusions differ from
21 those of Barnes and Wellburn (1998) who suggested that sequential exposures tended to result in
22 antagonistic effects (largely based on the summary by Bender and Weigel (1992)), whereas
23 simultaneous exposures were likely to lead to synergistic responses. With disagreements both
24 among the data and their interpretation, it is not possible to determine the circumstances under
25 which specific interactions of O3 and NO2 may occur, but there is no reason to doubt the validity
26 of the individual findings of each study. Far more systematic investigation is needed to clarify
27 the situation.
28 There appear to have been no studies of O3 interacting with HNO3 in the vapor phase.
29 However, in the southern California montane forests (Takemoto et al., 2001), in Sweden (Janson
30 and Granat, 1999), and elsewhere, significant amounts of N are deposited in this form because of
31 the vapor's high deposition velocity. As a consequence, although much of it ultimately reaches
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1 the ground through leaching and leaf fall and enters the soil as NO3 , it may also be used as a N
2 source by the foliage itself (Garten and Hanson, 1990; Hanson and Garten, 1992; Norby et al.,
3 1989). This nutritional role is independent of any contribution that HNO3 vapor may make to
4 acidic deposition. Indirect interactions with the effects of O3 through N-deposition of NOX,
5 HNO3, and NH3 are related to the interactions of O3 with N as a nutrient, and have recently been
6 examined in the review by Takemoto et al. (2001). The 1996 O3 AQCD (U.S. Environmental
7 Protection Agency, 1996) stated that the evidence available at that time led to estimates of total
8 forest dry deposition, including HNO3, ranging from 5.7 to 19.1 kg N ha"1 year1 (Taylor et al.,
9 1988). However, Takemoto et al. (2001) pointed out that in parts of the mid-elevation forests of
10 southern California, dry deposition rates may reach more than 40 kg N ha"1 year"1. As a result,
11 some locations have seen the conversion from N-limited to N-saturated forests. The concern for
12 California's forests is well stated by Takemoto et al.: "As potential modifiers of long-term forest
13 health, O3 is a stressor and N deposition is an enhancer of ponderosa/Jeffrey pine physiology and
14 growth (Grulke and Balduman, 1999). The progression toward a deciduous growth habit, higher
15 shoot:root biomass ratios, increasing depths of litter, tree densification, and elevated NO3 levels
16 in soil and soil solution, all point to the replacement of pine species with nitrophilous, shade- and
17 O3-tolerant tree species, such as fir and cedar (Minnich, 1999; Minnich et al., 1995)."
18 Few studies have been reported of interactions of O3 with NH3. The 1996 criteria
19 document made reference to the work on kidney bean by Tonneijck and Van Dijk (1994, 1998).
20 Although NH3 alone tended to increase growth and O3 alone to inhibit it, one interaction was
21 noted (Tonneijck and Van Dijk, 1994) on the number of injured leaves. Dueck et al. (1998)
22 studied the effects of O3 and NH3 on the growth and drought resistance of Scots pine.
23 Significant interactions were found for some growth features, but there were no consistent
24 patterns of the effects of NH3 on O3 response or vice versa. However, O3 was found to
25 ameliorate the enhancement of drought stress caused by NH3 on Scots pine.
26 At this time there is insufficient information to offer any general conclusions about the
27 interactive effects of O3 and NH3.
28
29 AX9.3.6.4 Hydrogen Fluoride and Other Gaseous Pollutants
30 Although HF and other fluorides are important local air pollutants associated with
31 aluminum smelting and superphosphate fertilizer manufacture, no studies of possible interactions
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1 with oxidants appear to have been reported since that of MacLean (1990). He found that HF
2 retarded the accelerated senescence and loss of chlorophyll resulting from O3 exposure in corn
3 seedlings. However, such an isolated observation cannot be taken to indicate that HF can reduce
4 the impact of O3 on other species or even that the effect would ultimately have led to an effect on
5 mature plants.
6
7 AX9.3.6.5 Acidic Deposition
8 The deposition of acidic species onto vegetation may elicit direct effects on the foliage or
9 indirect effects via changes induced in the soil. The 1996 O3 AQCD (U.S. Environmental
10 Protection Agency, 1996) included an extensive listing of investigations into the effects of O3
11 and acidic deposition (usually in the form of simulated acid rain, SAR) on plant growth and
12 physiology. The majority of studies found no effects of SAR or acidic mists or fogs at pH values
13 greater than about 3.0 and no interactive effects with O3. (In ambient air, pH values less than
14 3.0 have rarely been reported.) In the few reports in which significant interactions were found,
15 most were antagonistic and were explained as probably being the result of increased fertility due
16 to N(V and SO42" supplied in the SAR.
17 Although numerous reviews have recently appeared (e.g., Bussotti and Ferretti, 1998;
18 Fliickiger et al., 2002; Fox and Mickler, 1996; Nussbaum et al., 1999; and Sheppard and Cape,
19 1999), the shift in interest in air pollution effects away from acid deposition has resulted in little
20 new research having been reported over the past 10 or so years. In most of the reported studies,
21 no effects due to the O3 exposures, the SAR treatments used, or their combinations were
22 observed, e.g., Baker et al. (1994) on loblolly pine; Laurence et al. (1997), and Vann et al.
23 (1995) on red spruce; and Laurence et al. (1996) on sugar maple. Branch chamber studies of
24 12-year-old Ponderosa pine trees by Mom en et al. (1997, 1999) revealed no O3 effects or
25 interactions. With red spruce, Sayre and Fahey (1999) noted no effects of O3 on the foliar
26 leaching of Ca or Mg, which only became significant with SAR at pH 3.1. Izuta (1998)
27 observed no interactions with Nikko fir (Abies homolepis), although SAR at ph 4.0 reduced dry
28 matter. Shan et al. (1996) reported adverse effects of O3 but none attributable to SAR on the
29 growth ofPinus armandi.
30 With herbaceous species, Ashenden et al. (1995) noted significant antagonistic interactions
31 of O3 and acid mist in white clover, in which the adverse effect of low pH was countered by O3.
August 2005 AX9-144 DRAFT-DO NOT QUOTE OR CITE
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1 In contrast, Ashenden et al. (1996) found that, although pH 2.5 mist caused a significant
2 stimulation of the growth of ryegrass attributed to a fertilizer effect, and O3 caused reduced
3 growth, there was no interaction. Bentgrass (Agrostis capillaris) behaved similarly.
4 A study by Bosley et al. (1998) on the germination of spores of the moss, Polytrichum
5 commune, and the ferns, Athyrium felix-femina and Onoclea sensibilis, revealed no effect of O3
6 on moss spores, while SAR at pH <4.0 was completely inhibitory. With the ferns, germination
7 was progressively reduced by both increased O3 and acidity.
8 In summary, the few findings of interactions in these recent studies are consistent with the
9 previous conclusion regarding the likelihood of such interactions being antagonistic. However,
10 the interactions observed were in each case largely the result of the response to the lowest pH
11 used, which, in several cases, was below 3.0, and hence may not be relevant to most field
12 conditions.
13
14 AX9.3.6.6 Heavy Metals
15 As there appears to have been no further research into the interactions of oxidants with
16 heavy metal pollutants, our understanding is unchanged from at the time of the 1996 O3 AQCD
17 (U.S. Environmental Protection Agency, 1996). As noted therein, the limited data available
18 from early studies indicates varying degrees of enhancement of any adverse effects of O3 but
19 precludes the development of any response relationships.
20
21 AX9.3.6.7 Mixtures of Ozone with Two or More Pollutants
22 In many airsheds, the mixtures that occur, both concurrently and over time, may involve
23 three or more pollutants. Very little useful information exists on the effects of O3 with multiple
24 pollutants. As the 1996 criteria document and others have pointed out, most of the early studies
25 on such combinations can be discounted, because of their use of (1) high and environmentally
26 irrelevant exposure concentrations and (2) unrealistic, repetitive exposure profiles (Barnes and
27 Wellburn, 1998; U.S. Environmental Protection Agency, 1996).
28 The large investment in experimental facilities required to study these complex interactions
29 is a major deterrent. So, although the topic has been included in several reviews that have
30 appeared in the last decade, there appear to have been only two studies that have provided new
31 information on the effects of O3 in combination with more than one other pollutant stress.
August 2005 AX9-145 DRAFT-DO NOT QUOTE OR CITE
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1 Ashenden et al. (1996) studied the effects of O3 and/or (SO2 + NO2) with four acidities of SAR
2 applied to each gas treatment, on white clover and two pasture grasses (Lolium perenne and
3 Agrostis capillaris). With each species, the antagonism reported for the O3 x SAR interaction
4 (Section AX9.3.4.6.5) tended to be nullified by concurrent exposure to the other gases, while the
5 combination of the three gaseous pollutants resulted in the most severe growth inhibition,
6 regardless of the acidity of the SAR.
7 With such meager evidence, no clear conclusions can be drawn as to the ways in which the
8 effects of multiple airborne stressors could influence or be influenced by O3.
9
10 AX9.3.7 Interactions with Agricultural Chemicals
11 The review of interactions involving O3, plants, and various agricultural chemicals
12 presented in the 1996 O3 AQCD (U.S. Environmental Protection Agency, 1996) remains a valid
13 assessment of our limited knowledge of these interrelationships. Our knowledge is largely based
14 on the protection against O3 afforded to a range of crop species by applications of various
15 chemicals, particularly fungicides, such as benomyl (benlate; methyl-l-[butylcarbamoyl]-2-
16 benzimidazolecarbamate) and several carbamates and triazoles. A recent report has added
17 azoxystrobin (AZO) and epoxyconazole (EPO) to the list (Wu and Tiedemann, 2002). Foliar
18 sprays of either AZO or EPO provided 50 to 60% protection against O3 injury to barley
19 (Hordeum vulgare) leaves. Both had similar modes of action involving stimulation of the levels
20 of antioxidant enzymes such as SOD, ascorbate peroxidase, guaiacol peroxidase, and catalase.
21 In contrast, applications of herbicides have yielded variable results ranging from increased
22 sensitivity to protection from O3; the nature of the effect is usually species- or cultivar-
23 dependent. Although of less wide application, some plant growth retardants have also been
24 found to provide protection, but no insecticide appears to have been clearly shown to have
25 similar properties.
26 Despite the attraction of the use of permitted chemicals to provide crop protection, the
27 statement in the 1996 O3 AQCD (U.S. Environmental Protection Agency, 1996) is still valid:
28 "It is premature to recommend their use specifically for protecting crops from the adverse effects
29 of O3, rather than for their primary purpose."
30
August 2005 AX9-146 DRAFT-DO NOT QUOTE OR CITE
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1 AX9.3.8 Factors Associated with Global Climate Change
2 During the last decade, interest in the effects of climatic change on vegetation has replaced
3 concerns over the purported causes of forest decline and the effects of acidic deposition. Two
4 specific components of climate change have been singled out as the foci of most of the research
5 activity:
6 • the effects of increasing mean global CO2 concentrations in the lower atmosphere, and
7 • the effects of increasing levels of surface-level irradiation by UV-B (the result of
stratospheric O3 depletion).
8 In spite of the crucial role of temperature as a climatic determinant (Monteith and Elston, 1993),
9 the effects of increasing mean global temperatures and their interactions with increasing CO2
10 levels in particular have received less attention.
11 All of the biotic and chemical interactions with oxidants discussed in the preceding
12 sections may be modified by these climatic changes. However, research activities have largely
13 focused on the two-way O3 x CO2 interaction. Little if any experimental evidence exists related
14 to three-way interactions, such as O3 x CO2 x disease or O3 x CO2 x nutrient availability,
15 although such interactions cannot be predicted from the component two-way interactions.
16 Numerous reviews have appeared since the 1996 O3 AQCD (U.S. Environmental
17 Protection Agency, 1996) dealing with the issues involved. General reviews include
18 publications of IPCC (1996, 2001); and UNEP (1993, 1999); the volume by Wellburn (1994);
19 the volumes edited by Alscher and Wellburn (1994), De Kok and Stulen (1998), Singh (2000),
20 and Yunus and Iqbal (1996); and papers by Idso and Idso (1994), Krupa and Groth (2000), Luo
21 et al. (1999), Polle and Pell (1999), Poorter and Perez-Soba (2001), Runeckles (2002), and
22 Weubbles et al. (1999). Effects on agriculture and crop production, growth, and metabolism
23 have been reviewed by Groth and Krupa (2000), Rotter and Van De Geijn (1999), and Schnug
24 (1998); effects on forests have been reviewed by Bortier et al. (2000a); with focus on insect
25 pests, Docherty et al. (1997), Karnosky et al. (2001a,b), McLaughlin and Percy (1999), and Saxe
26 etal. (1998).
27 As background to the discussion of interactions with O3, it should be noted that the
28 increased levels of CO2 experienced since the mid-18th century are such that, without abatement
29 of the rates of increase, increased levels of from 540 to 970 ppm have been projected by the year
August 2005 AX9-147 DRAFT-DO NOT QUOTE OR CITE
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1 2100 (IPCC, 2001). Such increases in the concentration of CO2, the principal GHG released into
2 the atmosphere, will inevitably lead to increased global mean temperatures, evidence for which
3 is already available from oceanic, icepack, and other records. The latest estimates of the global
4 warming are for an increase in the range of 1.4 to 5.8 °C over this century, in contrast to the
5 0.6 °C rise experienced since 1900 (IPCC, 2001). However, considerable uncertainty is
6 associated with such projections of future increases in global temperature.
7 The use of elevated CO2 concentrations has been common practice for many years in the
8 production of many greenhouse crops. Much of our early knowledge of the effects of higher
9 than ambient CO2 levels on plant growth derives from this application, coupled with research of
10 plant physiologists on how CO2 concentrations affect the process of photosynthesis. Information
11 available about the effects of increased CO2 levels on photosynthesis and stomatal function, in
12 particular, has provided the underlying bases for numerous process models that simulate plant
13 growth under stress and in changed climates.
14 Although simple O3 x temperature interactions were discussed in Section AX9.3.4.2, the
15 close linkage between global CO2 levels and global mean temperatures in the context of climate
16 change requires that an assessment of the interactive effects with O3 should focus, as much as
17 possible, on interactions involving all three factors.
18
19 AX9.3.8.1 Ozone-Carbon Dioxide-Temperature Interactions
20 Idso and Idso (1994) reviewed several hundred reports published between 1982 and 1994
21 on the effects of increased CO2 on plant growth and net photosynthesis. Their survey covered a
22 wide range of temperate and tropical, herbaceous and perennial species, including coniferous
23 trees. They concluded that, for responses to a 300-ppm increase in CO2, somewhat less than a
24 doubling of present-day levels, but somewhat greater than the 540 ppm lower limit suggested by
25 the IPCC (IPCC, 2001), averaged across all species:
26 • light intensity had a negligible effect on net photosynthesis other than at limiting low
intensities under which the CO2-driven enhancement was increased;
27 • increased temperature tended to increase the CO2-driven enhancement of dry matter
accumulation (growth) and net photosynthesis;
28 • drought conditions tended to increase the CO2-driven enhancements of both growth and
net photosynthesis, but increased salinity had little effect;
August 2005 AX9-148 DRAFT-DO NOT QUOTE OR CITE
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1 • mineral nutrient deficiency (especially of nitrogen) tended to increase the CO2-driven
enhancement of growth; and
2 • in the presence of air pollutants (especially SO2 and NOX), the CO2-driven enhancement
of net photosynthesis tended to be increased.
3 It should be noted that the statement that CO2-enhanced growth increased with temperature
4 referred to total dry matter accumulation by the whole plant and not to the yield of grain, fruit, or
5 seed. Unfortunately, despite the existence of several reports at the time, the summary of
6 interactions with air pollutants contained only a single reference to O3, i.e., Pfirrmann and
7 Barnes (1993), who reported surprisingly that a doubling of CO2 levels led to a 27% increase in
8 dry weight of radish but that the combination with O3 led to a 77% increase.
9 The more recent reviews by Rudorff et al. (2000) and Olszyk et al. (2000) have
10 addressed CO2 x O3 interactions in detail, with the latter focusing on the implications for
11 ecosystems. They concluded that:
12 • the effects of both gases on stomatal closure were predominantly additive, with little
evidence of interaction;
13 • increased photosynthesis resulting from elevated CO2 may be canceled by exposures to
high O3 levels;
14 • foliar O3 injury is reduced by elevated CO2; and
15 • interactions between CO2 and O3 can affect storage carbohydrates, leaf free-radical
metabolism, and carbon allocation to shoots and roots.
16 Olszyk et al. (2000) also made specific note of the relative lack of information on below-ground
17 effects.
18 Much of the recently published information on the effects of increased CO2 and O3 levels
19 is summarized in Table AX9-12. Note that the table only lists the directions of O3-induced
20 effects and any modifications of these effects resulting from elevated CO2, not their magnitudes.
21 These directions are usually, but not necessarily, the same as the corollary effects of O3
22 on CO2-induced responses.
23 The bulk of the available evidence clearly shows that, under the various experimental
24 conditions used (which almost exclusively employed abrupt or "step" increases in CO2
25 concentration, as discussed below), increased CO2 levels may protect plants from the adverse
August 2005 AX9-149 DRAFT-DO NOT QUOTE OR CITE
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Table AX9-12. Effects of Increased Carbon Dioxide on Ozone-Induced Responses of Plants at the Metabolic,
Physiological, and Whole-Plant Levels
to
o
o
H
6
o
o
H
O
o
HH
H
W
Plant Response
Block emical/Metabolic
Ascorbate peroxidase
Catalase
Chlorophyll
Glutathione reductase
Glycolate oxidase
Hydroxypyravate
reductase
Rubisco
Co2 Effects:
O3 Response3
V
V
V
V
A
V
V
V
V
V
o
o
V
V
V
V
[A]
V
O3 Effects: V, Decrease; A, Increase; O, No Significant Effect.
A, Additive/Synergistic; T, Antagonistic/Ameliorative; O, No Significant Effect.
CO2 Modification"
T
OD
OD
OD
OD
OD
T
V
T
V
OD
OD
V
OD
T
T
T
V
Species
Wheat (T. aestivum)
Sugar maple (A. saccharum)
Trembling aspen (P.
tremuloides)
Wheat (T. aestivum)
Soybean (G. max)
Wheat (T. aestivum)
Potato (S. tuberosum)
Soybean (G. max)
Wheat (T. aestivum)
Sugar maple (A. saccharum)
Aspen (P. tremuloides)
Soybean (G. max)
Soybean (G. max)
Soybean (G. max)
Wheat (T. aestivum)
Trembling aspen
(P. tremuloides)
Sugar maple (A. saccharum)
Facility0
CSTR, P
CEC,P
FACE, G
CEC,P
CEQP
OTC,P
OTC, G
OTC, G
CSTR, P
CEQP
FACE, G
OTC,P
OTC,P
OTC,P
OTC, G
FACE, G
CEQP
Reference
Rao etal. (1995)
Niewiadomska et al. (1999)
Wustman etal. (2001)
McKeeetal. (1997b)
Niewiadomska et al. (1999)
Booker etal. (1997)
Donnelly et al. (2000); Ommen et al.
(1999)
Donnelly et al. (200 la)
Heagle et al. (1998); Reid and Fiscus
(1998); Reid etal. (1998)
Rao etal. (1995)
Niewiadomska et al. (1999)
Wustman etal. (2001)
Booker etal. (1997)
Booker etal. (1997)
Reid etal. (1998)
McKee et al. (2000)
Noormets et al. (2001)
Gaucher et al. (2003)
-------
Table AX9-12 (cont'd). Effects of Increased Carbon Dioxide on Ozone-Induced Responses of Plants at the
Metabolic, Physiological, and Whole-Plant Levels
to
o
o
O3 Effects: V, Decrease; A, Increase; O, No Significant Effect.
'~f> Co2 Effects: A, Additive/Synergistic; T, Antagonistic/Ameliorative; O, No Significant Effect.
VO
i
^
O
H
6
o
0
H
O
O
0
o
H
W
Plant Response O3 Response3 CO2 Modification"
Biochemical/Metabolic (cont'd)
Rubisco activity V V
V T
V [T]
V T
Superoxide dismutase O OD
O OD
Physiological
Stomatal conductance V A
V T
V A
V V
V [A]
A - OD T - OD
V A
Species
Soybean (G. max)
Wheat (T. aestivum)
Wheat (T. aestivum)
European beech (F. sylvatica)
Wheat (T. aestivum)
Sugar maple (A. saccharum)
Radish (R. sativus)
Soybean (G. max)
Soybean (G. max)
Bean (P. vulgaris)
White clover (T. repens) (O3-
sensitive)
White clover (T. repens)
(O3-tolerant)
Tomato (L. esculentum)
Facility0
OTC,P
CEC,P
OTC, G
CEC,P
CEC,P
CEC,P
CEC,P
OTC, G
OTC, P,G
OTC,P
CSTR, P
CSTR, P
CEC,P
Reference
Reid etal. (1998)
McKeeetal. (1995)
McKee et al. (2000)
Liitz et al. (2000)
McKeeetal. (1997b)
Niewiadomska et al. (1999)
Barnes and Pfirrmann (1992)
Mulchi etal. (1992)
Booker et al. (2005); Fiscus et al. (1997)
Heagle et al. (2002)
Heagle etal. (1993)
Heagle etal. (1993)
Hao et al. (2000)
-------
Table AX9-12 (cont'd). Effects of Increased Carbon Dioxide on Ozone-Induced Responses of Plants at the Metabolic,
Physiological, and Whole-Plant Levels
to
o
o
u\
VO
1
to
o
H
1
O
o
0
H
O
d
o
0
o
H
W
O3 Effects: V, Decrease; A, Increase; O, No Significant Effect.
Co2 Effects:
Plant Response O3 Response3
Physiological (cont'd)
Stomatal conductance V
(cont'd)
V
[V]
V
V
A
V
V
OD
OD
OD
OD
[A]
V
A, Additive/Synergistic; T, Antagonistic/Ameliorative; O, No Significant Effect.
CO2 Modification"
A
A
A
OD
O-A
OD
OD
OD
OD
OD
OD
OD
V
A
Species
Potato (S, tuberosum)
Wheat (T. aestivum)
Agropyron smithii
Koeleria cristata
Bouteloua curtipendula
Schizachyrium scoparium
Black cherry (P. serotina)
Green ash (F. pennsylvanica)
Yellow poplar (L. tulipifera)
Trembling aspen
(P. tremuloides)
Facility0
OTQG
CEQP
CEQP
OTQG
CEQP
CEQP
CEQP
CEQP
CEQP
CEQP
CSTR, P
CSTR, P
CSTR, P
CEQP
Reference
Finnan et al. (2002)
Balaguer etal. (1995)
Barnes etal. (1995)
McKee etal. (1995)
Mulholland et al. (1997b)
Donnelly etal. (1998)
Tiedemann and Firsching (2000)
Volin etal. (1998)
Volin etal. (1998)
Volin etal. (1998)
Volin etal. (1998)
Loats and Rebbeck (1999)
Loats and Rebbeck (1999)
Loats and Rebbeck (1999)
Volin etal. (1998)
-------
Table AX9-12 (cont'd). Effects of Increased Carbon Dioxide on Ozone-Induced Responses of Plants at the Metabolic,
Physiological, and Whole-Plant Levels
to
o
o
VO
1
OJ
O
^
H
6
o
0
H
O
o
0
o
H
W
O3 Effects: V, Decrease; A, Increase; O, No Significant Effect.
Co2 Effects:
Plant Response O3 Response3
Physiological (cont'd)
Stomatal conductance O -V
(cont'd)
OD
[A]
Photosynthesis V
V
V
V
\f
V
OD
OD
[V]
V
V
A, Additive/Synergistic; T, Antagonistic/Ameliorative; O, No Significant Effect.
CO2 Modification"
V
OD
V
V
T
T
T
OD
OD
OD
[T]
T
T
OD
Species
Red oak (Q. rubra)
Durmast oak (Q. petraea)
Radish (R. sativus)
Soybean (G. max)
Soybean (G. max)
Bean (P. vulgaris)
Cotton (G. hirsutum)
Tomato (L. esculentum)
Potato (S. tuberosum)
Wheat (T. aestivum)
Wheat (T. aestivum)
Facility0
FACE, G
CEQP
CEQP
CEQP
OTQP
OTC, G,P
OTQP
OTQP
CEQP
OTQG
OTQG
CEQP
OTQG
CEQP
CEQP
Reference
Noormets et al. (2001)
Volin etal. (1998)
Broadmeadow et al. (1999)
Barnes and Pfirrmann (1992)
Booker etal. (1997)
Mulchi et al. (1992)
Heagle et al. (2002)
Heagleetal. (1999b)
Hao et al. (2000)
Donnelly et al. (200 la)
Lawson etal. (200 Ib)
Barnes etal. (1995)
Donnelly et al. (2000);
Mulholland et al. (1997b)
Reid and Fiscus (1998)
Tiedemann and Firsching (2000)
Cardoso-Vilhena and Barnes (2001)
-------
Table AX9-12 (cont'd). Effects of Increased Carbon Dioxide on Ozone-Induced Responses of Plants at the Metabolic,
Physiological, and Whole-Plant Levels
to
o
o
VO
1
(_/l
r\
O
H
6
o
0
H
O
Co2 Effects:
Plant Response O3 Response3
Physiological (cont'd)
Photosynthesis (cont.) OD
\l
O-V
[V]
V
V
OD
[V]
OD
V
V
OD
V
O3 Effects: V, Decrease; A, Increase; O, No Significant Effect.
A, Additive/Synergistic; T, Antagonistic/Ameliorative; O, No Significant Effect.
CO2 Modification"
OD
T
OD
T
T
T
OD
T
OD
V
T
OD
T
Species
Agropyron smithii
Koeleria cristata
Bouteloua curtipendula
Schizachyrium scoparium
Ponderosa pine
(P. ponderosa)
Scots pine (P. sylvestris)
Black cherry (P. serotind)
Green ash (F. pennsylvanica)
Yellow poplar (L. tulipiferd)
Trembling aspen (P.
tremuloides)
European beech (F. sylvaticd)
Red oak (Q. rubrd)
Sugar maple (A. saccharum)
Facility0
CEQP
CEQP
CEQP
CEQP
CEQG
OTQG
CSTR, P
CSTR, P
CSTR, P
CEQP
CEQP
CEQP
CEQP
Reference
Volinetal. (1998)
Volinetal. (1998)
Volinetal. (1998)
Volinetal. (1998)
Olszyketal. (2001)
Kellomaki and Wang (1997a,b)
Loats and Rebbeck (1999)
Loats and Rebbeck (1999)
Loats and Rebbeck (1999)
Volinetal. (1998)
Grams etal. (1999)
Volinetal. (1998)
Gaucher et al. (2003)
O
HH
H
W
-------
Table AX9-12 (cont'd). Effects of Increased Carbon Dioxide on Ozone-Induced Responses of Plants at the Metabolic,
Physiological, and Whole-Plant Levels
to
o
o
O3 Effects: V, Decrease; A, Increase; O, No Significant Effect.
'~f> Co2 Effects: A, Additive/Synergistic; T, Antagonistic/Ameliorative; O, No Significant Effect.
VO
i
0,
o
>
H
6
o
0
H
O
O
0
o
H
W
Plant Response O3 Response3 CO2 Modification"
Physiological (cont'd)
Photorespiration A V
V A
Growth, Yield
Total biomass V V
V T
V T
V T
[V] OD
V V
OD OD
V V
OD OD
Species
Soybean (G. max)
Wheat (T. aestivum)
Parsley (P. sativum)
Bean (P. vulgaris)
Soybean (G. max)
Alfalfa (M. sativa)
White clover (T. repens)
(O3-sensitive)
White clover (T. repens)
(O3-tolerant)
Tomato (L. esculentum)
Potato (S. tuberosum)
Facility0
OTQP
CEQP
CEQP
CEQP
OTQP
OTC, G
CSTR, P
OTC, P,G
OTQP
CEQP
CSTR, P
CSTR, P
CEQP
CSTR, P
OTC, G
Reference
Booker etal. (1997)
McKeeetal. (1997b)
Cardoso-Vilhena et al. (1998)
Cardoso-Vilhena et al. (1998)
Heagle et al. (2002)
Mulchi etal. (1992)
Reinertetal. (1997)
Booker et al. (2005)
Booker et al. (2004)
Johnson etal. (1996a)
Heagle etal. (1993)
Heagle etal. (1993)
Hao et al. (2000)
ReinertandHo(1995)
Donnelly etal. (200 Ib);
Persson et al. (2003)
-------
Table AX9-12 (cont'd). Effects of Increased Carbon Dioxide on Ozone-Induced Responses of Plants at the Metabolic,
Physiological, and Whole-Plant Levels
to
o
o
O3 Effects: V, Decrease; A, Increase; O, No Significant Effect.
'~f> Co2 Effects: A, Additive/Synergistic; T, Antagonistic/Ameliorative; O, No Significant Effect.
VO
i
Ul
Oi
O
f?
H
1
O
o
0
H
O
o
0
o
H
W
Plant Response O3 Response3 CO2 Modification6 Species
Growth, Yield (cont'd)
Total biomass (cont'd) V OD
V T Mustard (S. alba)
V V Plantain (P. major)
V V Cotton (G. hirsutum)
V V Wheat (T. aestivum)
[ V ] T Wheat (T. aestivum)
V OD Wheat (T. aestivum)
V [T] Wheat (T. aestivum)
[ V ] T Timothy (P. pratense)
V T Agropyron smithii
V T Koeleria cristata
Facility0
OTC, G
CEC,P
CEC,P
OTC,P
CEC,P
OTC, G
OTC,P
CEC,P
OTC, G
CSTR, P
OTC, G
OTC, G
CEC,P
OTC, G
CEC,P
CEC,P
CEC,P
Reference
Lawsonetal. (200 la)
Cardoso-Vilhena et al. (1998)
Cardoso-Vilhena et al. (1998)
Booker (2000)
Heagleetal. (1999b)
Cardoso-Vilhena et al. (1998)
Fangmeier et al. (1996)
Heagle et al. (2000)
McKeeetal. (1997a)
Pleijel et al. (2000)
Rao etal. (1995)
Rudorffetal. (1996a)
Bender etal. (1999)
Mulholland et al. (1997a)
Cardoso-Vilhena et al. (1998)
Tiedemann and Firsching (2000)
Ewart and Pleijel (1999)
Johnson etal. (1996a)
Volin etal. (1998)
Volin etal. (1998)
-------
Table AX9-12 (cont'd). Effects of Increased Carbon Dioxide on Ozone-Induced Responses of Plants at the Metabolic,
Physiological, and Whole-Plant Levels
to
o
o
VO
1
ij
o
^
H
6
O
0
H
O
O
0
o
H
W
O3 Effects: V, Decrease; A, Increase; O, No Significant Effect.
Co2 Effects:
Plant Response O3 Response3
Growth, Yield (cont'd)
Total Biomass V
(cont'd)
V
OD
V
V
[V]
A
OD
[A]
V
V
V
OD
V
A, Additive/Synergistic; T, Antagonistic/Ameliorative; O, No Significant Effect.
CO2 Modification"
V
OD
OD
V
A
OD
T
OD
A
V
V
OD
OD
V
Species
Corn (Z. /ways)
Bouteloua curtipendula
Schizachyrium scoparium
Ponderosa pine (P. ponderosa)
Birch (B. pendula)
Black cherry (P. serotina)
Green ash (F. pennsylvanica)
European ash (F. excelsior)
Yellow poplar (L. tulipiferd)
Sugar maple (A. saccharum)
Trembling aspen
(P. tremuloides)
(O3-tolerant clone}
Trembling aspen
(P. tremuloides)
(O3-sensitive clone)
Red oak (Q. rubra)
Durmast oak (Q. petraea)
Facility0
OTC, G
CEQP
CEQP
CEC, G
CEQP
CSTR, P
CSTR, P
OTC, G
CSTR, P
CEQP
CEQP
OTQP
OTC, G
OTC, G
CEQP
CEQP
OTC, G
Reference
Rudorffetal. (1996a)
Volinetal. (1998)
Volinetal. (1998)
Olszyketal. (2001)
Kytoviita et al. (1999)
Loats and Rebbeck (1999)
Loats and Rebbeck (1999)
Broadmeadow and Jackson (2000)
Loats and Rebbeck (1999)
Gaucher et al. (2003)
Volinetal. (1998)
Dicksonetal. (1998)
Dicksonetal. (2001)
Dicksonetal. (2001)
Volinetal. (1998)
Broadmeadow et al. (1999)
Broadmeadow and Jackson (2000)
-------
-------
Table AX9-12 (cont'd). Effects of Increased Carbon Dioxide on Ozone-Induced Responses of Plants at the Metabolic,
Physiological, and Whole-Plant Levels
to
o
o
VO
1
VO
o
s
H
6
O
0
H
O
O
0
o
H
W
Co2 Effects:
Plant Response O3 Response3
Growth, Yield (cont'd)
Relative growth rate V
V
V
V
OD
OD
OD
V
[V]
V
OD
Specific leaf area-SLA V
V
V
V
O3 Effects: V, Decrease; A, Increase; O, No Significant Effect.
A, Additive/Synergistic; T, Antagonistic/Ameliorative; O, No Significant Effect.
CO2 Modification"
V
T
T
T
OD
OD
OD
V
V
T
OD
OD
T
T
OD
Species
Wheat (T. aestivum)
Agropyron smithii
Koeleria cristata
Bouteloua curtipendula
Schizachyrium scoparium
European ash (F. excelsior)
Trembling aspen (P.
tremuloides)
Red oak (Q. rubra)
Durmast oak (Q. petraea)
Scots pine (P. sylvestris)
Radish (R. sativus)
Soybean (G. max)
Cotton (G. hirsutum)
Facility0
CEQP
CEQP
CEQP
CEQP
CEQP
CEQP
OTQG
CEQP
CEQP
OTQG
OTQG
CEQP
OTQP
OTQP
OTQG
Reference
Barnes etal. (1995)
Cardoso-Vilhena and Barnes (2001)
Volin etal. (1998)
Volin etal. (1998)
Volin etal. (1998)
Volin etal. (1998)
Broadmeadow and Jackson (2000)
Volin etal. (1998)
Volin etal. (1998)
Broadmeadow and Jackson (2000)
Broadmeadow and Jackson (2000)
Barnes and Pfirrmann (1992)
Reid etal. (1998)
Booker (2000)
Mulchi etal. (1992)
-------
S-
to
o
Table AX9-12 (cont'd). Effects of Increased Carbon Dioxide on Ozone-Induced Responses of Plants at the Metabolic,
Physiological, and Whole-Plant Levels
H
6
o
o
H
/O
Co2 Effects:
Plant Response O3 Response3
Growth, Yield (cont'd)
Specific leaf area-SLA V
(cont'd)
A
A
A
OD
A
A
A
Root/shoot ratio V
V
V
OD
A
O3 Effects: V, Decrease; A, Increase; O, No Significant Effect.
A, Additive/Synergistic; T, Antagonistic/Ameliorative; O, No Significant Effect.
CO2 Modification"
A
A
A
A
OD
OD
A
A
V
A
T
OD
A
Species
White clover (T. repens)
(O3-sensitive)
White clover (T. repens)
(O3-tolerant)
Agropyron smithii
Koeleria cristata
Bouteloua curtipendula
Schizachyrium scoparium
Trembling aspen
(P. tremuloides)
Red oak (Q. rubra)
Radish (R. sativus)
AlfaUa (M. sativa)
White clover (T. repens)
(O3-sensitive)
White clover (T. repens)
(O3-tolerant)
Wheat (T. aestivum)
Facility0
CSTR, P
CSTR, P
CEC,P
CEQP
CEQP
CEQP
CEQP
CEQP
CEQP
CEQP
CSTR, P
CSTR, P
CEQP
Reference
Heagleetal. (1993)
Heagleetal. (1993)
Volinetal. (1998)
Volinetal. (1998)
Volinetal. (1998)
Volinetal. (1998)
Volinetal. (1998)
Volinetal. (1998)
Barnes and Pfirrmann (1992)
Johnson etal. (1996a)
Heagleetal. (1993)
Heagleetal. (1993)
McKeeetal. (1997a)
o
HH
H
W
-------
S-
to
o
VO
H
6
o
o
H
/O
o
HH
H
W
Table AX9-12 (cont'd). Effects of Increased Carbon Dioxide on Ozone-Induced Responses of Plants at the Metabolic,
Physiological, and Whole-Plant Levels
Plant Response
Growth, Yield (cont'd)
Root/shoot ratio
(cont'd)
Foliar injury
Co2 Effects:
O3 Response3
V
V
OD
OD
OD
OD
A
A
A
A
A
A
A
O3 Effects: V, Decrease; A, Increase; O, No Significant Effect.
A, Additive/Synergistic; T, Antagonistic/Ameliorative; O, No Significant Effect.
CO2 Modification"
V
OD
OD
OD
OD
OD
T
T
T
V
V
T
T
Species
Timothy (P. pratense)
Soybean (G. max)
Black cherry (P. serotina)
Green ash (F. pennsylvanica)
Yellow poplar (L. tulipiferd)
Aspen (P. tremuloides)
Potato (S. tuberosum)
Bean (P. vulgaris)
Soybean (G. max)
Cotton (G. hirsutum)
Wheat (T. aestivum)
Trembling aspen (P.
tremuloides)
European beech (F. sylvatica)
Facility0
CEQP
OTC, G,P
CSTR, P
CSTR, P
CSTR, P
OTC, G
OTC, G
OTC,P
OTC,P
OTC,P
CEQP
OTC, G
FACE, G
CEQP
Reference
Johnson etal. (1996a)
Booker et al. (2005)
Loats and Rebbeck (1999)
Loats and Rebbeck (1999)
Loats and Rebbeck (1999)
Dickson etal. (2001)
Donnelly et al. (200 Ib)
Persson et al. (2003)
Heagle et al. (2002)
Heagle etal. (1998)
Heagle etal. (1999b)
Barnes etal. (1995)
Mulholland et al. (1997a)
Karnosky etal. (1999)
Wustman etal. (2001)
Grams etal. (1999)
O3 m < 0.15 ppm.
b CO2-modifications of O3-effects resulting from ~2* present levels. (Trends are shown in brackets. Pronounced changes with ontogeny are, for example,
indicated thus: OD-T.)
0 Exposure facilities used: CEC: controlled environment chambers; CSTR: continuously stirred tank reactors (Heck etal., 1978); FACE: free air CO2
enrichment facilities; OTC: open-top chambers. G: plants rooted in the ground; P: plants grown in pots. All species are Q except corn, Bouteloua
and Schizachyrium.
-------
1 effects of O3 on growth. This protection may be afforded in part by CO2 acting together with O3
2 in inducing stomatal closure, thereby reducing O3 uptake, and in part by CO2 reducing the
3 negative effects of O3 on Rubisco and its activity in CO2-fixation. Although both CO2-induced
4 and O3-induced decreases in stomatal conductance have been observed primarily in short-term
5 studies, recent data clearly show a long-term and sustained reduction in stomatal conductance
6 under elevated CO2 for a number of species (Ainsworth and Long, 2005; Ellsworth et al., 2004;
7 Gunderson et al., 2002). Instances of increased stomatal conductance have also been observed in
8 response to O3 exposure, suggesting partial stomatal dysfunction after extended periods of
9 exposure (Maier-Maercker, 1998).
10 At the mechanistic level, Rubisco plays a key role in CO2-assimilation, and while both O3
11 and elevated CO2 per se can lead to reduced activity, CO2 can also reverse the O3-induced
12 inhibition of Rubisco activity and photosynthesis (Table AX9-12). However, in their review of
13 the possible mechanisms involved, Polle and Pell (1999) cautioned that Rubisco should not be
14 regarded "as a unique target for the interaction of the two gases." But it is clear from the bulk of
15 the evidence in Table AX9-12 that elevated CO2 levels can ameliorate the inhibition of growth
16 caused by O3 in many species, although the precise balance among the mechanisms involved
17 may well vary from species to species. Three important caveats must be raised with regard to
18 the findings presented in Table AX9-12:
19 • the applicability of results from experiments with an abrupt (step) increase in CO2 level
to understanding the consequences of the gradual increase in CO2 predicted for the
troposphere over the next hundred years;
20 • the validity of the findings in several long-term studies (particularly with tree species)
conducted using potted plants, because of possible added stressors imposed on their root
systems relative to trees growing in the field; and
21 • the relevance to understanding the effects of climate change of studies focused solely on
CO2 enrichment at current ambient conditions of temperature and precipitation patterns
that provide no insights into possible interactive effects as these other climatic variables
change concurrently with increasing CO2 (Intergovernmental Panel on Climate Change
(IPCC), 2001).
22 The first caveat concerns the distinctly different natures of the exposures to O3 and CO2
23 experienced by plants in the field. Changes in the ambient concentrations of these gases have
24 very different dynamics. In the context of climate change, CO2 levels increase relatively slowly
August 2005 AX9-162 DRAFT-DO NOT QUOTE OR CITE
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1 and may change little over several seasons of growth. On the other hand, O3 presents a
2 fluctuating stressor with considerable hour-to-hour and day-to-day variability (Polle and Pell,
3 1999). Almost all of the evidence presented in Table AX9-12 comes from experimentation
4 involving plants grown from the outset in, or subjected to, an abrupt or step increase to a higher
5 (more or less double), steady CO2 concentration. In contrast, the O3 exposure concentrations
6 usually varied from day to day. Luo and Reynolds (1999), Hui et al. (2002), and Luo (2001)
7 noted the difficulties in predicting the likely effects of a gradual CO2 increase from experiments
8 involving a step increase or those using a range of CO2 concentrations. Indeed, although using
9 the much accelerated timescale of an 80-day growing season, Hui et al. (2002) clearly showed
10 significant differences between the rates and magnitudes of various physiological and growth
11 responses of plantain (Plantago lanceolatd) to CO2 between gradual and step increase
12 treatments. The authors concluded that, even though there were major differences in most of the
13 parameters studied between the gradual and step treatments, "the convergence of the measured
14 parameters at the end of the experiment provides some encouragement for the applicability of
15 step-type experiments in the field; however, the study suggests caution in interpreting early
16 results from short-term studies."
17 In long-term studies, the matter of photosynthetic acclimation to elevated CO2 levels has to
18 be considered. Lawlor and Keys (1993) define acclimation in terms of long-term (days, weeks),
19 irreversible physiological changes, in contrast to regulation, which relates to more rapid
20 (minutes, hours), reversible changes. Each may be positive or negative, but many studies
21 indicate that, while positive acclimation to elevated CO2 levels initially led to enhanced
22 photosynthesis and growth, negative acclimation ultimately ensued and reduced CO2
23 assimilation and growth rates. However, the consensus from recent studies and reviews is that
24 such negative acclimation is most likely to occur in situations in which plants are grown under
25 some additional stress, induced, e.g., by limitations to growth posed by lack of resources such as
26 water or nutrients. The meta-analysis by Curtis (1996) revealed that slow or little negative
27 acclimation was noted in studies on unstressed tree species with unhindered opportunities for
28 root growth and development, a view originally suggested by Arp and Drake (1991) and largely
29 supported in the review by Eamus (1996). A nonwoody perennial, the rhizomatous wetland
30 sedge, Scirpus olneyi, grown in its natural environment with no edaphic limitations showed no
31 negative acclimation after 4 years; in fact, photosynthetic capacity increased by 31% (Arp and
August 2005 AX9-163 DRAFT-DO NOT QUOTE OR CITE
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1 Drake, 1991). No negative acclimation of well-watered, field-grown Ponderosa pine trees was
2 observed by Tissue et al. (1999) after 6 years of growth at 2x-ambient CO2 levels. Gifford and
3 Morison (1993) have summarized the situation thus: "Where the aerial or root environment for a
4 plant is restricted (as with inter-plant competition, for example), positive feedback is limited and
5 adjustments to the changed resource input balance under high CO2 can include 'down-
6 regulation' of leaf photosynthesis rate as an integral part of a positive growth response."
7 The influence of other environmental stressors is borne out by several long-term tree
8 studies. After 3 years in 565 ppm CO2 in the Duke Forest FACE facility in North Carolina,
9 maturing loblolly pine trees showed only a marginal CO2-induced carbon gain if grown on a
10 nutritionally moderate site, but zero gain if grown on a nutritionally poor site (Oren et al., 2001).
11 This is in sharp contrast to the substantially increased initial growth rates in elevated CO2
12 reported by DeLucia et al. (1999), but it is supported by the observations of Tognetti et al.
13 (2000) on five Mediterranean tree species growing for many years adjacent to geothermal
14 springs releasing CO2 sufficient to provide ambient levels averaging 700 ppm. No significant
15 differences in radial growth of the oaks (Quercus cerris, Q. ilex, and Q. pubescens), strawberry
16 tree (Arbutus unedo), and flowering ash (Fraxinus ornus) could be detected between trees at the
17 naturally enriched site and those at a nearby site exposed to normal ambient CO2 (-350 ppm).
18 The authors concluded that limited availability of water and nutrients may have counteracted any
19 positive effects of CO2 on growth at the enriched site or that the trees had acclimated to the
20 higher CO2 levels.
21 Because the ameliorative effects of CO2 on responses to O3 (Table AX9-14) were reported
22 mostly in short-term studies involving an abrupt increase in CO2 level, it is appropriate to ask
23 whether this amelioration is likely to persist to a time when the ambient CO2 concentration is
24 relatively stable at such levels. Regardless of any negative acclimation due to resource
25 limitations that may occur in the interim, steadily rising CO2 levels may well lead to natural
26 selection and genetic change. Nevertheless, it seems reasonable to expect that the amelioration
27 of O3 impact at elevated CO2 levels will be maintained in many situations, but the negative
28 acclimation that will probably occur in situations where other resources become limiting will
29 reduce the degree of protection.
30 Another caveat regarding the validity of some of the observations in Table AX9-12 is
31 related to the matter of stress-induced negative acclimation to elevated CO2 and concerns related
August 2005 AX9-164 DRAFT-DO NOT QUOTE OR CITE
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1 to using potted plants. Although much of the recent information on CO2 effects has come
2 from experiments with plants rooted in the ground, more than half of the studies listed in
3 Table AX9-12 used potted plants, whether in controlled environment and greenhouse chambers
4 or in OTCs. The degree to which pot-based studies resulted in similar patterns of response to
5 soil-grown plants appears to depend on the treatment conditions and plant growth conditions
6 used in the study. The use of potted plants was a confounding factor in the studies of Taylor
7 et al. (2001) of the differences in leaf growth of poplar (Populus) hybrids between plants
8 exposed to elevated CO2 in controlled environment chambers (potted plants), OTCs, or a FACE
9 facility. Loats and Rebbeck (1999) suggested that their lack of CO2 response in three broadleaf
10 species may have resulted from their use of pot-grown plants. In contrast, Heagle et al. (1999a)
11 found that the relative enhancement of soybean photosynthesis, growth, and yield by CO2
12 enrichment was similar in pots and in the ground. These findings were supported by Booker
13 et al. (2005). The recent meta-analysis of data on the effects of elevated CO2 on soybean
14 physiology and growth by Ainsworth et al. (2002) revealed a threefold smaller stimulation of
15 seed yield in pot-grown than in field-grown plants, even when large (>9 L) pots were used.
16 However, Ainsworth et al. (2002) included a wide range of treatment conditions (e.g., CO2
17 treatments ranging from 500 to!200 ppm) and plant growth conditions in the meta-analysis, so
18 caution is needed when generalizing conclusions about the applicability of pot-based studies.
19 Although the majority of the cases cited in Table AX9-12 indicate that O3 and CO2 act
20 additively or synergistically in causing stomatal closure, there are numerous exceptions.
21 Any reduction in stomatal aperture has consequences other than merely restricting O3 uptake and
22 the exchange of other gases. In particular, stomatal closure initially reduces the rate of
23 transpiration, although increased leaf temperature and VPD associated with stomatal closure can
24 offset decreases in transpiration. In instances where transpiration is reduced, water-use
25 efficiency may increase, however decreased transpirational flux may lead to decreased mineral
26 uptake, which could adversely impact growth over extended periods.
27 Hence, the final caveat regarding Table AX9-12 concerns the interactions of O3 and CO2
28 with other climatic variables, especially mean temperature. In light of the key role played
29 by temperature in regulating physiological processes and modifying plant response to
30 increased CO2 levels (Long, 1991; Morison and Lawlor, 1999) and the knowledge that
31 relatively modest increases in temperature may lead to dramatic consequences in terms of
August 2005 AX9-165 DRAFT-DO NOT QUOTE OR CITE
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1 plant development (Lawlor, 1998), it is unfortunate that much of the large investment in time
2 and resources spent on recent studies of the effects of climate change on vegetation have gone
3 into investigations limited to increasing our knowledge of the effects of higher levels of CO2 at
4 current ambient temperatures.
5 Some attention is now being paid to investigating the concurrent effects of CO2 increases
6 and warming (recently reviewed by Rowland-Bamford [2000] and Morison and Lawlor [1999]),
7 but the observed interactive effects on plant growth are inconsistent. For example, a FACE
8 study with ryegrass (Lolium perenne) showed that increased temperatures (provided by infrared
9 heaters) reduced the dry matter gain resulting from increased CO2 levels (Nijs et al., 1996). The
10 field studies by Shaw et al. (2002) on a California annual grassland dominated by the grasses
11 Avena barbata and Bromus hordeaceus and the forbs Geranium dissectum and Erodium botrys
12 involved free-air increased CO2 as well as increased temperature, precipitation, and N supply.
13 Not only did increased temperature reduce CO2-stimulated net primary productivity (NPP), but
14 increased CO2 itself, combined with other factors, was found to be able to reduce NPP.
15 There have been several investigations of effects on wheat. Batts et al. (1997) used plastic
16 tunnels to create temperature gradients and maintain elevated CO2 levels over field-grown wheat
17 and found that, in each of 4 years of study, a temperature rise of-1.5 °C consistently canceled
18 the growth and yield increases caused by a doubling of the CO2 level above ambient. Similar
19 findings were reported by Van Oijen et al. (1999) and Van Oijen and Ewart (1999) in OTC field
20 studies. Half of the chambers were cooled 1.6 to 2.4 °C below the uncooled chambers, to cancel
21 out the normal temperature increase over ambient, due to the so-called "chamber effect" (usually
22 a 1 to 3 °C increase above ambient temperature (Heagle et al., 1988). Although temperature had
23 no effect on CO2-enhanced assimilation rates, the CO2-enhanced growth and grain yields
24 observed in the cooled chambers were effectively canceled out in the warmer chambers. The
25 authors attributed this effect to accelerated phenology, a shorter period for grain filling, and a
26 lower leaf area index (LAI; total leaf area per unit ground area) in the warmer chambers.
27 Wheeler et al. (1996) observed that the benefit to wheat of doubling the CO2 level was offset by
28 a mean seasonal increase of only 1 to 1.8 °C. With the continuing use of OTCs for field
29 research, Runeckles (2002), has suggested that the temperature rise due to the chamber effect in
30 OTCs should be exploited (and measured) as a means of exploring temperature * CO2 as well as
31 temperature * CO2 * O3 interactions.
August 2005 AX9-166 DRAFT-DO NOT QUOTE OR CITE
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1 An indirect affirmation of the importance of temperature as a component of climate change
2 on wheat yield was provided by Van Oijen and Ewart (1999) using two simulation models,
3 AFRCWHEAT2-O3 and LINTULCC (Ewart et al., 1999). They analyzed data from the
4 ESP ACE-wheat program, which involved 25 OTC experiments in 1994, 1995, and 1996 at nine
5 European locations (Jager et al., 1999). Both models were able to predict control-treatment grain
6 yields closely (5.5 ±1.2 and 5.8 ±1.2 t.ha"1, respectively, versus the observed 5.9 ±1.9 t.ha"1),
7 and both indicated a predominantly negative effect of temperature on the yield response to
8 increased CO2 (a 3 °C rise reduced the gain in yield from 30 to 14%). However, neither model
9 had an R2 > 0.3, indicating that the models included other sources of variability among the sites
10 than the climatic factors. The multiple linear regression developed by Bender et al. (1999) based
11 on the same datasets also included temperature as a highly significant covariant. Both studies
12 are discussed more fully below.
13 Other studies, however, have found positive temperature-related growth effects, as
14 suggested by the early Idso and Idso (1994) analysis. In an OTC study using the perennial
15 grass Festucapratensis in which a temperature increase of 3 °C above ambient was combined
16 with CO2-enrichment to 700 ppm, both CO2 and temperature caused increases in total above-
17 ground biomass (Hakala and Mela, 1996). Studies with potato (Cao et al., 1994) and soybean
18 (Ziska and Bunce, 1997) using potted plants in controlled environment chambers also showed
19 temperature-enhanced increases in growth in enriched CO2 atmospheres. Read and Morgan
20 (1996) compared the effects of enriched CO2 and temperature on two grasses: cool-season
21 Pascopyrum smithii and warm-season Bouteloua gracilis. In the latter (a C4 species),
22 750 ppm CO2 resulted in increased dry matter production at daytime temperatures as high as
23 35 °C, but in P. smithii (a C3 species), CO2-stimulated growth was greatest at 20 °C. However,
24 the stimulation was progressively attenuated by increased temperature, so that at 35 °C, growth
25 in 750 ppm was only one third of that in 350 ppm CO2 at 20 °C.
26 Although the picture we have of temperature x CO2 interactions is inconsistent, Rowland-
27 Bamford (2000) has provided persuasive evidence that the nature of the response to temperature
28 in the grain yield of crops with as different temperature optima as rice and wheat will depend
29 upon whether the change is above or below the temperature optimum.
30 But what if we add O3 as another variable? Unfortunately, there have been very few
31 studies of the three-way interaction. With the information available on CO2 x O3 interactions
August 2005 AX9-167 DRAFT-DO NOT QUOTE OR CITE
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1 (Table AX9-12) and the limited information on temperature x O3 interactions (discussed in
2 Section AX9.3.4.2) simulation modeling can attempt to provide estimates of O3 x CO2 x
3 temperature effects, but experimental observation is still required to validate the models. The
4 questions that need to be answered are: if increased temperature can offset the gains in
5 productivity afforded by increased CO2 in important species such as wheat, and increased CO2
6 can offset the reductions in productivity caused by O3, will increased temperature modify this
7 protective effect? And if so, in what manner?
8 To date, the only information available appears to consist of the reports by Van Oijen and
9 Ewart (1999) and Bender et al. (1999) referred to above. In the former's simulation studies, the
10 overall yield depression of wheat caused by O3 was found to be 7 ± 4% for both
11 AFRCWHEAT2-O3 and LINTULCC models versus an observed 9 ±11%. The enhancements
12 due to CO2 were predicted to be 24 ± 9% and 42 ± 11%, respectively, which straddled the
13 observed 30 ± 22% gain. Based on the 13 experiments that included all four treatments (±O3,
14 ±CO2), an actual 10% yield loss due to O3 at ambient CO2 levels was reduced to a 4% loss by the
15 elevated CO2. The AFRCWHEAT2-O3 model predicted 7 and 4% losses, and LINTULCC
16 model predicted 8 and 5% losses due to the O3 and O3 + CO2 treatments. The actual and
17 simulated yield increases in response to CO2 increased further with increasing temperature, but
18 although temperature had no discernible effect on the observed depression of yield caused by O3
19 alone, both models suggested that the yield reduction was diminished both by higher
20 temperatures and higher CO2 levels.
21 The analysis of the ESPACE-wheat experiments by Bender et al. (Bender et al., 1999) led
22 to the following multiple linear regression:
7=1004.6*** + 0.588***[CO2] 1.908**[O3] 31.230***[r]+7.309[/] 1448.423***[H2O], (9-5)
23
24 where 7= grain yield, g • nT2; [CO2] = ppm CO2; [O3] = ppb O3, 12-h mean; [7] = °C; [/] = light
25 intensity, MJ • nT2/day; and [H2O] is a dummy variable: well watered = 1; limited water
26 supply = 2. (***, p < 0.001; **, p < 0.01; the coefficient for/was not significant). With R2 =
27 0.3983, adjusted for 258 degrees of freedom, a large part of the variability was still unaccounted
28 for by the five variables. However, this analysis suggests that CO2, O3, temperature, and water-
August 2005 AX9-168 DRAFT-DO NOT QUOTE OR CITE
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1 status are important codeterminants of wheat yield, but assumes no interactions. Substitution in
2 the model at summer light intensities and with well-watered plants indicates that, at 20° C,
3 a doubling of CO2 levels to 700 ppm alone would lead to a 29.5% increase in yield, while
4 50 ppb O3 alone would decrease yield by 10.9%. With both gases at those levels, the yield
5 would only increase 20%, but with a concurrent temperature rise of 2 °C, it would shrink to a
6 9.6% increase.
7 Both studies, therefore, indicate an amelioration of the effects of O3 by CO2, the magnitude
8 of which would be reduced at warmer temperatures. However, they relate to a single crop whose
9 response to CO2 is temperature-sensitive. Information about other species in which the effects
10 of CO2 and temperature are additive are limited. However, Wolf and Van Oijen (2003) recently
11 described a model (LPOTCO) simulating the effects of changes in climatic variables, CO2
12 and O3 on tuber yield potential of irrigated potato (S. tuberosum cv. Bintje) over locations within
13 the European Union ranging from Finland to Italy. They noted that although increased CO2, O3,
14 and light intensity were predominant controlling factors, increased temperature also influenced
15 potential yields substantially, with increases in northern latitudes (attributed to a longer growing
16 season) but decreases in southern latitudes (attributed to decreased assimilate production).
17 A clear understanding of the complex interactions of increased CO2 and temperature
18 with O3 must await further experimentation or simulations. However, it seems likely that any
19 CO2-induced amelioration of the adverse effects of O3 on aspects of growth other than seed or
20 grain yield may be lessened or increased by increased temperature, depending upon the
21 temperature optima for the species, along the lines suggested by Rowland-Bamford (2000).
22 Other crop simulation models which incorporate O3 and some of the various environmental
23 factors, including elements of climate change, have been reviewed by Kickert et al. (1999) and
24 Rotter and Van De Geijn (1999). However, to date, the applications tend to have focused on
25 interactions of O3 with factors such as soil moisture or nutrient availability.
26 With forest trees, the situation has the added complexity of a perennial growth form and
27 the inevitability, over time, of subjection to additional environmental stresses such as nutrient-
28 limitation. Here, too, although numerous models of tree growth have been described, there
29 appear to have been few applications to interactions of O3 and factors of climate change.
30 Constable et al. (1996) used TREGRO to model the growth of Ponderosa pine exposed to
31 three O3 levels (0.5*, 1.0*, and 2* ambient), two levels of CO2 (ambient and ambient +
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1 200 ppm CO2), and two temperature regimes (ambient and ambient + 4 °C). Plant growth was
2 predicted to be decreased 1, 19, and 39% by the three levels of O3, respectively. Increased CO2
3 reduced the loss at the highest O3 level to 7%; however, the combination of elevated CO2 with
4 the higher temperature more than overcame the adverse effects of O3, leading to a 4% increase,
5 largely attributed to increased fine root mass. The authors suggested that, in relation to the
6 baseline conditions used in the simulations (Corvallis, OR), higher concentrations of CO2 and O3
7 and a warmer climate will have little impact on total-tree growth, but they noted the importance
8 of undertaking multiple stress studies in order to be able to make accurate forecasts of the impact
9 of such changes on forest trees.
10 More recently, Constable and Friend (2000) compared the capabilities of six published
11 process-based models (CARBON, ECOPHYS, PGSM, TRE-BGC, TREGRO, and W91) for
12 simulating tree response to elevated CO2, O3, and temperature. They concluded that, although
13 these models were capable integrators of the effects of various environmental factors on
14 individual processes such as photosynthesis, they were less reliable when extrapolating to
15 growth.
16 Although most of the research emphasis has been on simple CO2 * O3 interactions, a few
17 isolated studies of interactions have involved O3, CO2, and biotic environmental factors. Heagle
18 et al. (1994) observed that both O3 and CO2 tended to be additive in encouraging the growth of
19 spider mite (Tetranychus urticae) populations on clover. Infection of wheat with leaf rust
20 (Puccinia reconditd) sensitized the plants to O3 injury, but its severity was significantly reduced
21 in elevated CO2 (Tiedemann and Firsching, 2000). The effects of O3 and CO2 on mycorrhizal
22 symbioses was studied by Kytoviita et al. (1999) who found that CO2 did not ameliorate the
23 adverse effects of O3 on the root growth of Aleppo pine and European white birch. In another
24 study with Aleppo pine, Kytoviita et al. (2001) noted that both O3 and elevated CO2 reduced
25 mycorrhiza-induced N-uptake by the roots. In Scots pine, Kasurinen et al. (1999) observed
26 transient effects of elevated CO2 and O3 on root symbiosis, but none of the effects persisted over
27 the 3 years of the study.
28 The soil water x O3 x CO2 interaction was experimentally investigated by Broadmeadow
29 and Jackson (2000) in Durmast oak, European ash, and Scots pine. No interactions were noted
30 with ash and pine; but with oak, elevated CO2 ameliorated and irrigation exacerbated the effects
31 of O3, although the resultant effects were essentially additive.
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1 Booker (2000) noted that soil nitrogen levels interacted only slightly with O3 and CO2 in
2 determining the composition of cotton leaves and roots. Carbon dioxide reversed the inhibition
3 of leaf growth caused by O3, but increased N-fertility tended to reduce this reversal.
4 Because of the small number of studies of possibly significant interactions of three or more
5 environmental factors, it is impossible to draw any sweeping conclusions as to how O3, in the
6 context of global climate change, may affect relationships among plants and insects, diseases,
7 and symbionts or among plants and nutrients or other air pollutants. The only interaction that
8 has some degree of general support is the amelioration of adverse O3 effects by elevated CO2.
9
10 AX9.3.8.2 Ozone-UV-B Interactions
11 As noted in the 1996 O3 AQCD (U.S. Environmental Protection Agency, 1996), depletion
12 of stratospheric O3 by halofluorocarbons has resulted in increased intensities of UV-B radiation
13 (280 to 320 nm wavelengths) at the Earth's surface. The situation is discussed more fully in
14 Chapter 10.
15 While stratospheric O3 depletion may result in increased surface UV-B irradiation,
16 absorption of UV-B is a property of the O3 molecule regardless of its location; surface UV-B
17 flux is, therefore, also reduced by O3 in the troposphere. Although only about 10% of the total
18 atmospheric O3 column occurs in the troposphere (Fishman et al., 1990), it contributes a
19 disproportionately greater absorption effect than stratospheric O3, because the UV radiation
20 penetrating the troposphere becomes increasingly diffuse as it reaches the surface, with a
21 consequent increase in mean path length (Briihl and Crutzen, 1989). Any benefits to vegetation
22 from reduced ambient O3 stress must, therefore, also be viewed in the context of possible
23 adverse effects due to increased UV-B irradiation. There are, thus, two distinct types of possible
24 interactions between surface level O3 and UV-B radiation:
25 • direct interactions involving simultaneous, sequential, or mixed exposures to O3 and
UV-B stresses; and
26 • effects on responses to UV-B itself resulting from changes in radiation intensity caused
by changes in surface-level O3 concentrations.
27 Only the first type of interaction is discussed here. The second type of interaction has broad
28 implications for both health and welfare and focuses on the impacts of UV-V radiation per se.
29 This topic is dealt with separately in Chapter 10.
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1 The most recent reviews specifically addressing the combined effects of tropospheric O3
2 and UV-B on plants are by Runeckles and Krupa (1994) and Krupa and Jager (1996), although
3 the topic has also been included in several more general reviews of O3 effects and factors of
4 climate change, such as those by Unsworth and Hogsett (1996), Krupa et al. (1998), Posthumus
5 (1998), and Krupa and Groth (2000).
6 However, little new information has become available since Runeckles and Krupa (1994)
7 noted that the scanty knowledge of the effects of UV-B and O3 combinations available at that
8 time was derived solely from studies of soybean. Miller et al. (1994) observed no interaction
9 and no effect of UV-B on yield, in contrast to a previous report by Teramura et al. (1990) using
10 the same cultivar, Essex. More recently, in a study of the saltmarsh grass Elymus athericus
11 subjected to reciprocal exposures to O3 and UV-B, Van De Staaij et al. (1997) observed no
12 interactive effects and no adverse effects of UV-B following 14-day exposures, even though an
13 earlier report showed that longer exposures to UV-B (65 days) could cause a 35% loss of
14 biomass (Van De Staaij et al., 1993). However, in a study in which ambient, high-altitude UV-B
15 levels were compared with near-zero levels, at ambient or 2x-ambient levels of O3, interactions
16 involving the levels of antioxidants in Norway spruce and Scots pine were reported by
17 Baumbusch et al. (1998). Schnitzler et al. (1999) subsequently reported that O3-induced injury
18 and adverse effects on photosynthesis were more pronounced with near-zero UV-B levels,
19 indicating an amelioration of the O3-response. A later study with Scots pine (Zinser et al., 2000)
20 revealed O3 x UV-B interactions at the gene expression and biochemical levels. In contrast,
21 Ormrod et al. (1995) reported that UV-B predisposed Arabidopsis thalliana to injurious growth
22 effects from O3 exposure.
23 At various organizational levels, Runeckles and Krupa (1994) identified several similarities
24 between plant response to O3 and UV-B, and at the level of gene expression, there have recently
25 been several reports of both similarities and distinctions. Willekens et al. (1994) reported similar
26 effects of O3, UV-B, and SO2 on the expression of antioxidant genes in Nicotiana
27 plumbaginifolia. In parsley (Petroselinum crispum), Eckey-Kaltenbach et al. (1994a) found
28 that O3 was a cross-inducer for both the UV-B-induced enhanced biosynthesis of flavonoids
29 and the pathogen-induced furanocoumarin phytoalexins, in keeping with the previously
30 observed O3-induction of fungal and viral defense reactions. In this regard, Yalpani et al. (1994)
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1 provided evidence that O3 and UV-B acted similarly in increasing disease-resistance via a
2 salicylate-mediated enhancement of defense proteins in tobacco. However, subsequent work
3 with tobacco led Thalmair et al. (1996) to conclude that exposure to UV-B did not lead to the
4 accumulation of pathogenesis-related proteins. In Scots pine, although O3 is known to induce
5 stilbene synthase and cinnamyl alcohol dehydrogenase, UV-B was found to enhance the former
6 but suppress the latter, revealing an interaction at the level of gene expression (Zinser et al.,
7 2000).
8 In summary, the present base of information about possible interactions between increased
9 UV-B radiation and O3 is insufficient to draw any firm conclusions in terms of gross effects, but
10 there is some evidence of similarities in the effects of O3 and UV-B individually and of the
11 mechanisms involved at the molecular level.
12
13 AX9.3.8.3 Interactions of Ozone with Multiple Climate Change Factors
14 Despite the need for experimental investigations of three-way or more complex
15 interactions among O3, CO2, UV-B, temperature, and other climate change factors, few studies
16 have been reported, even without O3 as a factor. In an isolated report, using tomato seedlings,
17 Hao et al. (2000) employed preexposure to UV-B (±CO2 enrichment) followed by exposure
18 to O3 (±CO2 enrichment). They observed that CO2 enrichment more than overcame the
19 inhibition of photosynthesis caused by O3, but pretreatment with UV-B reduced the resultant
20 increase.
21 In view of the unexpected observations made in their grassland study of the combined
22 effects of CO2, temperature, precipitation, and N-supply, Shaw et al. (2002) affirmed that
23 "Ecosystem responses to realistic combinations of global changes are not necessarily simple
24 combinations of the individual factors." The addition of O3 to the list of variables results in
25 further complexity.
26 Although computer simulation modeling may ultimately lead to improved understanding of
27 these complex issues, to date, no such models appear to have been applied to these interactions,
28 possibly because of the scarcity of experimental data for parameterization.
29
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1 AX9.3.9 Summary - Environmental Factors
2 Although O3 and other photochemical oxidants are phytotoxic, their actions on vegetation
3 may be modified by a host of biotic and abiotic factors in the environment; conversely, they may
4 modify plant response to these other factors. The extensive review of these biological, physical,
5 and chemical factors conducted for the 1996 O3 AQCD (U.S. Environmental Protection Agency,
6 1996) concluded with a statement that our understanding was too fragmented to permit drawing
7 many general conclusions. With today's increased awareness of the need for more complete
8 information on interactions, it is unfortunate that, in the interval since the 1996 criteria
9 document, rigorous, systematic investigations of interactions have been rare, and most of the
10 new information is as fragmented as before. This is inevitable, partly in view of the vast scope
11 of the possible interactions between O3 and other environmental variables and partly due to the
12 overall lack of funding for such research in these areas.
13 In the area of biotic interactions, new evidence with regard to insect pests and diseases has
14 done little to remove the uncertainties noted in the 1996 criteria document. Most of the large
15 number of such interactions that may affect crops, forest trees, and other natural vegetation have
16 yet to be studied. The trend suggested previously that O3 increases the likelihood and success of
17 insect attack has received some support from recent studies, but only with respect to chewing
18 insects. With the economically important group of sucking insects such as the aphids, no clear
19 trends have been revealed by the latest studies. Hence, although it seems likely that some insect
20 problems could increase as a result of increased O3 levels, we are still far from being able to
21 predict the nature of any particular O3 plant insect interaction, its likelihood, or its severity.
22 The situation is a little clearer with respect to interactions involving facultative
23 necrotrophic plant pathogens with O3, generally leading to increased disease. With obligate
24 biotrophic fungal, bacterial, and nematode diseases, there are twice as many reports
25 indicating O3-induced inhibitions than enhancements. The frequent reports that infection
26 by obligate biotrophs reduces the severity of O3-induced foliar injury should not be interpreted
27 as "protection", because of the negative effects on the host plant of the disease per se. With
28 obligate biotrophs, the nature of any interaction with O3 is probably dictated by the unique,
29 highly specific biochemical relationships between pathogen and host plant. At this time,
30 therefore, although some diseases may become more widespread or severe as a result of
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1 exposure to O3, it is still not possible to predict which diseases are likely to present the greatest
2 risks to crops and forests.
3 Several studies have indicated that the functioning of tree root symbioses with mycorrhizae
4 may be adversely affected by O3, but there is also evidence that the presence of mycorrhizae may
5 overcome root diseases stimulated by O3 and that O3 may encourage the spread of mycorrhizae
6 to the roots of uninfected trees. The latest studies, therefore, present no clearer picture of the
7 likely nature of simple interactions of O3 and root symbionts, but in view of the importance of
8 mycorrhizae as below-ground components of ecosystems, they are discussed more fully in
9 Section AX9.5.
10 The few recent studies of the impact of O3 on intraspecific plant competition have again
11 confirmed that grasses frequently show greater resilience than other types of plants. In grass-
12 legume pastures, the leguminous species suffer greater growth inhibition. And the suppression
13 of Ponderosa pine seedling growth by blue wild-rye grass was markedly increased by O3.
14 However, we are far from being able to predict the outcome of the impact of O3 on specific
15 competitive situations, such as successional plant communities or crop-weed interactions.
16 Light, a component of the plant's physical environment, is an essential "resource" whose
17 energy content drives photosynthesis and CO2 assimilation. It has been suggested that increased
18 light intensity may increase the sensitivity to O3 of light-tolerant species while decreasing that of
19 shade-tolerant species, but this appears to be an oversimplification with many exceptions.
20 Temperature affects the rates of all physiological processes based on enzyme-catalysis and
21 diffusion, and each process and overall growth (the integral of all processes) has a distinct
22 optimal temperature range. Although some recent field studies have indicated that O3 impact
23 significantly increases with increased ambient temperature, other studies have revealed little
24 effect of temperature. But temperature is unquestionably an important variable affecting plant
25 response to O3 in the presence of the elevated CO2 levels contributing to global climate change
26 (see below). In contrast, evidence continues to accumulate to indicate that exposure to O3
27 sensitizes plants to low temperature stress by reducing below-ground carbohydrate reserves,
28 possibly leading to responses in perennial species ranging from rapid demise to impaired growth
29 in subsequent seasons.
30 Although the relative humidity of the ambient air has generally been found to increase the
31 adverse effects of O3 by increasing stomatal conductance and thereby increasing O3 flux,
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1 abundant evidence indicates that the ready availability of soil moisture results in greater
2 sensitivity to O3. The partial "protection" against the adverse effects of O3 afforded by drought
3 has been observed in field experiments and modeled in computer simulations. There is also
4 compelling evidence that O3 can predispose plants to drought stress. Hence, the response will
5 depend to some extent upon the sequence in which the stresses occur, but, even though the
6 nature of the response is largely species-specific, successful applications of model simulations
7 will lead to larger-scale predictions of the consequences of O3 * drought interactions. However,
8 it must be recognized that regardless of the interaction, the net result on growth in the short-term
9 is negative, although in the case of tree species, other responses such as increased water use
10 efficiency could be a benefit to long-term survival.
11 Mineral nutrients in the soil, other gaseous air pollutants, and agricultural chemicals
12 constitute chemical factors in the environment. The evidence regarding interactions with
13 specific nutrients is still contradictory. Some experimental evidence indicates that low general
14 fertility increases sensitivity to O3, while simulation modeling of trees suggests that nutrient
15 deficiency and O3 act less than additively; however there are too many example of contrary
16 trends to permit any sweeping conclusions. Somewhat analogously with temperature, it appears
17 that any shift away from the nutritional optimum may lead to greater sensitivity, but the shift
18 would have to be substantial before a significant effect on response to O3 was observed.
19 Interactions of O3 with other air pollutants have received relatively little recent attention.
20 The situation with SO2 remains inconsistent, but seems unlikely to pose any additional risk to
21 those related to the individual pollutants. With NO and NO2, the situation is complicated by
22 their nutritional value as N sources. In leguminous species, it appears that NO2 may reduce the
23 impact of O3 on growth, with the reverse in other species, but the nature of the exposure pattern,
24 i.e., sequential or concurrent, also determines the outcome. Much more investigation is needed
25 before we will be able to predict the outcomes of different O3-NO-NO2 scenarios. The latest
26 research into O3 x acid rain interactions has confirmed that, at realistic acidities, significant
27 interactions are unlikely. A continuing lack of information precludes offering any
28 generalizations about interactive effects of O3 with NH3, HF, or heavy metals. More evidence
29 has been reported that the application of fungicides affords some protective effects against O3.
30 Over the last decade, considerable emphasis has been placed on research into O3
31 interactions with the components of global climate change: increased atmospheric CO2,
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1 increased mean global temperatures, and increased surface-level UV-B radiation. However,
2 most of these studies have tended to regard increased CO2 levels and increased mean
3 temperatures as unrelated phenomena. Experiments into the effects of doubled CO2 levels at
4 today's mean ambient temperatures are not particularly helpful in trying to assess the impact of
5 climate change on responses to O3. To date, the limited experimental evidence and evidence
6 obtained by computer simulation suggest that in a 600+ ppm world, although the enriched CO2
7 would more than offset the impact of O3 on responses as varied as wheat yield or the growth of
8 young Ponderosa pine trees, the concurrent increase in temperature would reduce, but probably
9 not eliminate, the net gain. A similar decrease in the net gain resulting from the complete
10 reversal by CO2 of the inhibition of photosynthesis caused by O3 has been reported for increased
11 UV-B irradiation. However, these are preliminary results based on minimal data.
12 In conclusion, although the increased use of computer simulations may be important in
13 suggesting outcomes of the many complex interactions of O3 and various combinations of
14 environmental factors, the results obtained will only be as reliable as the input data used for their
15 parameterization. The data needed for good simulations can only come from organized,
16 systematic study. For predicting the future, ignorance is as good as dependence on poor
17 simulations.
18
19
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1 AX9.4 EFFECTS-BASED AIR QUALITY EXPOSURE INDICES
2 AX9.4.1 Introduction
3 Indices are metrics that relate measured plant damage (i.e., reduced growth) to monitored
4 ambient O3 concentration over time. An index is needed to provide a consistent metric for
5 reviewing and comparing exposure-response effects obtained from various studies. Such indices
6 may also provide a basis for developing a biologically-relevant air quality standard for protecting
7 ecological resources. The determination of O3 impact on plants has been the effect on plant
8 growth and/or yield for purposes of the air quality standard setting process (U.S. Environmental
9 Protection Agency, 1986, 1996b). The quantifying function over some time frame has
10 frequently been referred to as "dose-response" and "exposure-response." The distinction is in
11 how the pollutant concentration is measured: "dose" is the measured pollutant concentration
12 absorbed by the leaf over some time period, whereas "exposure" is the ambient air concentration
13 measured near the plant over some time period.
14 A measure of plant O3 uptake from the ambient air (either rate of uptake or cumulative
15 seasonal uptake) is the ideal measure, because without O3 or its reactive product(s) reaching the
16 target tissue there is no effect (Tingey and Taylor, 1982). Uptake is controlled in part by stomata
17 (see Section AX9.2 for a detailed discussion). An uptake measure should integrate all those
18 environmental factors that influence stomatal conductance, e.g., temperature, humidity, soil
19 water status. A direct measure of the internal leaf concentration of O3, however, is technically
20 difficult and thus uptake values are generally obtained with simulation models that require
21 species- and site-specific variables. Because of this, a surrogate for uptake was sought early on
22 using statistical summaries of monitored ambient pollutant concentration over some integral of
23 time (Lee et al., 1988; Lefohn and Benedict, 1982; O'Gara, 1922; U.S. Environmental Protection
24 Agency, 1986, 1992, 1996b).
25 An index of exposure then must consider those abiotic and biotic factors known to modify
26 the plant response by altering O3 uptake (Hogsett et al., 1988; U.S. Environmental Protection
27 Agency, 1996b), including the temporal dynamics of exposure (e.g., concentration, frequency,
28 duration), plant phenology (see Section AX9.3), plant defense mechanisms (e.g., antioxidants)
29 (see Section AX9.2), and site climate and soil factors (e.g., temperature, VPD, soil moisture)
30 (see Section AX9.3). The development of such indices continues to be a challenge (U.S.
31 Environmental Protection Agency, 1996b).
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1 AX9.4.2 Summary of Conclusions from the Previous Criteria Document
2 The 1996 O3 AQCD (U.S. Environmental Protection Agency, 1996b) focused on the
3 research developing exposure indices to quantify growth and yield effects in crops, perennials,
4 and trees (primarily seedlings) and not foliar injury. The indices were various functional and
5 statistical summaries of monitored hourly O3 concentrations over designated time periods. The
6 testing of the adequacy of these indices to order the measured responses of growth and/or yield
7 in crops and tree species as seedlings was accomplished through regression analyses of earlier
8 exposure studies. No direct experimental testing of the adequacy of these indices was
9 accomplished. Their development focused on consideration and inclusion of some, but not all,
10 factors that affect O3 uptake and expression of effects (e.g., Lee et al., [1988]). The 1996
11 document (U.S. Environmental Protection Agency, 1996b) drew a number of conclusions that
12 built on even earlier conclusions (U.S. Environmental Protection Agency, 1992). Based on a
13 review of the research published since 1996, those conclusions are still valid.
14 Studies prior to 1996 indicated that the components of exposure, including concentrations,
15 temporal dynamics (e.g., time of day of peak events), frequency of occurrence, duration, and
16 respite time, were integral to developing indices of exposure related to growth response.
17 Evidence from the few direct experimental studies of varying exposure components indicate the
18 importance of peak concentrations, temporal pattern of occurrence, respite time and the
19 importance of cumulating the concentrations over the exposure period (Hogsett et al., 1985;
20 Musselman et al., 1983, 1986, 1994; U.S. Environmental Protection Agency, 1996b).
21 Exposure duration influences the degree of plant response. Single season, year-long, or
22 multiyear experimental results indicated that greater yield losses occurred when plants were
23 exposed for the longer duration and that a cumulative-type index was able to better describe the
24 exposure-yield relationship. Indices that do not consider duration, e.g., 7-h seasonal mean
25 concentration index, single peak event index, or the index that cumulated all concentrations (i.e.,
26 SUMOO), were unable to adequately describe the relationship. These single event or mean-type
27 indices do not consider the role of duration of exposure and either focus only on the peak event
28 or put too much focus on the lower hourly average concentrations (U.S. Environmental
29 Protection Agency, 1996b).
30 Higher hourly averaged concentrations had a greater effect on plant response. It was
31 concluded that cumulative indices that gave greater weight to higher concentrations related well
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1 with plant response (crops and tree seedlings) and ordered the treatment means in monotonically
2 decreasing fashion with increasing exposure, based on studies that applied two or more types of
3 exposure regimes with replicate studies of the same species. Examples of these indices, among
4 others, were: AOT60 (the seasonal sum of the difference between an hourly concentration above
5 the threshold value of 60 ppb, minus the threshold value of 60 ppb), SUM06 (the seasonal sum
6 of hourly concentrations at or above the threshold value of 60 ppb), W126 (a sigmoid functional
7 weighting of all hourly concentrations for the season), (U.S. Environmental Protection Agency,
8 1996b).
9 No studies before or after 1996, have enabled a discrimination among the various
10 weighted, cumulative indices. Various functional weighting approaches have been used,
11 including allometric, sigmoid, and threshold weighting, and compared for best statistical fit of
12 the plant growth or yield data; however, no one functional weighting was favored.
13 An exposure index that incorporated either the daily or seasonal temporal patterns of
14 higher concentration occurrence with the temporal pattern of individual species' stomatal
15 conductance was not reported in the 1996 O3 AQCD (U.S. Environmental Protection Agency,
16 1996b). Based on available data, it was unresolved how to proceed with weighting time of day
17 or temporal patterns of species conductance.
18 The relative importance of cumulative peak concentrations (>0.10 ppm) versus cumulative
19 mid-range concentrations (0.05 to 0.099 ppm) was questioned. Although controlled experiments
20 had provided important evidence that the higher hourly average concentrations should be given
21 greater weight than the mid-level values in developing indices, there was concern that, under
22 ambient conditions in the field, the higher concentrations did not occur at the time of maximum
23 plant uptake. This coincidence was considered to be the critical factor in determining peak
24 concentration impacts on plants. Based on the evidence at that time, it was not possible to
25 conclude whether the cumulative effects of mid-range concentrations were of greater importance
26 than those of peak hourly average concentrations in determining plant response (U.S.
27 Environmental Protection Agency, 1996b). No direct experimental studies, however, had
28 addressed this question prior to 1996, nor have any since.
29 The composite exposure-response functions for crops and tree seedlings were derived from
30 single and multiyear exposure studies that used modified or simulated ambient exposure profiles.
31 These profiles were typified by episodic occurrence of a large number of higher O3
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1 concentrations. This type of pattern is not atypical but is not found in all rural agricultural and
2 some forested areas in the United States. Selecting a concentration value from these crop and
3 seedling response models may result in an over- or underestimation of growth effects if applied
4 to regions of the country where a different type of temporal pattern of occurrence is prevalent
5 (U.S. Environmental Protection Agency, 1996b). A multicomponent index was suggested that
6 combined the concentration-weighted, cumulative index with the number of occurrences of
7 hourly averaged concentrations >0.10 ppm that might reduce the uncertainty associated with
8 selecting the exposure value for protection based on NCLAN-type studies (Lefohn and Foley,
9 1992; Musselman et al., 1994; U.S. Environmental Protection Agency, 1996b). No direct
10 experimental studies addressed this question prior to 1996, nor have any since.
11 Since 1996, additional research has focused on the time of day when the higher hourly
12 average concentrations occur, the time of day of maximum plant uptake, the diurnal variability
13 of plant defense mechanisms, and various suggestions as to including these factors in any one of
14 the cumulative, concentration-weighted exposure indices. A much broader literature has focused
15 since 1996 on relating O3 flux to plant response and how to use this as an index relating ambient
16 concentration to effects. These new developments are discussed in the sections that follow.
17
18 AX9.4.3 Evaluation of Various Exposure Indices for Describing Ambient
19 Exposure-Response Relationships
20 Mathematical approaches for summarizing ambient air quality information in biologically
21 meaningful forms that can serve as surrogates for dose for O3 vegetation effects assessment
22 purposes have been explored for more than 80 years (O'Gara, 1922; U.S. Environmental
23 Protection Agency, 1996b). Several indices have attempted to incorporate some of the
24 biological, environmental, and exposure factors (directly or indirectly) that influence the
25 magnitude of the biological response and contribute to observed variability (Hogsett et al.,
26 1988). In the 1996 O3 AQCD (U.S. Environmental Protection Agency, 1996b), the exposure
27 indices were arranged into five categories; (1) One Event, (2) Mean, (3) Cumulative,
28 (4) Concentration Weighted, and (5) Multicomponent, and were discussed in detail (Lee et al.,
29 1989). Figure AX9-16 illustrates how several of the indices weighted concentration and
30 accumulate exposure.
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Q.
Q.
0.15
0.12-
0.09-
o
Q.
X
LU
C
O
o
X
0.06-
0.03-
0.00
8 10
n m
0 00-
PF>m 2ndHDM-»-
M-7 = 0.05 ppm
024681
Day
Figure AX9-16. Diagrammatic representation of several exposure indices, illustrating
how they weight concentration and accumulate exposure, (a) SUM06:
the upper graphic illustrates an episodic exposure profile; the shaded
area under some of the peaks illustrates the concentrations greater than
or equal to 0.06 ppm that are accumulated in the index. The insert shows
the concentration weighting (0 or 1) function. The lower portion of the
graphic illustrates how concentration is accumulated over the exposure
period, (b) SIGMOID: the upper graphic illustrates an episodic
exposure profile; the variable shaded area under the peaks illustrates the
concentration-dependent weights that are accumulated in the index. The
insert shows the sigmoid concentration weighting function. The midpoint
of the sigmoid weighting scheme was 0.062 ppm. The lower portion of the
graphic illustrates how concentration is accumulated over the exposure
period, (c) 2ndHDM and M-7: the upper graphic illustrates an episodic
exposure profile. The lower portion of the graphic illustrates that the
2ndHDM considers only a single exposure peak, while the mean applies a
constant exposure value over the exposure period.
Source: Tingey et al. (1991).
1 Various components of the exposure-response relationship, including concentration, time
2 of day, respite time, frequency of peak occurrence, plant phenology, predisposition, etc., were
3 weighted with various functions and evaluated on their ability in ordering the regression of
4 exposure versus growth or yield response. The statistical evaluations for each of these indices
5 were accomplished using growth/yield response data from many earlier exposure studies (e.g.,
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1 NCLAN). This retrospective approach was necessary, because there were no studies specifically
2 designed to test the goodness of fit of the various indices. The regression approach selected
3 those indices that most properly ordered and spaced the treatment means to optimize the fit of a
4 linear or curvilinear model. This approach provided evidence for the best indices, albeit not as
5 defensible as that from studies with experimental designs and analyses that focus on specific
6 components of exposure.
7 Most of the early retrospective studies reporting regression approaches used data from the
8 NCLAN program or data from Corvallis, Oregon or California (Lee et al., 1987; Lee et al., 1988;
9 Lefohn et al., 1988; Musselman et al., 1988; U.S. Environmental Protection Agency, 1992; U.S.
10 Environmental Protection Agency, 1986). These studies were previously reviewed by the EPA
11 (U.S. Environmental Protection Agency, 1992, 1996b) and were in general agreement that the
12 best fit of the data were cumulative concentration-weighted exposure indices. Lee et al. (1987)
13 suggested that exposure indices that included all the 24-h data performed better than those that
14 used only 7 h of data; this was consistent with the conclusions of Heagle et al. (1987) that plants
15 receiving exposures for an additional 5-h/day showed 10% greater yield loss than those exposed
16 for 7-h/day. In an earlier analysis using the NCLAN data, Lee et al. (1988) found the "best"
17 exposure index was a phenologically-weighted cumulative index, with sigmoid weighting on
18 concentration and a gamma weighting function as a surrogate for plant growth stage. This index
19 was the best statistical fit, but it depended upon a greater knowledge of species and site
20 conditions making specification of weighting functions difficult for general use.
21 The next best fits were the several indices which only cumulated and weighted higher
22 concentrations (e.g., W126, SUM06, SUM08, AOT40). Amongst this group it was not possible
23 to distinguish a single best fit (Heagle et al., 1994; Lee et al., 1988; Musselman et al., 1988).
24 A statistical approach based on profile likelihoods was used to estimate parameters in
25 generalized exposure indices similar to the SUM06 and AOT40 indices (Blankenship and
26 Stefanski, 2001) using data from experiments conducted during 1993 at eight sites in the eastern
27 United States in which O3-sensitive and -tolerant white clover genotypes were grown using
28 methods developed by Heagle et al. (Heagle et al., 1994). The results showed that for the
29 SUMX family of indices, where X is a cutoff value, hourly O3 concentrations over -71 ppb
30 contribute the most to yield prediction. For the AOTX family of indices, the parameter was
31 54.4. These values are similar to those used in the SUM06 and AOT40 indices already in use.
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1 Furthermore, investigation of weighting for time of day confirmed the importance of the mid-
2 afternoon hours for this data set, unlike the results found for wheat in Sweden (Danielsson et al.,
3 2003;Pleijeletal.,2000a).
4 Other factors, including predisposition time (Hogsett et al., 1988; McCool et al., 1988) and
5 crop development stage (Heagle et al., 1991; Tingey et al., 2002), contributed to variation in the
6 biological response and suggested the need for weighting O3 concentrations to account for
7 predisposition time and phenology. However, the roles of predisposition and phenology in plant
8 response vary considerably with species and environmental conditions, so specification of a
9 weighting function for general use in characterizing plant exposure was not possible.
10 European scientists took a similar approach in developing indices describing growth and
11 yield loss in crops and tree seedlings, using OTCs with modified ambient exposures, but many
12 fewer species and study locations were employed in the European studies. There is evidence
13 from some European studies that a lower (Pleijel et al., 1997) or higher (Finnan et al., 1996,
14 1997) cutoff value may provide a better statistical fit to the experimental data. Finnan et al.
15 (1997) used seven exposure studies of spring wheat to confirm that cumulative exposure indices
16 emphasizing higher O3 concentrations were best related to plant response and that cumulative
17 exposure indices using weighting functions, including cutoff concentrations, allometric and
18 sigmoidal, provided a better fit and that the ordering of these indices differed with different
19 linear or Weibull dose-response models. Weighting those concentrations associated with
20 sunshine hours in an attempt to incorporate a element of plant uptake did not improve the index
21 performance (Finnan et al., 1997). A more recent study using data from several European
22 studies of Norway spruce, analyzed the relationship between relative biomass accumulation and
23 several cumulative, weighted indices, including the AOT40 and the SUM06 (Skarby et al.,
24 2004). All the indices performed relatively well in regressing biomass and exposure index, with
25 the AOT20 and AOT30 doing slightly better (r2 = 0.46-0.47). In another comparative study of
26 four independent data sets of potato yield and different cumulative uptake indices with different
27 cutoff values, a similarly narrow range of r2 was observed (0.3 -0.4) between the different
28 cumulative uptake of O3 indices (Pleijel et al., 2002).
29 In both the United States and Europe, the adequacy of these statistical summaries of
30 exposure in relating biomass and yield changes have, for the most part, all been evaluated using
31 data from studies not necessarily designed to compare one index to another (Lee et al., 1988,
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1 1989; Skarby et al., 2004). But given the available data, the cumulative, concentration-weighted
2 indices perform better than the peak or mean indices. It is not yet possible, however, to
3 distinguish differences between the cumulative, concentration-weighted indices with direct
4 experimental studies.
5 The main conclusions from the 1996 O3 AQCD (U.S. Environmental Protection Agency,
6 1996b) regarding an index based on ambient exposure are still valid. No information has come
7 forth in the interim to alter those conclusions significantly, and in fact, some recent studies have
8 further substantiated them. These key conclusions can be restated as follows:
9 • O3 effects in plants are cumulative;
10 • higher O3 concentrations appear to be more important than lower concentrations in
eliciting a response;
H • plant sensitivity to O3 varies with time of day and crop development stage;
12 • exposure indices that accumulate the O3 hourly concentrations and preferentially weight
the higher concentrations have better statistical fits to growth/yield response than do the
mean and peak indices.
13 Following the 1996 criteria review process (U.S. Environmental Protection Agency,
14 1996a,b), the EPA proposed an alternative form of the secondary NAAQS for O3 using a
15 cumulative, concentration-weighted exposure index to protect vegetation from damage (Federal
16 Register, 1997). The EPA considered three specific concentration-weighted indices: the cutoff
17 concentration weighted SUM06 and AOT60 and the sigmoid-weighted W126 exposure index
18 (U.S. Environmental Protection Agency, 1996a). All three indices performed equally well in
19 predicting the exposure-response relationships observed in the crop and tree seedlings studies
20 conducted during the previous 20 years (Lee et al., 1989). In a workshop convened to consider
21 the science supporting these indices, the participants agreed that all the cumulative
22 concentration- weighted indices considered were equally capable of predicting plant response
23 (Heck and Cowling, 1997).
24 The cutoff concentration-weighted index AOT40 was selected for use in Europe in
25 developing exposure-response relationships based on OTC studies of a limited number of crops
26 and trees (Griinhage and Jager, 2003). The United Nations Economic Commission for Europe
27 (UNECE, 1988) adopted the critical levels approach for assessment of O3 risk to vegetation
28 across Europe. As used by the UNECE, the critical levels are not air quality regulatory standards
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1 in the U.S. sense, but rather planning targets for reductions in pollutant emissions to protect
2 ecological resources. Critical levels for O3 are intended to prevent long-term deleterious effects
3 on the most sensitive plant species under the most sensitive environmental conditions, but not to
4 quantify O3 effects. A critical level was defined as "the concentration of pollutant in the
5 atmosphere above which direct adverse effects on receptors, such as plants, ecosystems, or
6 materials may occur according to present knowledge" (UNECE, 1988). The nature of the
7 "adverse effects" was not specified in the original definition, which provided for different levels
8 for different types of harmful effect (e.g., visible injury or loss of crop yield). There are also
9 different levels for crops, forests, and seminatural vegetation. The caveat, "according to present
10 knowledge," is important because critical levels are not rigid; they are revised periodically as
11 new scientific information becomes available. For example, the original critical level for O3
12 specified concentrations for three averaging times, but further research and debate led to the
13 current critical level being stated as the cumulative exposure (concentration x hours) over a
14 cutoff concentration of 40 ppb (AOT40) (Fuhrer et al., 1997). The level of 3 ppm-hr was
15 selected, corresponding to a 5% yield loss in spring wheat as determined from 15 OTC studies.
16 The critical level was defined for a 3-month period calculated for daylight hours. This value is
17 currently used for all crops, because it is the best supported value and because the limited data
18 from other crop species do not provide strong evidence that a more stringent value is required
19 (Fuhrer et al., 1997). "Level I" critical levels are currently used to map and identify areas in
20 Europe in which the levels are exceeded, and that information is then used to plan optimized and
21 effects-based abatement strategies. In the 1990s, areas of exceedance were mapped, but analyses
22 of many exposure studies led to the conclusion that the simple, exposure-based approach led to
23 the overestimation of effects in some regions and underestimation in others (Fuhrer et al., 1997;
24 Karenlampi and Skarby, 1996). The "Level I" approach did not differentiate between plant
25 species, and it did not include modifying site and micrometeorological factors of O3 uptake such
26 VPD, water stress, temperature, and light and variation in canopy height.
27 A decision was made to work towards a flux-based approach for the critical levels
28 ("Level II"), with the goal of modeling O3 flux-effect relationships for three vegetation types:
29 crops, forests, and seminatural vegetation (Griinhage and Jager, 2003). Progress has been made
30 in modeling flux (see Section AX9.4.5 (Ashmore et al., 2004a,b) and the Mapping Manual is
31 being revised (Ashmore et al., 2004a,b; Grennfelt, 2004; Karlsson et al., 2003). The revisions
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1 may include a flux-based approach for three crops: wheat, potatoes, and cotton. However,
2 because of a lack of flux-response data, a cumulative, cutoff concentration-based (AOTx)
3 exposure index will remain in use for the near future for most crops and for forests and
4 seminatural herbaceous vegetation (Ashmore et al., 2004a).
5
6 AX9.4.4 Identifying Exposure Components That Relate to Vegetation Effects
7 The efficacy of exposure indices in predicting biological responses requires that
8 researchers identify a relationship between measured growth and/or yield effects and exposure
9 components and those environmental and site factors that control pollutant uptake by the plant
10 effects. A number of these relationships were identified and discussed in the 1996 O3 AQCD
11 (U.S. Environmental Protection Agency, 1996a). A significant, but in some instances,
12 unquantified role was identified for (1) concentration; (2) duration of exposure; (3) the diurnal
13 and seasonal patterns of exposure, e.g., time of day of peak event, season of higher exposures,
14 seasons of high precipitation and humidity, the frequency of occurrence of peak events to respite
15 time (peak to valley ratios); (4) plant phenology; (5) plant canopy structure; (6) meteorological
16 and site factors, e.g., light, humidity; and (7) plant defense mechanisms.
17
18 AX9.4.4.1 Role of Concentration
19 A significant role of higher concentrations was established earlier, based on several
20 experimental studies (U.S. Environmental Protection Agency, 1996b). Several studies since the
21 last review (Nussbaum et al., 1995; Oksanen and Holopainen, 2001; Yun and Laurence, 1999a)
22 have added support for the important role that peak concentrations, as well as the pattern of
23 occurrence, plays in plant response to O3. Oksanen and Holopainen (2001) found that the peak
24 concentrations and the shape of the O3 exposure (i.e., duration of the event) were important
25 determinants of foliar injury in European white birch saplings, but growth reductions were found
26 to be more related to total cumulative exposure. Based on air quality data from 10 U.S. cities,
27 three 4-week exposure treatments having the same SUM06 value were constructed by Yun and
28 Laurence (1999a). They used the regimes to explore effects of treatments with variable versus
29 uniform peak occurrence during the exposure period. The authors reported that the variable peak
30 exposures were important in causing injury, and that the different exposure treatments, although
31 having the same SUM06, resulted in very different patterns of foliar injury. Nussbaum et al.
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1 (1995) also found peak concentrations and the pattern of occurrence to be critical in determining
2 the measured response. The authors recommended that to describe the effect on total forage
3 yield, peak concentrations >0.11 ppm must be emphasized by using an AOT with higher
4 threshold concentrations.
5 A greater role for higher concentrations affecting plant growth might be inferred based on
6 recent air quality analyses for the Southern California area (Lee et al., 2003; Tingey et al., 2004).
7 In the late 1960s and 1970s, extremely high O3 concentrations had impacted the San Bernardino
8 NF. However, over the past 15 plus years, significant reductions in O3 exposure have occurred
9 in the San Bernardino National Forest (Davidson, 1993; Lee et al., 2003; Lefohn and Shadwick,
10 2000; Lloyd et al., 1989). An illustration of this improvement in air quality is shown by the
11 37-year history of O3 air quality at a site in the San Bernardino Mountains (Figure AX9-17)
12 (Lee et al., 2003). The O3 exposure increased from 1963 to 1979 concurrent with increased
13 population and vehicular miles, followed by a decline to the present mirroring decreases in
14 precursor emissions. The pattern in exposure was evident in various exposure indices including
15 the cumulative concentration weighted (SUM06), as well as maximum peak event (1-h
16 peak), and the number of days having hourly averaged O3 concentrations >95 ppb (i.e., the
17 California O3 standard). The number of days having hourly averaged O3 concentrations >95 ppb
18 declined significantly from 163 days in 1978 to 103 days in 1997. The changes in ambient O3 air
19 quality for the site were reflected in the changes in the frequency and magnitude of the peak
20 hourly concentration and the duration of the exposure (Figure AX9-17). Considering the role of
21 exposure patterns in determining response, the seasonal and diurnal patterns in hourly O3
22 concentration did not vary appreciably from year to year over the 37-year period (Lee et al.,
23 2003).
24 The inference for a role of higher concentrations comes both from results of ground
25 measures of tree conditions on established plots and from results of model simulations. Across a
26 broad area of the San Bernardino NF, the Forest Pest Management (FPM) method of injury
27 assessment indicated an improvement in crown condition from 1974 to 1988; and the area of
28 improvement in injury assessment is coincident with an improvement in O3 air quality (Miller
29 and Rechel, 1999). A more recent analysis of forest changes in the San Bernardino NF using an
30 expanded network of monitoring sites has verified significant changes in growth, mortality rates,
31 basal area, and species composition throughout the area since 1974 (Arbaugh et al., 2003).
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Figure AX9-17.
Trends in May to September 12-h SUM06, peak 1-h O3 concentration
and number of daily exceedances of 95 ppb for Crestline in 1963 to 1999
in relation to trends in mean daily maximum temperature for Crestline
and daily reactive organic gases (ROG) and oxides of nitrogen (NOX)
for San Bernardino county. Annual ROG and NOX emissions data for
San Bernardino county were obtained from Alexis et al. (2001) and the
California Air Resource Board's emission inventory available at
http://www/arb/ca.gov/emisinv/emsmain/emsmain.htm.
Source: Lee et al. (2003).
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1 A model simulation of Ponderosa pine growth over the 40- year period in the San Bernardino NF
2 showed a significant impact of O3 exposure on tree growth and indicates improved growth with
3 improving air quality. The improvement in growth was assigned to improved air quality, but no
4 distinction was made regarding the relative role of mid-range and higher hourly concentrations,
5 only that improved growth tracked decreasing SUM06, maximum peak concentration, and
6 number of days of hourly O3 >95 ppb (Tingey et al., 2004). A summary of air quality data from
7 1980 to 2000 for the San Bernardino NF area of the number of "mid-range" hourly
8 concentrations indicated no dramatic changes over this 20-year period, ranging from about 1500
9 to 2000 hours per year (Figure AX9-18). There was a slow increase in the number of mid-range
10 concentrations from 1980 to 1986, which corresponds to the period after implementation of the
11 air quality standard. Another sharper increase was observed in the late 1990s. This pattern of
12 occurrence of mid-range hourly concentrations suggests a lesser role for these concentration
13 ranges compared to the higher values in either of the ground-level tree injury observations of the
14 model simulation of growth over the 40-year period.
15
16 AX9.4.4.2 Role of Duration
17 Recent studies have called into question the period of time over which concentrations are
18 accumulated and the form of the exposure index. Heagle and Stefanski (2000) reported that the
19 form of the exposure index was important only for 24-h indices for which SUMOO (cumulated all
20 hourly concentrations with no concentration weighting) provided the poorest fit. The authors
21 reported that the SUMOO, SUM06, W95 (Lefohn and Runeckles, 1987), W126, and AOT40
22 produced similarly good fits of the foliage biomass data for 6-, 5-, and 4-h midday accumulating
23 periods. The study pooled data from San Bernardino (CA) and Riverside (CA) with data from
24 Amherst (MA), Corvallis (OR), Kennedy Space Center (FL), Raleigh (NC), and Blacksburg
25 (VA). Ozone exposures were much higher at the two California sites (indicated by high W126,
26 SUM06, W95, and AOT40 values) compared to the other locations. Because of the pooling of
27 the data, the large number of high hourly average O3 concentrations that occurred at the
28 California sites may have resulted in the exposure indices being highly correlated with one
29 another and made it difficult to identify one optimal index.
30 In another study in California, Arbaugh et al. (1998) reported that the SUMOO exposure
31 index performed better for describing visible injury than the SUM06, W126, number of
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Crestline, San Bernardino, CA
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Figure AX9-18. The number of hourly average concentrations between 50 and 89 ppb
for the period 1980 to 2000 for the Crestline, San Bernardino, CA
monitoring site.
Source: U.S. Environmental Protection Agency (2003).
1 hours >0.08 ppm, and the number of days between measurement periods (U.S. Environmental
2 Protection Agency, 1996b). These exposure indices were originally developed and tested using
3 only growth/yield data, not foliar injury (U.S. Environmental Protection Agency, 1996b). This
4 distinction is critical in comparing the efficacy of one index to another. However, for many
5 locations in California, a large number of higher hourly average concentrations occur; thus the
6 SUMOO could be highly correlated with the frequency of elevated hourly average concentrations
7 and could be a good predictor of vegetation effects.
8
9 AX9.4.4.3 Patterns of Exposure
10 A significant factor in developing exposure indices is the temporal patterns of O3
11 occurrence over a day, a month, and a year, as well as seasonally overlaying the daily and
12 seasonal temporal patterns of those influential climatic and site factors. The coincidence of
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1 peak O3 with maximal stomatal conductance and detoxification processes is key to affecting
2 plant growth response (Musselman and Minnick, 2000).
3
4 Daily Patterns
5 The diurnal patterns of coincidence of the maximal leaf/needle conductance and
6 occurrence of higher ambient concentrations are relevant to the question of which hours during
7 the day over a season that hourly concentrations should be cumulated for those indices that
8 cumulate and weight concentration.
9 A 12-h daylight period for cumulating exposure was proposed following the 1996 O3
10 AQCD based primarily on the assumption that most species probably do not have significant
11 conductance at night (U.S. Environmental Protection Agency, 1996a). An extensive review of
12 the literature, however, reported that a large number of species had varying degrees of nocturnal
13 stomatal conductance (Musselman and Minnick, 2000). The role of nighttime stomatal
14 conductance and O3 exposure was demonstrated experimentally as well. Grulke et al. (2003)
15 showed that the stomatal conductance at night for Ponderosa pine in the San Bernardino NF
16 (CA) ranged from one tenth to one fourth that of maximum daytime gas exchange. In June, at
17 the high-elevation site, 11% of the total daily O3 uptake of pole-sized trees occurred at night. In
18 late summer, however, O3 uptake at night was negligible. Birch seedlings exposed to O3 at night
19 show greater reductions in growth than those exposed to O3 in daylight (Matyssek et al., 1995).
20 Field mustard (Brassica rapa L.) plants exposed to O3 during the day or night showed little
21 significant difference in the amounts of injury or reduced growth response to O3 treatment,
22 although the stomatal conductance was 70 to 80% lower at night (Winner et al., 1989). Tissue
23 biomass of Ponderosa pine seedlings was significantly reduced when seedlings were exposed to
24 either daytime or nighttime episodic profiles (Lee and Hogsett, 1999). However, the biomass
25 reductions were much greater with daytime peak concentrations than with nighttime peak
26 concentrations.
27 Although stomatal conductance was lower at night than during the day for most plants,
28 nocturnal conductance could result in some measurable O3 flux into the plants. In addition,
29 plants might be more susceptible to O3 exposure at night than during the daytime, because of
30 possibly lower plant defenses at night (Musselman and Minnick, 2000). Nocturnal O3 flux also
31 depends on the level of turbulence that intermittently occurs at night. Massman (2004)
August 2005 AX9-210 DRAFT-DO NOT QUOTE OR CITE
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1 suggested that nocturnal stomatal O3 uptake accounted for about 15% of the cumulative daily
2 effective O3 dose that was related to predicted injury. Based on a review of the literature
3 regarding plant nocturnal stomatal conductance, Musselman and Minnick (2000) recommended
4 that any O3 exposure index used to relate air quality to plant response should use the 24-h
5 cumulative exposure period for both exposure-response and effective flux models. However, in
6 an evaluation of a very large number of indices that described the O3 impact on spring wheat,
7 Finnan et al. (1997) did not find any improvement in performance of the cumulative
8 concentration-weighted indices by weighting those concentrations occurring during sunlight
9 hours.
10 Stomatal conductances are species-dependent and linked to both diurnal and seasonal
11 meteorological activity as well as to soil/site conditions (e.g., soil moisture). Daily patterns of
12 leaf/needle conductance were often highest in midmorning, whereas higher ambient O3
13 concentrations generally occurred in early to late afternoon when stomata were often partially
14 closed and conductances were lower. Total O3 flux depends on atmospheric and boundary layer
15 resistances, both of which exhibit variability throughout the day. Recent experimental studies
16 with tree species demonstrated the decoupling of ambient O3 exposure, peak occurrence, and gas
17 exchange, particularly in areas of drought (Panek, 2004). Several recent studies have suggested
18 that Ponderosa pine trees in the southern and northern Sierra Nevada Mountains may not be as
19 susceptible to high O3 concentrations as to lower concentrations, due to reduced needle
20 conductance and O3 uptake during the period when the highest concentrations occur (Arbaugh
21 et al., 1998; Bauer et al., 2000; Panek et al., 2002; Panek and Goldstein, 2001). Panek et al.
22 (2002) compared direct O3 flux measurements into a canopy of Ponderosa pine and demonstrated
23 a lack of correlation of daily patterns of conductance and O3 occurrence, especially in the late-
24 season drought period; they concluded that a consideration of climate or season was essential,
25 especially considering the role of soil moisture and conductance/uptake. In contrast, Grulke
26 et al. (2002) reported high conductance when O3 concentrations were high in the same species,
27 but under different growing site conditions. The decoupling of conductance and higher ambient
28 O3 concentration would hold true for more mesic environments as well as xeric landscapes. The
29 longer-term biological responses reported by Miller and Rechel (1999) for Ponderosa pine in the
30 same region, and the general reduction in recent years in ambient O3 concentrations, suggest that
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1 stomatal conductance alone may not be a sufficient indicator of potential vegetation injury or
2 damage.
3 The generalized models of stomatal conductance may provide a means to link patterns
4 of O3 occurrence with climatic and site factors that affect O3 uptake, provided conductance is
5 modeled by regions of similar seasonal moisture and by similar canopy structure (Emberson
6 et al., 2000a, 2000b) (Griinhage et al., 2000b) (Massman, 2004).
7
8 Seasonal Patterns
9 Several of the recent studies measuring O3 flux to pine canopies also reported on the
10 importance of seasonal patterns in relating exposure to response (Bauer et al., 2000). These
11 seasonal patterns can be early- versus late-season occurrence of higher O3 concentrations,
12 reflecting climate and precursor availability. The patterns also reflected seasonal drought and
13 the role soil moisture played in stomatal conductance and O3 uptake. Recently, studies looked
14 directly at this linkage. Panek et al. (2002) compared direct O3 flux measurements into a canopy
15 of Ponderosa pine with a number of exposure indices and demonstrated a lack of correlation,
16 especially in the late-season drought period; the authors concluded that a consideration of
17 climate, especially soil moisture, was essential. They suggested that a better metric for a
18 seasonally drought-stressed forests would be one that incorporates forest physiological activity,
19 through mechanistic modeling, by weighting ambient O3 concentrations by stomatal
20 conductance, or by weighting O3 concentrations by site moisture conditions. Panek (2004)
21 demonstrated a decoupling of O3 exposure and uptake seasonally as well, via seasonal drought
22 influence. Maximum O3 uptake occurred at the beginning of the season and in the winter,
23 whereas the pines were nearly dormant during August to September.
24 Using TREGRO, a process-based tree growth model, Tingey et al. (2004) simulated long-
25 term growth of Ponderosa pine over a 37-year period. The simulation showed a high degree of
26 association between O3 exposure and O3-induced reductions in tree growth (R2 = 0.56). The
27 scatter about the line, however, indicated that other factors beside O3 are required to describe the
28 association between exposure and response. Incorporating annual temperature and precipitation
29 increased the R2 to 0.67. In keeping with the observations of Panek (2004) on the decoupling of
30 peak O3 occurrence and maximal conductance, the remaining unexplained variation is attributed
31 to differences in timing of peak O3 uptake and peak O3 exposure over the years.
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1 AX9.4.4.4 Frequency of Occurrence of Peak Concentrations
2 Several earlier studies demonstrated the greater effect of episodic occurrence of O3 peaks
3 compared to daily peak events (U.S. Environmental Protection Agency, 1996b). Since the
4 1996 O3 AQCD, a few studies have corroborated the importance of this pattern in growth
5 response (Kollner and Krause, 2003; Yun and Laurence, 1999a; Nussbaum et al., 1995). Kollner
6 and Krause (2003) reported that, under equal exposure conditions, the most pronounced effects
7 on the yield of sugar beet (Beta vulgaris L.) and soybeans occurred with those regimes that
8 emphasized the episodic occurrence of peak events. Similarly, Yun and Laurence (1999b) used
9 exposure regimes constructed from 10 U.S. cities to demonstrate that variable peak occurrence
10 versus uniform occurrence was important in causing injury in tree seedlings. Nussbaum et al.
11 (1995) compared the effects of different patterns of peak occurrences with similar AOT40 values
12 and reported a stronger effect on total forage yield from the episodic treatment.
13
14 AX9.4.4.5 Canopy Structure
15 Another factor important in either O3 exposure or uptake, is how canopy structure
16 affects O3 concentration in and under forest canopies. There have been several investigations
17 of O3 concentrations under tree canopies (Enders, 1992; Fontan et al., 1992; Fredericksen et al.,
18 1995; Joss and Graber, 1996; Kolb et al., 1997; Lorenzini and Nali, 1995; Neufeld et al., 1992;
19 Samuelson and Kelly, 1997). In general, they indicated a reduction in O3 of-20 to 40% in the
20 area below the canopy but above the shrub/herb layers. An essential component in the
21 determination of the AOT40 as a critical level was the height at which the O3 concentration was
22 measured. The measurement heights are related to the O3 concentration measured at the top of
23 the canopy, i.e., upper surface boundary of the (quasi-) laminar layer (Griinhage and Jager,
24 2003). This location is presumably more related to stomatal uptake. Weighting the O3
25 concentration at this location takes into account stomatal opening and, if weighted with the
26 Jarvis-Steward factors for radiation, temperature, and soil moisture, the "lexicologically"
27 effective AOT40 is obtained (Griinhage and Jager, 2003). A question exists however as to
28 whether this "canopy" O3 concentration is clearly connected to stomatal O3 uptake. During site
29 conditions that limit stomatal conductance (e.g., low soil moisture, high VPD) at the top of the
30 canopy, high concentrations of O3 can occur with minimal risk.
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1 In a study that considered those factors important in O3 uptake that are also spatially
2 distributed as a result of canopy structure, Davison et al. (2003) reported that the variation in
3 visible injury in coneflower (Echinacea spp.) populations was unlikely to be due to differences
4 in O3 flux and more likely due to variation in PAR. At a height of 50 cm above ground, PAR
5 was reduced by almost 90%, whereas the O3 varied from about 15 to 90% of ambient. Ozone
6 injury was not solely related to O3 flux. Although there have been studies of the effects of
7 different light levels on O3 response, there have been few at the very low levels that occur in
8 canopies of tall herbaceous stands or in the ground layer of forests. Davison et al. (2003)
9 reported that conductance was not related to diurnal changes in light. The O3 levels were still
10 about 90% of the O3 concentration above the canopy when light was less than 5%. Light
11 intensity dropped to 1.5% of open at 130 cm from the edge of the canopy, while O3 dropped to
12 only 42%. The study, although reporting on the adequacy of visible foliar injury as an indicator
13 of O3 effects, suggested that consideration of other factors such as light were important in
14 predicting response. How this might be included in developing exposure-response indices was
15 not considered.
16
17 AX9.4.4.6 Site and Climate Factors
18 Soil moisture is a critical factor in controlling O3 uptake through its effect on plant water
19 status and stomatal conductance. In an attempt to relate uptake, soil moisture, and ambient air
20 quality to identify areas of potential risk, available O3 monitoring data for 1983 to 1990 were
21 used along with literature-based seedling exposure-response data from regions within the
22 southern Appalachian Mountains that might have experienced O3 exposures sufficient to inhibit
23 growth (Lefohn et al., 1997). In a small number of areas within the region, O3 exposures and
24 soil moisture availability were sufficient to possibly cause growth reductions in some O3-
25 sensitive species (e.g., black cherry). The conclusions were limited, however, because of the
26 uncertainly in interpolating O3 exposures in many of the areas and because the hydrologic index
27 used might not reflect actual water stress.
28
29 AX9.4.4.7 Plant Defense Mechanism - Detoxification
30 The non-stomatal component of plant defenses are the most difficult to quantify, but some
31 studies are available (Barnes et al., 2002; Chen et al., 1998; Massman and Grantz, 1995; Plochl
August 2005 AX9-214 DRAFT-DO NOT QUOTE OR CITE
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1 et al., 2000), and a larger discussion can be found in Section AX9.3. Massman et al. (2000)
2 developed a conceptual model of a dose-based index to determine how plant injury response
3 to O3 relates to the traditional exposure-based parameters. The index used time-varying-
4 weighted fluxes to account for the fact that flux was not necessarily correlated with plant injury
5 or damage. Their model applied only to plant foliar injury, and suggested that application of
6 flux-based models for determining plant damage (yield or biomass) would require a better
7 understanding and quantification of the injury and damage relationship.
8
9 AX9.4.5 Ozone Uptake or Effective Dose as an Index
10 Another approach in developing an index that relates growth response to ambient O3 is
11 based on determining the O3 concentration going from the atmosphere into the leaf, or flux.
12 Interest has been increasing in recent years, particularly in Europe, in using mathematically
13 tractable flux models for O3 assessments at the regional and national scale (Emberson et al.,
14 2000a,b). Reducing uncertainties in flux estimates for areas with diverse surface or terrain
15 conditions to within ±50% requires "very careful application of dry deposition models, some
16 model development, and support by experimental observations" (Wesely and Hicks, 2000). As
17 an example, the annual average deposition velocity of O3 among three nearby sites in similar
18 vegetation was found to vary by ±10%, presumably due to terrain (Brook et al., 1997).
19 Moreover, the authors stated that the actual variation was even greater, because stomatal uptake
20 was unrealistically assumed to be the same among all sites, and flux is strongly influenced by
21 stomatal conductance (Brook et al., 1997). This uptake-based approach to quantify the
22 vegetation impact of O3 requires inclusion of those factors that control the diurnal and
23 seasonal O3 flux to vegetation (e.g., climate patterns and species and/or vegetation-type factors
24 and site-specific factors). The models have to distinguish between stomatal and non-stomatal
25 components of O3 deposition to adequately estimate actual concentration reaching the target
26 tissue of a plant to elicit a response. Determining this O3 uptake via canopy and stomatal
27 conductance by necessity relies on models to predict flux and ultimately the "effective" flux
28 (Griinhage et al., 2004; Massman et al., 2000; Massman, 2004). "Effective flux" has been
29 defined as the balance between the O3 flux and the detoxification process (Dammgen et al.,
30 1993; Griinhage and Haenel, 1997; Musselman and Massman, 1999). The time-integrated
31 "effective flux" is termed "effective dose". The uptake mechanisms and the resistances in this
August 2005 AX9-215 DRAFT-DO NOT QUOTE OR CITE
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1 process, including stomatal conductance and biochemical defense mechanisms, are discussed in
2 the previous Section AX9.3. The flux-based index is the goal for the "Level II" critical level for
3 assessment of ozone risk to vegetation and ecosystems across Europe (Ashmore et al., 2004a).
4
5 AX9.4.5.1 Models of Stomatal Conductance
6 Only a limited number of studies have measured O3 concentration or its reaction products
7 within the leaf (e.g., Moldau and Bichele (2002); see Section AX9.4.3). Altimir et al. (2002)
8 described an enclosure technique for measuring O3 flux to foliage at the shoot level that allowed
9 determination of partitioning and seasonality of the removal pathways on the foliage. The loss
10 of O3 to the wall material of the chamber was great and required a correction when the stomatal
11 activity was low. Only a few instances of direct measures of O3 flux to foliage in the field are
12 reported. Most measures of O3 flux are from canopy measurements made with
13 micrometeorological techniques, but a number of assumptions are necessary and there are
14 limitations due to landscapes (Griinhage et al., 2000a; Wesely and Hicks, 2000). Comparison of
15 simulated and measured O3 flux densities show good agreement in the mean (Griinhage et al.,
16 2000a). Comparison, however, of continuous O3 concentrations and fluxes measured over a
17 5-year period by the gradient method (Fowler et al., 1989) in a 30-year-old Norway spruce stand
18 demonstrated a correlation over 5 years but were not correlated on a diurnal or seasonal basis.
19 The correlation was based on two uncoupled processes inside and outside the stomata, i.e., the
20 destruction of O3 outside the stomata in the canopy was influenced by those same factors
21 (temperature, light, humidity) that control the diurnal opening and closing of the stomata.
22 A similar lack of correlation of measured concentration and estimated flux, daily and seasonally,
23 into Norway spruce and cembran pine (Pinus cembrd) at six sites was due mostly to the control
24 of stomatal conductance by those same microenvironmental factors (temperature, humidity,
25 irradiance) (Emberson et al., 2000b). Seasonal variation of flux was attributed to the
26 temperature course. During the growing season, the leaf-air VPD was the environmental factor
27 controlling stomatal conductance and O3 flux into the needles.
28 Given the limitations of actual measures of flux and the lack of correlation between
29 measured concentrations and flux, the effort is to develop an uptake or flux-based response index
30 using models that consider site, climatic, meteorological, and species-specific (e.g.,
31 detoxification reactions) factors. Models of O3 conductance into plant tissue are available
August 2005 AX9-216 DRAFT-DO NOT QUOTE OR CITE
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1 (Griinhage et al., 1997; Massman, 1993; Wesely, 1989). The European Monitoring and
2 Evaluation Program (EMEP) developed an O3 deposition model for application across Europe in
3 conjunction with the EMEP photochemical model as a tool for the critical levels program
4 (Emberson et al., 2000a). The model was developed to estimate vegetation type-specific O3
5 deposition and stomatal flux, calculated according to a standard three-resistance formulation
6 incorporating atmospheric, boundary layer, and stomatal resistances (Emberson et al., 2000a).
7 The model used a multiplicative algorithm of the stomatal conductance of O3 (Jarvis, 1976) and
8 has been parameterized for 10 European tree species, seven crop species, and one type of
9 seminatural vegetation. The model calculates conductance as a function of leaf phenology,
10 temperature, photosynthetic flux density (PFD), VPD, and soil moisture deficit (SMD). The
11 environmental variables are site-specific (or regionally-specific). The most important factors
12 limiting O3 with this model were VPD, SMD, and phenology (Emberson et al., 2000a). These
13 factors demonstrate the critical linkage of high VPD and stomatal closure, which typically co-
14 occur with high O3 concentrations.
15 A number of recent model-based studies have investigated the relationship of flux and
16 plant growth response in several crop and forest tree species (Karlsson et al., 2004a,b; Pleijel
17 et al., 2004; Altimir et al., 2004; Bassin et al., 2004; Elvira et al., 2004; Emberson et al., 2000b;
18 Gerosa et al., 2004; Matyssek et al., 2004; Mikkelsen et al., 2004; Soja et al., 2004; Tuovinen
19 et al., 2004; Wieser and Emberson, 2004). The studies used earlier exposure experiments as well
20 as explicitly designed field studies, but no clear associations emerged to provide a basis for a
21 flux-based index. Griinhage and Jager (2003) emphasized the need for chamber-less
22 experiments to develop flux-effect relationship based on flux estimates at canopy height.
23 The complexity of using flux as an index of O3 exposure for growth response is shown in
24 field studies that measured O3 flux into Norway spruce and cembran pine (Emberson et al.,
25 2000b). They demonstrated that stomatal conductance was the main limiting factor for O3
26 uptake and showed the dependence of that measure on crown position, needle age, and altitude.
27 Consideration of the role of climate illustrates the importance of a flux measure. Pleijel et al.
28 (2000b) reported the improved relationship of yield in spring and winter wheat grown in OTCs
29 in many areas across Europe when it was related to the cumulative stomatal O3 uptake during the
30 grain-filling period. Compared to the AOT40, the cumulative uptake index estimated larger
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1 yield losses in the relatively humid parts of western and northern Europe, while smaller yield
2 loss was estimated for the dry summer climates in southern and central Europe.
3 Danielsson et al. (2003) compared the ability of two different stomatal models to relate
4 grain yield in field-grown spring wheat to cumulated O3 uptake and an exposure index of AOT40
5 and found that the cumulated O3 uptake determined with either model performed better in
6 relating exposure to yield than did the cumulative exposure index of AOT40.
7 Cumulative O3 uptake (CU) was modeled for three deciduous and two coniferous species
8 growing at different sites and elevations and compared with the AOT40 exposure measure at
9 these sites (Matyssek et al., 2004). A general linearity was demonstrated between the two
10 measures of O3 exposure, and, at any given AOT40, there was a 25 ± 11% variation in CU.
11 Although no correlation of growth alterations was observed with either the exposure or the
12 uptake measure, the modeled cumulative uptake was able to describe the variation in tree size
13 and site location, making for a better measure in risk assessment of O3 (Matyssek et al., 2004).
14 Karlsson et al. (2004b) compared the biomass-response relationship in young trees at seven
15 experimental sites across Europe using modeled cumulative O3 uptake and AOT40. A weaker
16 dose-response relationship was reported for the cumulative uptake metric compared to the
17 AOT40 (Karlsson et al., 2004b).
18 Concern about the complexity of the stomatal models and the data needed to model O3
19 uptake has led some researchers to offer modified accumulated exposure indices that consider
20 the meteorological factors controlling uptake (Gerosa et al., 2004; Karlsson et al., 2004a). In a
21 study of subterranean clover in Austria, Belgium, and southern Sweden, Karlsson et al. (2004a)
22 reported on the performance of a modified accumulated exposure over the threshold (mAOT)
23 which was based on solar radiation and VPD. This index improved the relationship for observed
24 visible injury. But when modeled uptake of O3 was derived from a simple stomatal conductance
25 model considering solar radiation, VPD, and air temperature, this index gave an even greater
26 improvement in the relationship to visible injury than did the ambient exposure index of AOT40
27 (Karlsson et al., 2004a). The added value of the mAOT was worthwhile, as was its lower degree
28 of complexity and data requirements compared to simulating O3 uptake with stomatal models.
29 Based on a study of O3 fluxes over a barley (Hordeum vulgare L.) field in Italy, a similar
30 modified exposure index was reported and referred to as "effective exposure" (Gerosa et al.,
31 2004). Their approach was similar in its consideration of physiological aspects in conjunction
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1 with monitored O3 concentrations. It also addressed the shortcomings of the data needs for
2 modeled O3 uptake.
3 Models that partition O3 uptake into stomatal and non-stomatal components are also now
4 available and predict a significant non-stomatal component in calculating O3 flux (Altimir et al.,
5 2004; Bassin et al., 2004; Mikkelsen et al., 2004; Nikolov and Zeller, 2003; Nussbaum et al.,
6 2003; Zeller and Nikolov, 2000). Altimir et al. (2004) compared the relative contributions of
7 stomatal and non-stomatal sinks at the shoot level for Scots pine. Using the EMEP model with a
8 revised parameterization for Scots pine, they demonstrated that a major removal of O3 was due
9 to the non-stomatal component; when a non-stomatal term was introduced dependent on ambient
10 relative humidity, the non-stomatal contribution to the total conductance was about 50%. Zeller
11 and Nikolov (2000) demonstrated a large non-stomatal O3 uptake (41% of the total annual flux)
12 in subalpine fir at a site in southern Wyoming using the biophysical model FORFLUX. In a
13 5-year study of measured O3 flux to a Norway spruce canopy, Mikkelsen et al. (2004) showed
14 monthly patterns of non-stomatal and stomatal deposition as part of total deposition to the
15 canopy. Their study demonstrated that daily means of O3 concentration and fluxes averaged
16 over 5 years correlated well, but the correlation was based on two different noncoupled
17 processes outside and inside the stomata. The destruction of O3 in the canopy was influenced by
18 temperature, light, and humidity, and these same factors influence stomatal opening, e.g.,
19 midday and night closure. Consequently, the diurnal O3 concentration and O3 flux do not
20 correlate at all during the growing season. The study estimated yearly stomatal uptake to be a
21 minimum of 21% of total deposition (i.e., non-stomatal uptake was as high as 80% of total).
22 The stomatal uptake was highest May to August (30 to 33%) and lowest November to February
23 (4 to 9%).
24
25 AX9.4.5.2 Nonlinear Response and Developing Flux Indices
26 If O3 flux were used as the only metric to predict vegetation injury or damage, the
27 prediction might be overestimated, because of nonlinear relationships between O3 and plant
28 response (Amiro et al., 1984; Amiro and Gillespie, 1985; Bennett, 1979, 1996b, 1986; U.S.
29 Environmental Protection Agency, 1978). The nonlinearity in the response surface suggests the
30 existence of a biochemical threshold. Musselman and Massman (1999) suggested that those
31 species having high amounts of detoxification potential might show less of a relationship
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1 between O3 stomatal uptake and plant response. More recently, nonlinear relationships
2 between O3 flux and yield were shown for potato (Pleijel et al., 2002) and spring wheat
3 (Danielsson et al., 2003). The relationship between O3 flux and potato yield led to the use of
4 an instantaneous flux threshold to overcome the nonlinear relationship (Pleijel et al., 2002).
5 However, the authors did not report a substantial improvement in the mathematical fitting of the
6 model when applying the threshold. Most of the flux was accumulated below 0.06 ppm.
7 However, Danielsson et al. (2003) showed an improved relationship between O3 uptake and
8 yield of spring wheat using a threshold of 5 nmoles m"2 sec"1 (0.24 mg m"2 sec"1). These results
9 suggest not all O3 entering the stomata contribute to a reduction in yield, which depends to some
10 degree on the amount of internal detoxification occurring for each particular species (see Section
11 AX9.2). The cellular detoxification reactions and repair processes which both detoxify oxidants
12 as well as play central role in the carbon economy of the plant are another level of resistance
13 to O3 reaching the target tissue (see Section AX9.2). The magnitude of the response is
14 determined by the amount of the pollutant reaching the target site and the ability of the plant to
15 reestablish homeostatic equilibrium. Thus, one would expect to observe a decoupling of O3
16 uptake with vegetation effects, which would manifest as a nonlinear relationship between O3 flux
17 and injury or damage.
18 Additional factors for inclusion in flux-based models to predict vegetation effects would be
19 the defense and repair mechanisms. However, the fact that the defense and repair mechanisms
20 vary diurnally as well as seasonally may make it extremely difficult to apply a mathematically
21 determined threshold to instantaneous flux measurements to calculate cumulative flux. The
22 threshold models do not allow for the temporal (i.e., daily and seasonal) variability of defense
23 mechanisms. Specifically, the relationship between conductance, O3 concentration, and
24 defense/repair mechanisms needs to be included. Recently, Massman (2004) illustrated that the
25 combination of stomatal conductance, O3 concentration, and diurnal variation of defense
26 mechanisms showed the daily maximum potential for plant injury (based on effective dose)
27 coincided with the daily peak in O3 mixing ratio. Massman et al. (2000) stressed that the product
28 of the overlapping mathematical relationships of conductance, concentration, and defense
29 mechanisms results in a much different picture of potential impact to vegetation than just the use
30 of conductance and concentration in predicting vegetation effects.
31
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1 AX9.4.5.3 Simulation Models
2 Another approach for determining O3 uptake and relating growth response to ambient O3
3 exposure may be the use of physiologically-based simulation models. Several of these have
4 been used in various contexts, comparing O3 response in a number of tree species with varying
5 climate and site factors (e.g., soil moisture) (Hogsett et al., 1996; Laurence et al., 2001; Ollinger
6 et al., 1997, 1998; Weinstein et al., 2001, 2002). These process-based models provide for an
7 integration of species, climate, and site factors controlling O3 uptake with long-term growth.
8 One of the important considerations in applying simulation modeling is to carefully assess the
9 uncertainties associated with the modeling predictions. Further efforts need to be made to
10 exercise the models so that they predict past growth losses associated with changes in O3
11 exposures that can be verified with on-the-ground surveys.
12
13 AX9.4.6 Summary
14 A large number of studies pertinent to the development of exposure indices have been
15 published since 1996, and these are predominantly focused on the development of a flux-based
16 index to relate ambient O3 to effects. There were only a few such studies prior to 1996 and these
17 were reviewed in the 1996 O3 AQCD (U.S. Environmental Protection Agency, 1996b). The few
18 studies published since 1996 on the role of O3 exposure components (including concentration,
19 duration, and exposure patterns) in describing growth response to O3 exposures have
20 substantiated earlier conclusions of the importance of higher concentration, shape of the peak,
21 and the episodicity of peak occurrence in the plant response to O3 exposure. An inferred role of
22 peak concentrations is possible from consideration of improved air quality in regions such as the
23 San Bernardino Mountains in southern California. Studies provide the basis for focusing on the
24 higher O3 concentrations, while including the lower levels, when estimating the effects of
25 emission reductions on vegetation.
26 A few studies have demonstrated the potential disconnection of the temporal patterns of
27 peak events and maximal stomatal conductance. In addition, a few other studies have
28 demonstrated the uptake of O3 during nighttime hours, suggesting the need to cumulate O3
29 exposure 24 h per day and not just during daylight hours.
30 Several studies since 1996 have demonstrated another critical concern in developing an
31 index for exposure. The concern is that peak O3 events and maximum stomatal conductance
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1 may be temporally separate. This disconnection introduces uncertainty in assessing O3 impact
2 when using the current ambient exposure based cumulative, concentration-weighted indices.
3 If stomatal conductance is relatively low, as in the late afternoon in arid climates, and that is the
4 same time as the peak O3 concentrations, then use of an exposure index that does not consider
5 this disconnect will overestimate the effect of the exposure. This concern is especially apparent
6 when assessing the impact of O3 across all the varied climatic regions of the United States or
7 Europe. Some studies use stomatal models to predict uptake (Ashmore et al., 2004a) or
8 physiological process-based models (Laurence et al., 2001) to integrate those species, climate,
9 and site factors that drive this temporal pattern of stomatal conductance and exposure, and thus
10 reduce some of the uncertainty in regional and national assessments of effects. These
11 approaches, however, are still limited by being species-dependent.
12 The results of these studies and reviews indicate the need to continue to develop indices
13 that are more physiologically and meteorologically connected to the actual dose of O3 the plant
14 receives. The cumulative concentration-weighted exposure indices are acknowledged surrogates
15 for effective dose and are simple conceptually and easy to measure. They do not fully
16 characterize the potential for plant uptake and resulting effects associated with O3, because the
17 indices, being measures of ambient concentration, do not include the physical, biological, and
18 meteorological processes controlling the transfer of O3 from the atmosphere through the leaf and
19 into the leaf interior (U.S. Environmental Protection Agency, 1996b). Use of such indices is
20 especially limited in spatial risk characterizations, because of the lack of linkage between
21 meteorology and species- and site-specific factors influencing O3 uptake. The flux-based
22 approach should provide an opportunity to improve upon the concentration-based (i.e., exposure
23 indices) approach. A cautionary argument was advanced in a few publications centered around
24 the nonlinear relationship between O3 uptake and plant injury (not growth alteration) response.
25 The concern was that not all O3 stomatal uptake results in a reduction in yield, which depends to
26 some degree on the amount of internal detoxification occurring with each particular species;
27 species having high amounts of detoxification potential may show less of a relationship
28 between O3 stomatal uptake and plant response.
29 The European approach and acceptance of flux-based critical values is a recognition of this
30 problem; a concerted research effort is needed to develop the necessary experimental data and
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1 modeling tools that will provide the scientific basis for such critical levels for O3 (Dammgen
2 et al., 1994; Fuhrer et al., 1997; Griinhage et al., 2004).
3 At this time, based on the current state of knowledge, exposure indices that differentially
4 weight the higher hourly average O3 concentrations but include the mid-level values represent
5 the best approach for relating vegetation effects to O3 exposure in the United States. A large
6 database exists that has been used for establishing exposure-response relationships. Such a
7 database does not yet exist for relating O3 flux to growth response. The pattern disconnects
8 between period of uptake and peak occurrence, as well as the potential for nocturnal uptake,
9 should be considered by adding some weighting functions into the currently used exposure
10 indices. Of particular consideration would be their inclusion in regional-to-national estimations
11 of O3 impacts on vegetation. Another useful approach to regional assessment for certain species
12 is to simulate growth effects with process-based models that account for seasonal climate and
13 site factors that control conductance.
14 It is anticipated that, as the overlapping mathematical relationships of conductance,
15 concentration, and defense mechanisms are better defined, O3-flux-based models may be able to
16 predict vegetation injury and/or damage at least for some categories of canopy-types with more
17 accuracy than the exposure-response models.
18
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36
37
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1 AX9.5 OZONE EXPOSURE-PLANT RESPONSE RELATIONSHIPS
2 AX9.5.1 Introduction
3 Ambient O3 concentrations have long been known to cause visible symptoms, decreases in
4 photosynthetic rates, decreases in plant growth, and decreases in the yield of marketable organs
5 (U.S. Environmental Protection Agency, 1978, 1986, 1996). Yet, despite considerable research
6 in the U. S. and other countries during the past three decades, quantifying the effects of ambient
7 O3 exposure on vegetation remains a challenge. Numerous studies have related O3 exposure to
8 plant responses, with most effort focused on the yield of crops and the growth of tree seedlings.
9 Most experiments exposed individual plants grown in pots or soil under controlled conditions to
10 known concentrations of O3 for a segment of daylight hours for some portion of the plant's life
11 span (Section AX9.1). The response of a plant species or variety to O3 exposure depends upon
12 many factors discussed in previous sections, including genetic characteristics (Section AX9.3.2),
13 biochemical and physiological status (Section AX9.3), and previous and current exposure to
14 other stressors (Sections AX9.3, AX9.4). Section AX9.3 describes how O3 moves from the
15 atmosphere into the leaf and the subsequent biochemical and physiological responses of plants.
16 The current section focuses on the quantitative responses of plants to seasonal or multiyear
17 exposures to known amounts of O3. Quantitative responses include foliar symptoms and
18 decreased growth of whole plants or decreased harvestable portions of them. Because of the
19 available information, most of this section focuses on the response of individual plants,
20 especially crop plants and tree seedlings, with limited discussion of mixtures of herbaceous
21 species. The responses of natural ecosystems are discussed in Section AX9.6.
22 This section will pay particular attention to studies conducted since the publication of the
23 1996 AQCD (U.S. Environmental Protection Agency, 1996). However, because much O3
24 research was conducted prior to the 1996 AQCD, the present discussion of vegetation response
25 to O3 exposure is largely based on the conclusions of the 1978, 1986, and 1996 criteria
26 documents (U.S. Environmental Protection Agency, 1978, 1986, 1996). To provide a context for
27 the discussion of recent research, the key findings and conclusions of those three documents are
28 summarized below.
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1 AX9.5.2 Summary of Key Findings/Conclusions from Previous
2 Criteria Documents
3 Experimental data reviewed in the 1978 and 1986 O3 AQCDs dealt primarily with the
4 effects of O3 on agricultural crop species (U.S. Environmental Protection Agency, 1978, 1986).
5 The chapter on vegetation effects in the 1978 O3 AQCD (U.S. Environmental Protection Agency,
6 1978) emphasized foliar symptoms and growth effects, but not those effects that affected yield,
7 an emphasis dictated by the kind of data available at the time. The 1986 O3 AQCD reviewed a
8 substantial new body of evidence based on OTC experiments (see Section AX9.1) showing that
9 ambient O3 exposures reduced the growth and yield of herbaceous plants, again with a focus on
10 major crop species. In the 1986 and 1996 O3 AQCDs, data were presented from regression
11 studies conducted to develop exposure-response functions for estimating yield loss of major crop
12 species in different regions of the United States. The 1996 O3 AQCD included results from
13 additional herbaceous crop species as well as shrub and tree species. For a number of tree
14 species, biomass growth of seedlings was related to growing season O3 exposures to produce
15 response functions for estimating O3 exposures that reduce growth by 10 or 30%. Also, in the
16 1986 and 1996 O3 AQCDs, data from studies using EDU as a protectant were reviewed. The
17 1978, 1986, and 1996 O3 AQCDs also reviewed data on the response to O3 exposures of forest
18 ecosystems in the San Bernardino Mountains of southern California (U.S. Environmental
19 Protection Agency, 1978, 1986, 1996). Because this region is exposed to high concentrations of
20 O3 and has shown evidence of ecosystem-level changes, it remains an important study area (see
21 Section AX9.6).
22 Ozone can cause a range of effects, beginning with individual cells, leaves, and plants, and
23 proceeding to plant populations and communities. These effects may be classified as either
24 "injury" or "damage". Injury encompasses all plant reactions, such as reversible changes in
25 plant metabolism (e.g., altered photosynthetic rate), altered plant quality, or reduced growth that
26 does not impair yield or the intended use or value of the plant (Guderian, 1977). In contrast,
27 damage includes all effects that reduce or impair the intended use or value of the plant. Damage
28 includes reductions in aesthetic values as well as losses in terms of weight, number, or size of the
29 plant part that is harvested (yield loss). Yield loss also may include changes in crop quality, i.e.,
30 physical appearance, chemical composition, or the ability to withstand storage. Losses in
31 aesthetic values are difficult to quantify. Although foliar symptoms cannot always be classified
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1 as damage, their occurrence indicates that phytotoxic concentrations of O3 are present, and,
2 therefore, studies should be conducted to assess the risk to vegetation.
3 Visible symptoms due to O3 exposures reduce the market value of certain crops and
4 ornamentals for which leaves are the product, e.g., spinach, petunia, geranium, and poinsettia.
5 The concept of limiting values used to summarize foliar symptoms in the 1978 O3 AQCD (U.S.
6 Environmental Protection Agency, 1978) was also considered valid in the 1986 O3 AQCD (U.S.
7 Environmental Protection Agency, 1986). Jacobson (1977) developed limiting values by
8 assessing the available scientific literature and identifying the lowest exposure
9 concentration/duration reported to cause foliar symptoms in a variety of plant species. Graphical
10 analyses presented in those documents indicated that the limit for reduced plant performance was
11 an exposure to 50 ppb for several hours per day for more than 16 days. Decreasing the exposure
12 period to 10 days increased the concentration required to cause symptoms to 100 ppb; and a
13 short, 6-day exposure further increased the concentration required to cause symptoms to
14 300 ppb. These limiting values established in 1978 were still deemed appropriate in the 1986
15 and 1996 O3 AQCDs. Such foliar symptoms are caused by O3 concentrations that occur in the
16 United States as shown in Table AX9-13 (adapted from U.S. Environmental Protection Agency,
17 (U.S. Environmental Protection Agency, 1996).
18 The 1986 O3 AQCD emphasized that, although foliar symptoms on vegetation are often an
19 early and obvious manifestation of O3 exposure, O3 effects are not limited to foliar symptoms.
20 Other effects include reduced growth of many organs (including roots), changes in crop quality,
21 and alterations in plant susceptibility to biotic stressors and sensitivity to abiotic stressors. The
22 1986 O3 AQCD also emphasized that O3 exerts phytotoxic effects only if a sufficient amount
23 of O3 reaches sensitive sites within the leaf (Section AX9.2). Ozone injury will not occur if the
24 rate of O3 uptake is low enough that the plant can detoxify or metabolize O3 or its metabolites or
25 if the plant is able to repair or compensate for the effects (Tingey and Taylor, 1982; U.S.
26 Environmental Protection Agency, 1986). Cellular disturbances that are not repaired or
27 compensated for are ultimately expressed as foliar symptoms, reduced root growth, or reduced
28 yield of fruits or seeds.
29 Beginning in the 1986 O3 AQCD and continuing in the 1996 O3 AQCD, OTC studies that
30 better quantified the relationship between O3 exposure and effects on crop species were
31 reviewed, with a focus on yield loss. These studies can be grouped into two types, depending on
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Table AX9-13. Summary of Ozone Exposure Indices Calculated for 3- or 5-Month
Growing Seasons from 1982 to 1991a
3-Month Growing Season (June-August)
No of
Year Sites"
1982 99
1983 102
1984 104
1985 117
1986 123
1987 121
1988 139
1989 171
1990 188
1991 199
Among Years
No. of
Year Sites
1982 88
1983 87
1984 95
1985 114
1986 118
1987 116
1988 134
1989 158
1990 172
1991 190
Among Years
HDM2C
ppm
Mean CVd
0.114 23.7%
0.125 24.9%
0.117 24.6%
0.117 24.6%
0.115 21.8%
0.119 22.9%
0.129 21.3%
0.105 23.1%
0.105 21.6%
0.106 22.0%
0.113 11.1%
M7
ppm
Mean
0.048
0.051
0.048
0.048
0.048
0.050
0.054
0.047
0.049
0.050
0.049
M7
ppm
Mean
0.05
0.06
0.05
0.05
0.05
0.06
0.06
0.05
0.05
0.05
0.05
5-Month
CV
18.7%
21.9%
18.2%
17.1%
19.1%
17.6%
17.8%
17.5%
18.3%
18.4%
10.0%
SUMOO
ppm-h
Mean
82.9
86.1
84.1
84.6
85.3
86.9
97.6
86.4
85.7
87.7
87.0
CV
19.1%
22.1%
19.9%
18.0%
18.0%
17.3%
19.6%
19.9%
21.0%
21.3%
9.9%
SUM06
ppm-h
Mean CV
26.8 68.8%
34.5 58.1%
27.7 58.4%
27.4 59.6%
27.7 65.0%
31.2 56.4%
45.2 46.8%
24.8 78.7%
25.8 76.2%
28.3 74.2%
29.5 42.1%
SIGMOID
ppm-h
Mean
26.3
33.0
27.4
27.4
27.7
30.4
42.9
25.8
26.6
28.9
29.4
CV
56.7%
52.3%
47.9%
47.6%
51.8%
46.8%
42.4%
59.4%
59.2%
59.5%
31.0%
Growing Season (May-September)
SUMOO
ppm-h
CV
20.6%
22.1%
18.0%
18.4%
20.3%
20.3%
18.7%
18.6%
19.8%
19.8%
9.8%
Mean
122.9
129.6
126.2
124.5
123.3
128.7
141.7
127.8
129.4
130.6
129.0
CV
22.3%
24.4%
19.1%
19.4%
21.4%
20.4%
22.0%
22.5%
22.7%
23.6%
9.9%
SUM06
ppm-h
Mean
37.3
44.4
36.7
36.2
34.9
42.2
58.0
32.7
34.6
36.8
38.7
CV
70.9%
61.9%
60.8%
63.8%
70.7%
62.0%
50.5%
87.8%
82.7%
80.7%
42.5%
SIGMOID
ppm-h
Mean
37.1
43.8
37.6
37.0
35.6
41.8
55.6
35.2
37.0
38.8
39.6
CV
57.8%
52.7%
46.9%
50.3%
55.7%
50.3%
45.0%
64.1%
62.1%
62.9%
29.8%
1 Updated and additional years from data given in Table III of Tingey et al. (1991), where the spatial and temporal variation
in ambient O3 exposures is expressed in terms of several exposure indices.
b Indicates the number of separate monitoring sites included in the analysis; fewer sites had 5 months of available data than
had 3 months of available data.
c The 2HDM index is calculated for sites with at least 3 months of available data. SUMOO, SUM06, M7, SIGMOID, and
2HDM are the cumulative sum above 0.0 ppm, the cumulative sum above 0.06 ppm, the 7-h seasonal mean, the sigmoid
weighted summed concentration, and the second highest daily maximum 1-h concentration, respectively.
d C V = coefficient of variation.
Source: Table 5-30 from U.S. Environmental Protection Agency (1996) based on Tingey et al. (1991).
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1 the experimental design and statistical methods used to analyze the data: (1) studies that
2 developed predictive equations relating O3 exposure to plant response, and (2) studies that
3 compared the effects of discrete treatment level(s) to a control. The advantage of the regression
4 approach is that exposure-response models can be used to interpolate results between treatment
5 levels.
6 Discrete treatment experiments were designed to test whether specific O3 treatments were
7 different from the control rather than to develop exposure-response equations, and the data were
8 analyzed using analyses of variance. When summarizing these studies using discrete treatment
9 levels, the lowest O3 concentration that significantly reduced yield was determined from
10 analyses done by the original authors. Often, the lowest concentration used in a study was the
11 lowest concentration reported to reduce yield; hence, it was not always possible to estimate a no-
12 effect exposure concentration. In general, the data indicated that 100 ppb O3 (frequently the
13 lowest concentration used in the studies) for a few hours per day for several days to several
14 weeks usually caused significant yield reductions of 10 to 50%.
15 By the time the 1986 O3 AQCD was prepared, much new information concerning the
16 effects of O3 on the yield of crop plants had become available through EPA's NCLAN research
17 program and through research funded by other agencies. The NCLAN project was initiated by
18 the EPA in 1980 primarily to improve estimates of yield loss under field conditions and to
19 estimate the magnitude of crop losses caused by O3 throughout the United States (Heck et al.,
20 1982; Heck et al., 1991). The cultural conditions used in the NCLAN studies approximated
21 typical agronomic practices. The primary objectives were:
22 (1) to define relationships between yields of major agricultural crops and O3 exposure
as required to provide data necessary for economic assessments and development
ofO3NAAQS;
23 (2) to assess the national economic consequences resulting from O3 exposure of major
agricultural crops; and
24 (3) to advance understanding of cause-and-effect relationships that determine crop
responses to pollutant exposures.
25 Using NCLAN data, the O3 concentrations predicted to cause 10 or 30% yield loss were
26 estimated using linear or Weibull response functions. The data in Table AX9-14 are from the
27 1996 document and were based on yield-response functions for 38 species or cultivars developed
28 from studies using OTCs of the type developed by Heagle et al. (1973) (see Section AX9.1).
August 2005 AX9-234 DRAFT-DO NOT QUOTE OR CITE
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Table AX9-14. Ozone Exposure Levels (Using Various Indices) Estimated to Cause at Least
10% Crop Loss in 50 and 75% of Experimental Cases"
50th PERCENTILE"
SUM06 SEC
SIGMOID SE
M7
SE
2HDM SE
NCLANData(n
NCLAN Data (n
= 49; wet
= 39; wet
and dry)d
only)
24.4
22.3
3.4
1.0
21,
19,
.5
.4
2.0
2.3
0.049
0.046
0.003
0.003
0.094
0.090
0.006
0.010
NCLAN Data (n = 54; wet and dry)6 26.4
NCLAN Data (n = 42; wet only)6 23.4
NCLAN Data (n = 10; wet) 25.9
NCLAN Data (n = 10; dry) 45.7
Cotton Data (n = 5) 23.6
Soybean Data (n = 13) 26.2
Wheat Data (n= 6) 21.3
3.2
3.1
4.5
23.3
2.3
5.4
15.2
23.5
22.9
23.4
40.6
19.3
22.6
19.3
2.4
4.7
3.2
0.1
2.3
3.6
12.7
NA
NA
0.041
0.059
0.041
0.044
0.061
NA
NA
0.001
0.014
0.001
0.005
0.018
0.099
0.089
0.110
0.119
0.066
0.085
0.098
75th PERCENTILE"
0.011
0.008
0.042
0.017
0.032
0.013
0.059
Cotton Data (n =
Soybean Data (n
Wheat Data (n =
5)e
= 15)e
7)e
30.
23.
25.
,0
,9
,9
12.7
6.5
10.5
27,
22,
21
.2
.0
.4
12.8
8.0
9.4
NA
NA
NA
NA
NA
NA
0.075
0.088
0.097
0.012
0.008
0.028
NCLAN Data (n = 49; wet and dry)
NCLAN Data (n = 39; wet only)
NCLAN Data (n = 54; wet and dry)6
NCLAN Data (n = 42; wet only)6
NCLAN Data (n = 10; wet)
NCLAN Data (n = 10; dry)
Cotton Data (n = 5)
Soybean Data (n = 13)
Wheat Data (n = 6)
Cotton Data (n = 5)e
Soybean Data (n=15)e
Wheat Data (n = 7)e
14.2
14.3
16.5
17.2
16.4
24.0
21.8
14.2
11.7
21.1
15.3
5.1
4.2
2.7
4.3
3.0
3.7
0.8
5.0
0.1
2.5
6.0
4.1
2.6
11.9
12.6
14.5
14.7
13.7
22.3
17.5
12.4
10.9
16.7
13.4
8.5
5.6
2.3
3.2
2.4
3.2
0.1
2.8
0.1
2.4
5.7
4.1
3.4
0.040
0.039
NA
NA
0.040
0.053
0.041
0.041
0.054
NA
NA
NA
0.007
0.005
NA
NA
0.001
0.022
0.001
0.006
0.032
NA
NA
NA
0.051
0.056
0.073
0.070
0.080
0.093
0.065
0.069
0.062
0.070
0.078
0.054
0.010
0.006
0.006
0.006
0.032
0.003
0.014
0.004
0.035
0.034
0.007
0.027
aSee Appendix A for abbreviations and acronyms.
bThe numbers in parentheses are the number of cases used in deriving the various exposure levels.
Standard error (SE).
dNCLAN data refers to studies conducted as part of the NCLAN project. Wet and dry refer to watering regimes
used in the studies, wet being well-watered, and dry meaning some level of drought stress was imposed.
e24-h exposure statistics reported in Lee et al. (1994b). Relative yield loss for 2HDM is relative to yield at 40 ppb rather
than 0 ppb as was used in Tingey et al. (1991).
Source: U.S. Environmental Protection Agency (1996) modified from Tingey et al. (1991).
August 2005
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1 Composite exposure-response functions for both crops and tree seedlings as a function of O3
2 exposure expressed as SUM06 are shown in Figure AX9-19. Review of these data indicate that
3 10% yield reductions could be predicted for more than 50% of experimental cases when:
4 (1) 12-h SUM06 values exceeded 24.4 ppm-h, (2) SIGMOID values exceeded 21.5 ppm-h, or
5 (3) 7-h seasonal mean concentrations were 50 ppb. The SIGMOID index is very similar to the
6 W126 index (see Section AX9.4 for further information about O3 indices). Much lower values
7 are required for each index to protect 75% of experimental cases (Table AX9-14). Grain crops
8 were generally found to be less sensitive than other crops. The data summarized in the 1996
9 criteria document also indicated that the variation in sensitivity within species may be as great as
10 differences between species.
11 The chemical protectant, EDU, was also used to provide estimates of yield loss. The
12 impact of O3 on yield was determined by comparing the yield data from plots treated with EDU
13 versus untreated plots. Studies indicated that yields were reduced by 18 to 41% when daytime
14 ambient O3 concentrations exceeded 80 ppb for 5 to 18 days over the growing season. For this
15 approach to be credible, the effects of EDU itself on a particular species must be preestablished
16 under conditions without O3 exposure (Kostka-Rick and Manning, 1992).
17 The 1996 O3 AQCD reviewed several experiments demonstrating that the seedlings of
18 some tree species such as poplars (Populus spp.) and black cherry are as sensitive to O3 as are
19 annual plants, in spite of the fact that trees are longer-lived and generally have lower rates of gas
20 exchange, and, therefore, a lower uptake of O3. The 1996 document also reviewed data showing
21 that O3 exposures that occur at present in the United States are sufficient to affect the growth of a
22 number of trees species. For example, exposure-response functions for 51 cases of tree seedling
23 responses to O3, including 11 species representing deciduous and evergreen growth habits,
24 suggest that a SUM06 exposure for 5 months of 31.5 ppm-h would protect hardwoods from a
25 10% growth loss in 50% of the cases studied (Table AX9-15). Similarly, a SUM06 exposure of
26 42.6 ppm-h should provide the same level of protection for evergreen seedlings. However, these
27 results do not take into the account the possibility of effects on growth in subsequent years.
28 Because multiple-year exposures may cause a cumulative effect on the growth of some trees
29 (Simini et al., 1992; Temple et al., 1992), it is likely that a number of species are currently being
30 affected even at ambient exposures (Tables AX9-13, AX9-21).
31
August 2005 AX9-236 DRAFT-DO NOT QUOTE OR CITE
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o
'•B
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
a. Crops
75th Percentile
50th Percentile
25th Percentile
\
10
I
20
I
30
I
40
I
50
60
24-h SUM06 (ppm-h)
^_^
«"
o
w
03
E
o
5
•a
•2
1
£
100%-q
90% -1
80% -|
70% -I
60% -I
50% -1
40% -1
30% -|
20% -i
10%-|
n% -
b. Tree Seedlings
75th Percentile
50th Percentile
~~ 25th Percentile
\
10
\
20
\
30
\
40
50 60
24-h SUM06 (ppm-h) (adjusted to 92 days)
Figure AX9-19.
Distribution of biomass loss predictions from Weibull and linear
exposure-response models that relate biomass to O3 exposure.
Exposure is characterized with the 24-h SUM06 statistic using data
from (a) 31 crop studies from National Crop Loss Assessment
Network (NCLAN) and (b) 26 tree seedling studies conducted at U.S.
Environmental Protection Agency's Environmental Research Laboratory
in Corvallis, OR; Smoky Mountains National Park, TN; Houghton,
Michigan; and Delaware, Ohio. Separate regressions were calculated
for studies with multiple harvests or cultivars, resulting in a total of
54 individual equations from the 31 NCLAN studies and 56 equations
from the 26 seedling studies. Each equation was used to calculate the
predicted relative yield or biomass loss at 10, 20, 30, 40, 50, and 60 ppm-h,
and the distributions of the resulting loss were plotted. The solid line is
the calculated Weibull fit at the 50th percentile.
Source: U.S. Environmental Protection Agency (1996); Hogsett et al. (1995).
August 2005
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Table AX9-15. SUM06 Levels Associated with 10 and 20% Total Biomass
Loss for 50 and 75% of the Seedling Studies
(The SUM06 value is adjusted to an exposure length of 92 days per year.)a
Weibull Equations (all 51 seedling studies):
50th Percentile PRYLb = 1 - exp(-[SUM06/176.342]** 1.34962)
75th Percentile PRYL = 1 - exp(-[SUM06/104.281]** 1.46719)
Weibull Equations (27fast-growing seedling studies):
50th Percentile PRYL = 1 - exp(-[SUM06/150.636]** 1.43220)
75th Percentile PRYL = 1 - exp(-[SUM06/89.983]** 1.49261)
Weibull Equations (24 slow-to-moderate growing seedling studies):
50th Percentile PRYL = 1 - exp(-[SUM06/190.900]** 1.49986)
75th Percentile PRYL = 1 - exp(-[SUM06/172.443]**l.14634)
Weibull Equations (28 deciduous seedling studies):
50th Percentile PRYL = 1 - exp(-[SUM06/142.709]** 1.48845)
75th Percentile PRYL = 1 - exp(-[SUM06/87.724]** 1.53324)
Weibull Equations (23 evergreen seedling studies):
50th Percentile PRYL = 1 - exp(-[SUM06/262.911]** 1.23673)
75th Percentile PRYL = 1 - exp(-[SUM06/201.372]**1.01470)
Levels Associated with Prevention of a 10 and 20% Total Biomass Loss
for 50 and 75% of the Seedlings
All 51 Seedling Cases
Percent of Seedlings
Relative
Biomass Loss
27 Fast-Growing Seedling Cases
Relative
Biomass Loss
10%
20%
10%
20%
50%
33.3
58.0
Percent
50%
31.3
52.9
75%
22.5
37.5
of Seedlings
75%
19.4
32.4
August 2005 AX9-23 8 DRAFT-DO NOT QUOTE OR CITE
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Table AX9-15 (cont'd). SUM06 Levels Associated with 10 and 20% Total
Biomass Loss for 50 and 75% of the Seedling Studies
(The SUM06 value is adjusted to an exposure length of 92 days per year.)a
24 Slow-to-Moderate-Growth Seedling Cases
Percent of Seedlings
50% 75%
Relative 70% 42.6 24.2
Biomass Loss 20% 70.2 46.6
28 Deciduous Seedling Cases
Percent of Seedlings
Relative
Biomass Loss
23 Evergreen Seedling Cases
10%
20%
50%
31.5
52.1
75%
20.2
33
Percent of Seedlings
Relative
Biomass Loss
70%
20%
50%
42.6
78.2
75%
21.9
45.9
aSee Appendix [XXX] for abbreviations and acronyms.
bPRYL = predicted relative yield (biomass) loss
Source: U.S. Environmental Protection Agency (1996), based on Hogsett et al. (1995).
1 In 1986, the EPA (U.S. Environmental Protection Agency, 1986) established that 7-h per
2 day growing season mean exposures to O3 concentrations above 50 ppb were likely to cause
3 measurable yield loss in agricultural crops. At that time, few conclusions could be drawn about
4 the response of deciduous or evergreen trees or shrubs, due to the lack of information about
5 response of such plants to season-long exposures to O3 concentrations of 40 to 60 ppb and above.
6 However, the 1978 and 1986 O3 AQCDs (U.S. Environmental Protection Agency, 1978, 1986)
7 indicated that the limiting value for foliar symptoms on trees and shrubs was 60 to 100 ppb for
8 4 h. From 1986 to 1996, extensive research was conducted, establishing the sensitivity of many
9 tree species. Based on research published since the 1986 O3 AQCD (U.S. Environmental
August 2005 AX9-239 DRAFT-DO NOT QUOTE OR CITE
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1 Protection Agency, 1986), a number of conclusions were drawn in 1996 O3 AQCD (U.S.
2 Environmental Protection Agency, 1996):
3 (1) An analysis of 10 years of monitoring data from more than 80 to almost 200
nonurban sites in the United States established ambient 7-h growing season
average concentrations of O3 for 3 or 5 months of 51 to 60 ppb and 47 to 54 ppb,
respectively. The SUM06 exposures ranged (a) from 24.8 to 45.2 ppm-h for
3 months and (b) from 32.7 to 58.0 ppm-h for 5 months (Tingey et al. (1991),
Table AX9-13).
4 (2) The results of OTC studies that compared yields at ambient O3 exposures with
those in filtered air and retrospective analyses of crop data (Table AX9-14)
established that ambient O3 concentrations were sufficient to reduce the yield of
major crops in the United States. Research results since 1978 did not invalidate
EPA conclusions (U.S. Environmental Protection Agency, 1978, 1986) that foliar
symptoms due to O3 exposures reduce the market value of certain crops and
ornamentals where leaves are the product (such as spinach, petunia, geranium, and
poinsettia) and that such damage occurs at ambient O3 concentrations observed in
the United States.
5 (3) A 3-month SUM06 exposure of 24.4 ppm-h, corresponding to a 7-h mean of 49
ppb and a 2HDM of 94 ppb O3 may prevent a 10% loss in 50% of the 49
experimental cases analyzed by Tingey et al. (1991). A 12-h growing season
mean of 0.045 ppb should restrict yield losses to 10% in major crop species
(Lesser et al., 1990).
6 (4) Depending on duration, concentrations of O3 and SUM06 exposures currently in
the United States are sufficient to affect the growth of a number of tree species.
Given the fact that multiple-year exposures may cause a cumulative effect on the
growth of some trees (Simini et al., 1992; Temple et al., 1992), it is likely that a
number of species currently are being impacted, even at ambient O3 exposures
(Tables AX9-13, AX9-21).
7 (5) Exposure-response functions for 51 cases of seedling response to O3 (Hogsett
et al., 1995), including 11 species representing deciduous and evergreen growth
habits, suggest that a SUM06 exposure for 5 months of 31.5 ppm-h would protect
hardwoods from a 10% growth loss in 50% of the cases studied. A SUM06
exposure of 42.6 ppm-h should provide the same level of protection for evergreen
seedlings. Note that these conclusions do not take into the account the possibility
of effects on growth in subsequent years, an important consideration in the case of
long-lived species.
8 (6) Studies of the response of trees to O3 have established that, in some cases (for
instance, poplars and black cherry), trees are as sensitive to O3 as are annual
plants, in spite of the fact that trees are longer-lived and generally have lower gas
exchange rates, and, therefore, lower O3 uptake.
August 2005 AX9-240 DRAFT-DO NOT QUOTE OR CITE
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1 (7) Use of the chemical protectant, EDU, is of value in estimating O3-related losses in
crop yield and tree growth, provided that care is exercised in establishing
appropriate EDU dosages to protect the plants without affecting growth.
2 The major question to be addressed in the remainder of this section is whether new
3 information supports or alters the 1996 criteria document conclusions summarized above.
4 In particular, this section evaluates whether the response of plants to experimental treatments at
5 or near O3 concentrations characteristic of ambient levels in many areas of the United States
6 (Tables AX9-13, AX9-21) can be compared to a control or reduced O3 treatment to establish a
7 potential adverse effect. Before evaluating new information from the literature on O3 effects on
8 vegetation, O3 exposure indices used in O3 studies and trends in O3 exposure patterns during the
9 past two decades are briefly reviewed.
10
11 AX9.5.3 Ozone Indices and Ambient Exposure
12 As recognized in both the 1986 and the 1996 criteria documents, the characterization and
13 representation of the exposure of vegetation to O3 is problematic, because the specific aspects of
14 pollutant exposure that cause injury or damage are difficult to quantify. This issue was
15 addressed in Section AX9.4, and only a few points will be discussed here in order to provide a
16 context for interpreting data on exposure-response relationships. The most important effects
17 of O3 on vegetation occur due to uptake of O3 through stomata, with subsequent oxidative injury
18 that appears to be rather nonspecific (Section AX9.2). As has been discussed by numerous
19 authors during the last three decades, from a toxicological and physiological view, it is much
20 more realistic to relate effects to internal (absorbed) O3 dose rather than to exposure near the leaf
21 or canopy (Fowler and Cape, 1982; Fuhrer et al., 1992; Griinhage et al., 1993, 1999; Legge et al.,
22 1995; Massman et al., 2000; Musselman and Massman, 1999; Pleijel et al., 1995; Runeckles,
23 1974; Taylor et al., 1982; Tingey and Taylor, 1982) (see also Section AX9.4). Theoretically,
24 flux estimates should improve the assessment of O3 effects, but despite recent attention to this
25 topic, particularly in Europe, it remains difficult to estimate flux in the field outside of
26 experimental sites where continuous measurements of wind speed and other environmental
27 conditions are made. This topic is discussed further below in Section AX9.5.4.5.
28 No simple exposure index can accurately represent all of the numerous factors operating at
29 different timescales that affect O3 flux into plants and subsequent plant response (Section
August 2005 AX9-241 DRAFT-DO NOT QUOTE OR CITE
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1 AX9.4). Indices of peaks, such as the 2HDM, are not well suited for discerning exposure-
2 response relationships, because they do not capture the effects of lower O3 concentrations nor the
3 cumulative effects of O3 on vegetation (Heck and Cowling, 1997; U.S. Environmental Protection
4 Agency, 1996). Therefore, peak indices have not been used in recent decades to develop
5 exposure-response relationships for vegetation. Fortunately, other simple indices have shown
6 substantial correlation with responses such as crop yield under experimental conditions. During
7 the 1980s, the most commonly used indices for expressing O3 exposure were 7-, 8-, or 12-h
8 daytime average values over the duration of O3 exposure, which was often 3 months or
9 somewhat less for experimental studies with crops. These indices perform reasonably well for
10 interpreting experimental data on the response of vegetation to O3, particularly for individual
11 experiments, although they do not explain all of the variation among experiments in
12 retrospective analysis of multiple experiments (Lesser et al., 1990).
13 Since the 1980s, cumulative indices such as the SUM06, AOT40, or W126 that
14 preferentially weight higher concentrations have been used in conjunction with mean indices for
15 developing exposure-response relationships (Tables AX9-14 and AX9-15, and Figure AX9-18).
16 Such indices are often more suitable than mean values, because they are cumulative and because
17 they preferentially weight higher concentrations. Thus, these indices generally provide
18 somewhat better fits to experimental data than do mean indices, especially in retrospective
19 analyses of multiple experiments on multiple species (Lee et al., 1994a; Lee et al., 1994b;
20 Lee and Hogsett, 1999; Tingey et al., 1991). Unfortunately, no single index has been used
21 consistently even in the recent literature, making it difficult to compare results among and
22 between experiments and with ambient exposure data. However, Tables AX9-13 and AX9-21
23 provide summaries of ambient exposure data for several indices that can be compared to the
24 experimental results reviewed in the remainder of this section. Of the cumulative indices that
25 preferentially weight higher concentrations, the SUM06 index has been used most commonly in
26 the U.S. literature, and it was selected in a meeting of scientific experts on O3 effects on
27 vegetation as suitable for a secondary standard to protect vegetation (Heck and Cowling, 1997).
28 However, it should be noted that the W126 index has been selected for use in protecting
29 vegetation in Class 1 areas (Federal Land Manager's Air Quality Related Values Workgroup
30 (FLAG), 2000). Even in recent studies, O3 data are often presented using only a seasonal
31 mean index value, and so mean values are frequently presented in this section. Such reporting of
August 2005 AX9-242 DRAFT-DO NOT QUOTE OR CITE
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Table AX9-16. Summary of Selected Studies of Ozone Effects on Annual Species
_-
C/3
to
o
VO
1
OJ
O
rr5
H
1
o
o
2;
0
H
O
o
w
o
o
H
W
Species Facility
Bean, cv. OTC
Pros
Bean, cv. Lit OTC
Bean , cv. OTC
Bush Blue
Lake 290
Bean , cvs. OTC
Tenderette,
S156
Bean , cvs. OTC
R123,
Oregon-91,
S156
Corn OTC
Cotton, cv. OTC
Deltapine
Cotton, cv. OTC
Deltapine
Oat, cv. Vital OTC
Location
The Netherlands
Germany
Corvallis, OR
Raleigh, NC
Raleigh, NC
Beltsville, MD
Raleigh, NC
Raleigh, NC
Ostad, Sweden
O3 Concentration
(units are ppb unless
otherwise specified)1 Duration
CFtoCF75: 62 days
9-hmean = 3-70,
AOT40 = Oto 17.7ppm«h
CF, NF, CF- 1 x , CF-2 x : 3 months
mean=l, 14,15,32
CF, +O3: 63-65 days
SUM06 = 0.0, 75.7 or 68.4
ppm«h; AOT40 = 0.0, 50.9 or
46.4 ppm«h; 7-h mean = 7, 89
or 85 (early and late season
experiments)
CF, 1.4x: 1 years
12-hmean = 23,72
CF, NF, AA: 2 years
12-hmean=31,51,49in
year 2000; 25, 46, 47 in year
2001
CF, +40: 7-h mean = 20, 70 1 years
CF, 1.5x: 1 years
12-hmean = 21,71
CF,NF, 1.5x, 1 years
12-h mean = 24, 51,78
CF, NF: 7-h mean = 12, 27 1 years
Variable
Green pod
yield
Pod yield
Pod dry
weight
Pod dry
weight
Pod dry
weight
Grain yield
Seed-cotton
weight
Seed-cotton
weight
Grain yield
Response
(Decrease from
lowest, %)
29 at 9-h mean = 44
(AOT40 = 3.6ppm«h)
56 (CF, 2x)
51, 57 (early and late
season experiments)
n.s. for Tenderette, 90
for SI 56
n.s. forR123 n.s. for
Oregon-91 in 2001,
27 in 2001; 21, 45 for
S156
13
22
21,49(NF, 1.5x)
+2 (n.s.)
Reference
Tonneijck and
Van Dijk (1998)
Brunschon-Harti
etal. (1995)
Tingey et al.
(2002)
Heagle et al.
(1999)
Heagle et al.
(1999)
Mulchi et al.
(1995)Rudorff
etal. (1996c)
Heagle et al.
(1999)
Heagle et al.
(1999)
Pleijel et al.
(1994a)
-------
Table AX9-16 (cont'd). Summary of Selected Studies of Ozone Effects on Annual Species
a
S-
to
o
o
VO
1
[V
o
§
H
6
O
2;
0
H
O
O
H
W
0
^
o
1 — I
H
W
Species
Potato, cvs.
Superior,
Dark Red
Norland
Potato2
Rape, oilseed
Rice, cvs.
Koshi-hikari,
Nippon-bare
Soybean
Soybean, cv.
Essex
Soybean, cvs.
Forrest, Essex
Soybean, cv.
Essex
Soybean, cv.
Essex
Soybean, cv.
Holladay
Soybean, cv.
NK-6955
Soybean, 3
cvs.
Facility
OTC
OTC
Open Air
OTC
OTC
OTC
OTC
OTC
OTC
OTC
OTC
OTC
Location
Raleigh, NC
6 sites N. Europe
Northumberland,
UK
Japan
Beltsville, MD
Raleigh, NC
Maryland
Raleigh, NC
Raleigh, NC
Raleigh, NC
Raleigh, NC
Raleigh, NC
O3 Concentration
(units are ppb unless
otherwise specified)1
CF,NF, 1.5x:
12-h mean = 15,45,80
AOT40 = 6-27 ppm«h
AA, +O3: 7-h mean for 17
days Aug. -Sept = 30, 77, for
32 days in
May-June = 31, 80
CF, lx, 1.5x,2x,2.75x:7-h
mean =13. 5-93. 4
CF, +40: 7-h mean = 25, 72
CF, 1.5x: 12-hmeanfor3
years = 23, 82
CF, +40: 7-h mean = 24 and
24, 63 and 62 for each year
CF,NF, 1.5x:
12-h mean = 20, 50,79
CF,NF, 1.5x:
12-h mean = 18,42,69
CF,NF, 1.5x:
12-h mean = 18,42,69
CF,NF, 1.5x:
12-h mean = 18,42,69
CF,NF, 1.5x:
12-h mean = 14,36,64
Duration
1 years
2 years (1 year
at 2 sites)
17 days in fall,
overwinter, 32
days in spring
3 years
2 years
3 years
2 years
1 year
1 year
1 year
1 year
3 months
Variable
Tuber yield
Tuber yield
Seed yield
Grain yield
Seed yield
Seed yield
Seed yield
Seed yield
Seed yield
Seed yield
Seed yield
Seed yield
Response
(Decrease from
lowest, %)
15,31 for Norland in
NFandl.Sx; 11 for
Superior in 1.5x
4% average for all
experiments
14
3 to 10 at 40 ppb
25
41
10, 32 (2 cvs.)
16, 37 (NF, 1.5x)
15, 40 (NF, 1.5x)
22, 36 (NF, 1.5x)
+46, +4(NF, 1.5x)
At ambient = +14, 11,
16 for 3 cvs.
Reference
Heagle et al.
(1999)
Craigon et al.
(2002)
Ollerenshaw
etal. (1999)
Kobayashi et al.
(1995)
Mulchi et al.
(1995)
Fiscus et al.
(1997)
Chernikova
et al. (2000)
Heagle et al.
(1998)
Heagle et al.
(1998)
Heagle et al.
(1998)
Heagle et al.
(1998)
Miller et al.
(1994)
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Table AX9-16 (cont'd). Summary of Selected Studies of Ozone Effects on Annual Species
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Species Facility Location
Soybean, 3 OTC Raleigh, NC
cvs.
Soybean, cv. OTC Raleigh, NC
Essex
Soybean, cvs, OTC Beltsville, MD
Essex, Forrest
Soybean, cv. OTC Raleigh, NC
Essex
Timothy OTC Sweden
Watermelon OTC Spain
Wheat1, cv. OTC 8 sites in N
Minaret Europe
Wheat1 OTC Sweden
Wheat, cv. OTC SE Ireland
Promessa
Wheat, cv. OTC SE Ireland
Promessa
O3 Concentration
(units are ppb unless
otherwise specified)1
CF,NF, 1.5x: 12-h mean =
24, 49, 83
CF,NF, 1.5x:
12-h mean = 20, 50, 79
CF,NF+: 7-h mean = 24, 58
CF, 1.5x:
12-h mean = 24, 75(1999);
22, 67 (2000)
AOT40 = 10, 20, 340;
12-h mean = 20, 152
CF(O3 = 0),NFml988
AOT40 = 5.96 ppm«h,
SUM06 = 0.29 ppm«h, in
1989 AOT40 =18.92 ppm«h,
SUM06 = 4.95ppm«h
12-h mean (SD) low = 26.3
(12.2), 12-h mean (SD)-high
= 51.4 (18.3)
AOT40 mean (SD)
low = 6.18(8.54)ppm«h,
AOT40 mean (SD)
high = 28.23 (23.05) ppm«h
AOT40 0 to 15 ppnHi
CF, +50:
12-h total = 5.6, 32.6 ppm«h
CF, +25:
12-h total = 6.2, 33.4 ppm«h
Duration
4 months
4 months
134 days
164 d (1999),
149 d (2000)
1 year
2 expts of 1 year
13 studies of 1
year each
7 years
3 h/day, 5
d/week, 7 weeks
6 h/day, 5
d/week, 7 weeks
Variable
Seed yield
Seed yield
Seed yield
Seed yield
Biomass
Fruit yield
Grain yield
Grain yield
Grain yield
Grain yield
Response
(Decrease from
lowest, %)
At ambient = 17, 13,
18 (3 cvs.)
ll,22(amb., 1.5x)
Essex = +11 (n.s.),
Forrest = 21
24 (1999), 39 (pots in
2000), 41 (ground in
2000)
58
19, 3 9 (2 expts)
13 (n.s.)
23 at AOT40 = 15
ppm«h
53
+17
Reference
Miller et al.
(1994)
Miller et al.
(1998)
Robinson and
Bntz (2000)
Booker et al.
(2005)
Danielsson et al.
(1999)
Gimeno et al.
(1999)
Bender et al.
(1999)Hertstein
etal. (1999)
Danielsson et al.
(2003)
Finnan et al.
(1996)
Finnan et al.
(1996)
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Table AX9-16 (cont'd). Summary of Selected Studies of Ozone Effects on Annual Species
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Species Facility Location
Wheat, cv. OTC SE Ireland
Promessa
Wheat, cvs. OTC Beltsville, MD
Massey,
Saluda
Wheat, cv. OTC Germany
Turbo
Wheat, cv. OTC Germany
Turbo
Wheat, cv. OTC Germany
Turbo
Wheat, OTC Raleigh, NC
winter, 8 cvs.
Wheat, OTC Raleigh, NC
winter, 8 cvs
Wheat, cv. OTC Finland
Drabant
Wheat, cv. Open Air Northumberland,
Riband UK
Values for ambient or NF treatments are indicated in bold.
Bold indicates that multiple experiments (more than just 2
O3 Concentration
(units are ppb unless
otherwise specified)1
CF, +25, +50:
12-h total = 6. 7, 34, 34
ppm«h
CF, +40:
Duration
+25 = 6 h/day, 5
d/week,
+50 = 3 h/day, 5
d/week,
both 7 weeks
2 years
Variable
Grain yield
Grain yield
Response (Decrease
from lowest, %)
Amb + 25 = 3 (n.s.);
Amb + 50 = 17
20
7-h mean = 1 9, 20 and 6 1 , 65
(2 years)
8-h mean = 5.9, 61.2, 92.5
8-h mean = 4. 7, 86.4
7-h mean = 5, 41,73
12-h mean = 27, 47, 90
12-h mean = 22, 38, 74
1992: 12-h mean =14, 30,
61; AOT40 = 16.3, 34.8,
54.6ppm«h. 1993:
12-h mean =9, 21,45;
AOT40 = 10.2, 24.8, 40.6
ppm«h
AOT40 for Mar to
Aug93 = 3.5, 6.2ppm«h
years at a single site) were included
1 year
1 year
1 year
2 months
2 months
2 years
1 year,
overwinter
in the analysis.
Grain yield
Grain yield
Grain yield
Grain yield
Grain yield
Grain yield
Grain yield
14,40
(mid, high O3)
20
35
5 (n.s.)
16 (n.s.)
At highest O3 = 13
each year
13
Reference
Finnan et al.
(1996)
Mulchi et al.
(1995)Rudorff
etal. (1996b)
Bender et al.
(1994)
Bender et al.
(1994)
Fangmeier et al.
(1994)
Heagle et al.
(2000)
Heagle et al.
(2000)
Ojanpera et al.
(1998)
Ollerenshaw and
Lyons (1999)
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Table AX9-17. Summary of Selected Studies of the Effects of Ozone on Perennial Herbaceous Plants
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Species Facility
Alfalfa, cvs. OTC
Apica, Team
Bahia grass OTC
Bent grass OTC
(Capillaris sp.)
Blackberry Large
OTC
Clover, white Ambient
air
Clover, white Ambient
air
Clover, white OTC
Clover, white OTC
and red
Clover, white, OTC
cv. Menna
Location
Quebec,
Canada
Auburn, AL
United
Kingdom
Alabama
MA, OR, NC,
CA (2 sites)
andVA
14 European
sites
United
Kingdom
Switzerland
Italy
O, Concentration
J
(units are ppb unless otherwise
specified)1
12-hmean: 1991 = 6,39,49,110;
1992 = 0,34,42,94
12-hmean = 22, 45, 91
AOT40 = 0.8-15.0 ppm-h
1994: AOT40 = 2-1 12 ppm-h,
SUM06= l-162ppm-h,
1995: AOT40 = 3-83 ppm-h,
SUM06 = 0-132ppm-h
SUM06 for 6-h/day = 10.2-39.4
ppm-h, AOT40 for 12-h/day =
0.6-50.1 ppm-h
AOT40 for 28 d = 0-12 ppm-h
AOT40 = 0.8-15.0ppm-h
CF, NF, NF+. NF++:
12-hmean = 21, 39, 47, 65
CF,NF: AOT40 = 0.1;
8.9 ppm-h, 7-h mean = 24, 53
Duration
3 months in
each of 2
years
24 weeks
8 h/day for 3
months
7 months in
1994,6
months in
1995
2 growing
seasons
3 growing
seasons
8 h/day for
3 months
3.5 months/
year for
2 years
2 months
Variable
Biomass
Biomass at ambient O3 for
1 st, 2nd cutting of early
and late season plantings.
Biomass, in competition
with 3 other spp. Total
biomass in uncut pots,
aboveground biomass
in cut pots (cut every
14 days).
Percent canopy cover
(grown in old field
community), biomass ripe
fruit number.
Biomass ratio
(sensitive/resistant)
Biomass ratio
sensitive/resistant)
Biomass, in competition
3 other spp. Total biomass
in uncut pots, aboveground
biomass in cut pots
(cut every 14 days).
Biomass, in managed
pasture
Biomass
Response (Decrease
from lowest, %)
ForNF: Apica = 31,
21; Team = 14,2
(n.s.)
34,29(n.s.),+6(n.s.),
9 (n.s.)
8 (uncut), +18 (cut)
+124 for cover, n.s.
for biomass, 28% for
ripe fruit number but
sig. chamber effect.
4 at 6-h SUMO6 =
39.4 ppm-h; 12 h
AOT40 = 50.1ppm-h
5 at AOT40 for 28
days = 0.9-1.7 ppm-h
18 (uncut), 40 (cut)
24, 26, 52
20
Reference
Renaud et al.
(1997)
Muntifering
et al. (2000)
Ashmore and
Ainsworth
(1995)
Barbo et al.
(1998)
Chappelka
(2002)
Heagle and
Stefanski
(2000)
Mills et al.
(2000)
Ashmore and
Ainsworth
(1995)
Fuhrer et al.
(1994)
Fumagalli
etal. (1997)
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Table AX9-17 (cont'd). Summary of Selected Studies
Species Facility
Fescue, red OTC
Lespedeza, OTC
Sericea
Little bluestem OTC
Phleum alpinum OTC
Speedwell, OTC
Germander
Strawberry OTC
Sumac, winged OTC
Timothy OTC
Location
United
Kingdom
Auburn, AL
Auburn, AL
Sweden
United
Kingdom
United
Kingdom
Alabama
Sweden
O3 Concentration
(units are ppb unless otherwise
specified)1
AOT40 = 0.8-15.0 ppm-h
CF,NF,2x: 12-h mean = 23, 40,
83, SUM06 = 0.2, 9.1, 61.0,
AOT40 = 0.6, 7.0, 39.8
CF, NF, 2x: 12-h mean = 23, 40,
83 ppb, SUM06 = 0.2, 9.1, 61.0,
AOT40 = 0.6, 7.0, 39.8
AOT40 = 0.01, 0.02, 0.34 ppm-h;
12-h mean = 20, 152
AOT40 = 0.8-15.0 ppm-h
8-h mean = 27, 92;
AOT40 for +O3 = 24.59 ppm-h
SUM06 = Otol32ppm-h
CF, NF, CF+:
AOT40 = 0.0, 1.3, 20.3 ppm-h;
12-h mean = 20, 68, 152
of the Effects of Ozone on Perennial Herbaceous Plants
Duration Variable
8 h/day for 3 Biomass, in competition
months with 3 other spp. Total
biomass in uncut pots,
aboveground biomass in
cut pots (cut every 14
days).
10 weeks Biomass
10 weeks Biomass
1 year biomass
8 h/day for 3 Biomass, in competition
months with 3 other spp. Total
biomass in uncut pots,
aboveground biomass in
cut pots (cut every 14
days).
69 days Fruit size, yield
6 months Percent canopy cover
(grown in old field
community)
1 year Biomass
Response (Decrease
from lowest, %) Reference
+ 30 (uncut), +13 Ashmore and
(cut) Ainsworth
(1995)
n.s. Powell et al.
(2003)
n.s. Powell et al.
(2003)
87 Danielsson
etal. (1999)
14 (uncut), 26 (cut) Ashmore and
Ainsworth
(1995)
Size = 14, Drogoudi and
yield = (n.s.) Ashmore
(2000)
95 Barbo et al.
(1998)
n.s. in NF, 58 in CF+ Danielsson
etal. (1999)
1 Values for ambient or NF treatments are indicated in bold.
2 Bold indicates that multiple
experiments (more
than just 2 years at a single site) were
included in the analysis.
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Table AX9-18. Summary of Selected Studies of Ozone Effects on Deciduous Trees and Shrubs
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Species Age Facility
Ash, OTC
European
Ash, Seedling OTC
European
Aspen Cutting, OTC
Seedling
Aspen Cutting OTC
Aspen Cutting FACE
Aspen Cutting Large OTC
Aspen First year OTC
Beech, OTC
European
Beech, Seedling OTC
European
O3 Concentration
(units are ppb unless
Location otherwise specified)1
Hampshire, UK NF, NF+: Mean = 17.7,
44.1;AOT40for24h=1.9,
59.9 ppm'h
Switzerland 0.5 x, 0.85x, lx; 0.5x+30:
AOT40 = 0.1, 3.4, 7.1, 19.7
ppm-h
Michigan CF, lx, 2x:
3 months 7-h mean for
1990=7,43,63;
for 1991 (square wave
exposure) = 11,45,66
Michigan CF, lx,2x:
SUMOO= 11,58, 71 ppm-h
Wisconsin Ambient, 1.5 x: 4-year
ambient 12-h mean = 34.6,
36.9, 36.0, 36.6; 4-year
1.5x 12-h mean = 54.5,
51.1,48.9,52.8
New York lx, 1.7x,3x:
SUM06 = 1, 20, 62 ppm-h;
9-h mean = 40, 74, 124
Pennsylvania 8-h mean = 39, 73
Switzerland 0.5x, 0.85x, lx; 0.5x+30:
AOT40 = 0.1,3.4, 7.1,
19.7 ppm'h
Belgium CF, NF, +30:
8-h mean =5. 29, 33;
AOT40 = 0. 4.06,
8.88 ppm-h
Duration
3 years for
day 100 to
day 162
5 months
98 days
98 days
7 years
(only 4
years of O3
data
reported)
92 days
1 1 weeks
5 months
23 April -
30 Sept
Variable
Growth and
biomass of
organs
Biomass
Total
biomass of 3
clones and
seedlings
Total
biomass
Volume
(d2*h)
Shoot
biomass
Biomass
Biomass
Growth
Response
(Decrease from
lowest, %)
n.s.
26 at lx, 50 at
O.Sx + 30
For 1990: 2-22
at lx for clones
(mean= 16), 14
for seedlings.
For 1991: 23-39
at lx for clones
(mean = 30)
25-38 at 1 x
21 after 3 years,
14 after 7 years
at 1.5x
14, 25 for 2
clones at 1.7*
14-30 for 3 of 6
N treatments
6 at lx, 30 at
O.Sx+30
No effect
Reference
Broadmeadow
and Jackson
(2000)
Landolt et al.
(2000)
Kamosky et al.
(1996)
Dickson et al.
(2001)
Isebrands et al.
(2001)
Yunand
Laurence
(1999)
Pell et al.
(1995)
Landolt et al.
(2000)
Border et al.
(2000a)
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Table AX9-18 (cont'd). Summary of Selected Studies of Ozone Effects on Deciduous Trees and Shrubs
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Species Age Facility Location
Beech, European Seedling Growth chamber Belgium
Beech, European 0-3 years OTC Switzerland
Beech, Japanese 4 years Growth chamber Japan
Birch, silver Sapling FACE Finland
Birch, silver Sapling OTC Sweden
Birch, [B. Seedling Chamber in Norway
pubescens] glasshouse
Birch, paper Sapling FACE Wisconsin
Cherry, black 2 years OTC Norris, TN
O3 Concentration
(units are ppb unless
otherwise specified)1
CF, CF+40, CF+100:
SumO = 0.48, 8.93,25.14
ppm-h; AOT40of
NF+100 = 13.91 ppm-h;
uptake = 159,29657095
molm2
AOT40for24h/days =
4-73 ppm'h
CF, +60 ppb for 7 h/day
AOT40= l,15ppm-h;
7-h mean = 26, 40
NF, NF+, NF++, daylight
mean 1997 = 29, 37, 54;
1998 = 25, 42, 71 ppb;
AOT40 1997 = 2.4, 6.9,
35.1; 1998 = 0.6,19.6,
74.7 ppm-h
AOT40 = 0.1,2.5, 7.1,
7.4, 17.8, 19.8 ppm-h
Ambient, 1.5x: 4 y
Ambient 12-h mean =
34.6, 36.9, 36.0, 36.6; 4y
1.5x 12-h mean = 54.5,
51.1,48.9,52.8
CF, lx,2x:
7-h mean = 2 1,50, 97
Duration Variable
7 episodes of 5 Biomass,
days diameter
1-3 years Total
biomass
156 days Total
biomass
5 years Biomass
2 years Total
biomass
40 days Biomass
7 years (only 4 Volume
yofO3data (d2*h)
reported)
April to August Biomass
Response
(Decrease from
lowest, %)
No effect
20atAOT40
for 24 h = 32
ppm'h
19
34 for root, n.s.
for stem
Total biomass
n.s. atNF+,22
atNF++;root
biomass 30 at
NF++
Sig. decrease in
rootatAOT40
= 2.5 ppm-h,
shoot at 7. 1
ppm'h
No effect
No effect
Reference
Border et al.
(2001)
Braun and
Fluckiger
(1995)
Yonekura et al.
(2001)
Oksanen et al.
(2001)
Karlsson et al.
(2003)
Mortensen
(1998)
Isebrands et al.
(2001)
Samuelson,
1994)
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Table AX9-18 (cont'd). Summary of Selected Studies of Ozone Effects on Deciduous Trees and Shrubs
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Cherry, black Seedling OTC
Cherry, black Seedling OTC
O3 Concentration
(units are ppb unless
Location otherwise specified)1 Duration
GSMNP1 CF, lx, 1.5x,2x: 76 days
SUM06 = 0-40.6 ppm-h,
AOT40 = 0.03-28.3
ppm-h
GSMNP1 CF, 0.5x, Ix5l.5x52x: 140 days
SUM06 = 0-53.7 ppm-h;
Response
(Decrease from
Variable lowest, %)
Biomass n.s. at lx and
1.5x, 38at2x
Biomass n.s. at lx and
1.5x, 59at2x
Reference
Neufeld et al.
(1995)
Neufeld et al.
(1995)
AOT40 = 0-40.4 ppm-h
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Cherry, black 1 year OTC Delaware, OH CF, 0.5, 1, 1.5, 2x:
SUMOO in 1990 = 17-107
ppm-h, in 1991 = 31-197
ppm-h
Cherry, black Seedling OTC Pennsylvania CF, 0.75x, 0.97x:
7-h mean = 39 to 46,
SUM06 = 0-10.34 ppm-h
Cottonwood, Cutting Ambient, in buried In and within 100 12 h mean = 23-49 ppb
Eastern pots with km of New York
irrigation City, NY
Grape 3 years OTC Austria CF, lx, +30, +50:
(AOT40 = 0-50 ppm-h
Oak Seedling OTC Hampshire, UK NF,NF+:
Mean = 17.7, 44,
AOT40for24h=1.9,
59.9 ppm'h
Oak, red Seedling OTC Norris, TN SUM06 for 3 years = 0,
29, 326 ppm-h; SUMOO
for 3 years = 147, 255 and
507 pprn-h
2 years (in 1990
for 3.5 months,
1991 for 4
months)
3 years for 17
weeks
2 months each
year, 3 10-years
experiments
2 years
(preflowering,
past harvest)
3 years for day
100 to day 162
3 years
Total
biomass
Total
biomass
Total
biomass
Fruit yield
Biomass of
organs
Total
biomass
no effect at 1 x
andl.Sx, 32 at
2x
6 at 0.75 x, 14 at
0.97x
33% decrease at
38 ppb
compared to 23
ppb
Calculated 10 at
AOT40 = 27
pprn-h
30 for total
biomass
n.s.
Rebbeck
(1996)
Kouterick
et al. (2000)
Gregg et al.
(2003)
Soja et al.
(1997)
Broadmeadow
and Jackson
(2000)
Samuelson
etal. (1996)
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Species Age Facility
Oak, red 30 years OTC
Maple, red 2 years OTC
Maple, sugar 1 year OTC
Maple, sugar Seedling Large OTC
Maple, sugar Seedling Large OTC
Maple, sugar Sapling FACE
Plum, Casselman Sapling Large OTC
Poplar, black Seedling OTC
O3 Concentration
(units are ppb unless
Location otherwise specified)1
Norris, TN SUM06 for 3 years = 0,
29, 326 ppm-h; SUMOO
for 3 years = 147, 255 and
507 ppm-h
Norris, TN CF, lx,2x:
7-h mean = 2 1,50, 97
ppm-h
Delaware, OH CF, 0.5, 1.5, 2x:
UMOO in 1990 =17 to 107
ppm-h, in 1991 = 31 to
1 97 ppm'h
Ithaca,NY CF, lx, 1.5x,2x: 3 years
SUMOO = 148 to 591
ppm-h; daytime mean =
19.7 to 40.7
Ithaca,NY lx,1.7x, 3x:
3 years 12-h mean = 38,
69,117
Wisconsin Ambient, 1.5 x; 4 y
Ambient 12-h mean =
34.6, 36.9, 36.0, 36.6; 4y
1.5xl2-hmean=54.5,
51.1,48.9,52.8
Fresno, CA CF, lx,+O3:
12-h mean =31, 48, 91
Belgium CF, NF, +30:
8-h mean = 5, 29, 33;
AOT40 = 0, 4,8.9 ppm-h
Duration
3 years
April to August
2 years (in 1990
for 3.5 months,
1991 for 4
months)
3 years for 134,
128, 109 days
3 years for 109,
143, 116 days
7 years (only 4
y of O3 data
reported)
4 years
23 April - 30
Sept
Variable
Stem
increment
Biomass
Total
biomass
Biomass
Total
biomass
Volume
(d2*h)
Stem
increment,
fruit yield
Diameter,
height
Response
(Decrease from
lowest, %)
n.s. despite 50%
reduction in net
photosynthesis
No effect
n.s., but linear
trend
No effect
For 1.7x and
3x: 21, 64 in
low light, 26
and 41 in high
light
18
Fruit yield 16 at
lx, stem +14 at
+03
29 for diameter
in NF+, no
effect on height
Reference
Samuelson
etal. (1996)
Samuelson
(1994)
Rebbeck
(1996)
Laurence et al.
(1996)
Topa et al.
(2001)
Isebrands et al.
(2001)
Retzlaffetal.
(1997)
Bortier et al.
(2000b)
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Table AX9-18 (cont'd). Summary of Selected Studies of Ozone Effects on Deciduous Trees and Shrubs
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Species Age Facility
Poplar, hybrid 0 year FACE
(P. tremuloides x
P. tremula)
Poplar, hybrid Cutting OTC
Poplar, hybrid Cutting OTC
Yellow-poplar 1 year OTC
Yellow-poplar 1-7 year Large OTC
O3 Concentration
(units are ppb unless
Location otherwise specified)1
Finland AOT40 = 0.07, 1.6
ppm'h; 7-h mean = 30, 38
Michigan CF, CF+100:
12, 48 ppm-h
Michigan CF, CF+100:
12, 48 ppm-h
Delaware, OH CF, 0.5x, 1.5x, 2x:
SUMOOinl990 = 17to
107ppm-h, in 1991 =31
to 1 97 ppm-h
Delaware, OH CF, 0.5x, 1.5x,:
SUMOO= 145, 583, 861
ppm-h; SUM06 = 0.3,
228.7, 661.8 ppm-h over 5
years
Duration
2 months
60 days
60 days
2 years (in 1990
for 3.5 months,
1991 for 4
months)
5 years
Variable
Biomass,
height
Total
biomass
Total
biomass
Total
biomass
Total
biomass
Response
(Decrease from
lowest, %)
n.s. for biomass,
6 for height
46 for average
of 5 clones
46 for average
of 5 clones
No effect
No effect
Reference
Oksanen et al.
(2001)
Dickson et al.
(1998)
Dickson et al.
(1998)
Rebbeck
(1996)
Rebbeck
(1996)
H
6
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1 Values for ambient or NF treatments are indicated in bold.
2 Bold indicates that multiple experiments (more than just 2 years at a single site) were included in the analysis.
o
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-------
Table AX9-19. Summary of Selected Studies of Ozone Effects on Evergreen Trees and Shrubs
S-
to
o
ui Species
Fir,
Douglas
Hemlock,
eastern
Pine,
loblolly
Pine,
loblolly
Age Facility Location
Seedling Open air British
Columbia
Seedling OTC GSMNP1,
TN
12 weeks OTC Oak Ridge,
TN
1 year OTC Alabama
O3 Concentration
(units are ppb unless
otherwise specified)1
12 tits:
12-h mean 1988 =18-41;
1989 = 27-66
CFto2x:
SUM06 = 0.2-108.1 ppm«h,
AOT40 = 0.2-63. 9 ppm«h
CFto2x:
24-h summer = 74, 137, 169,
206, 284 ppm«h
1994:
AOT40 = 2-112ppm«h,
SUM06 = 10-162 ppm«h,
Duration
1988 = 92
days; 1989 =
101 days
3 years
3 months
2 years, April
to October
Variable
Second flush
biomass
Biomass
Biomass
Dry weight,
height,
diameter
Response
(decrease from
lowest, %) Reference
Calculated 55 at Runeckles and
highest exposure Wright (1996)
No effect Neufeld et al.
(2000)
14 in 1 x (avg for all McLaughlin
families) etal. (1994)
n.s. Barboetal.
(2002)
VO
iw:>:
AOT40 = 3-83 ppm«h,
SUM06 = 0-132ppm«h
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Pine, 3 years OTC Raleigh, NC Ambient, CF, NF, 1.5 x.,
loblolly 2.5x: 12-h mean = 54, 29, 47,
76,98
Pine, 4 weeks Large Auburn, AL CF, lx,2x:
loblolly OTC 12-h mean = 13, 47,, 98
ppm«hin!998; 12,44,97
Pine, Seedling OTC Corvalhs, For CF 12-h SUM06 = 0
ponderosa OR ppm«h; for +03 12-h
SUMO6 = 22, 27, 31 ppm«h
for 3 years
5 months
2 12 -week
experiments
3 years: 16
weeks, 16
weeks, 14
weeks
Height,
diameter,
needle length
Shoot biomass,
root biomass,
foliar injury
Total biomass
No effect on stem
height or diameter,
decrease in needle
length
Shoot = 15 in lx, 22
in 2x in both years;
Root = 26 in 2x in
both years; Foliar
sig. greater in lx in
1999 and 2x in both
years.
No effect without
grass, 25 with grass
present
Anttonen et al.
(1996)
McLaughlin
etal. (1994)
Andersen et al.
(2001)
-------
Table AX9-19 (cont'd). Summary of Selected Studies of Ozone Effects on Evergreen Trees and Shrubs
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to
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Species Age Facility
Pine, 39 to 45 Ambient
ponderosa years gradient
Pine, Seedling OTC
ponderosa
Pine, OTC
Scots
Pine, 3 -6 years Free air
Scots
Pine, Seedling OTC
Scots
Pine, 3 years OTC
Scots
Pine, 3 years Free air
Scots
Pine, Seedling OTC
Table
Mountain
Pine, Seedling OTC
Virginia
O3 Concentration
(units are ppb unless
Location otherwise specified)1
CA 24-h mean for 3 weeks late
July and early August for
1993 and 1994 = 70-90 ppb
CA CF, lx,2x:
24-h mean
approx. 20, 60, 120
Hampshire, NF, NF+:
UK Mean = 17.7, 44.1; 24-h
AOT40 = 1.9, 59.9 ppm«h
Finland Amb, +O3:
AOT40 = 0-1, 2-13 ppm«h
Switerzland 0.5x, 0.85x, lx, 0.5x+30:
AOT40 = 0.1, 3.4,7.1,
19.7 ppm«h
Finland CF, lx,+O3:
24 h AOT40 for
2 years = 0.5, 6, 73 ppm«h
Finland 1X,+O3:
24 h AOT40 for 2 years = 2,
37 ppm«h
GSMNP2, CFto2x:
TN SUM06 = 0.2-116.4 ppm«h,
AOT40 = 0.2-71. 7 ppm«h
GSMNP2, CFto2x:
TN SUM06 = 0.1-32.8, 47.9,
56.2 ppm«h;
AOT40 = 0.1-19.3, 27.1,
34.4 ppm«h
Duration Variable
Ambient Fine and
gradient medium root
growth
Total biomass
3 years for 62 Total biomass
days
3 years Biomass
5 months Biomass
2 years (4 Biomass
months each)
3 years Root and shoot
(3-4 months biomass
each)
3 years Biomass
1 -2 years (3 Biomass
expts)
Response
(decrease from
lowest, %)
85 at most polluted
site.
n.s.
15
No effect
14 at lx, 22 at
0.5x+30
No effect
32 only for root
biomass in high N
treatment
Slight decrease in
older needle mass
only
No effect
Reference
Grulke et al.
(1998)
Takemoto et al.
(1997)
Broadmeadow
and Jackson
(2000)
Kainulainen
et al. (2000)
Landolt et al.
(2000)
Utriainen et al.
(2000)
Utriainen and
Holopainen
(2001)
Neufeld et al.
(2000)
Neufeld et al.
(2000)
-------
Table AX9-19 (cont'd). Summary of Selected Studies of Ozone Effects on Evergreen Trees and Shrubs
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Species
Sequoia,
giant
Spruce,
Norway
Spruce,
Norway
Spruce,
Norway
Spruce,
Norway
Spruce,
Norway
Spruce,
red
1 Values
2 Great
Age
125 years
4-7 years
3 -7 years
Seedling
0-3
years
3 to 7
years
Saplin
9
Facility Location
Branch California
chamber
Open air Finland
OTC Sweden
OTC Switerzland
OTC Switzerl
and
OTC Sweden
Large Ithaca,
OTC NY
for ambient or NF treatments
O3 Concentration Response
(units are ppb unless (decrease from
otherwise specified)1 Duration Variable lowest, %)
0.25x, lx; 2x, 3x; 61 days Branch growth No effect
24-h SUMOO approx. 10, 85,
180, 560ppm«h
Amb, +O3: 3 years Biomass No effect
AOT40 = 01, 2-13ppm«h
CF, 1.5 x; 4 years Total biomass 8
12-h mean for 4 years = 12,
44; AOT40 = 2, 23 ppm«h
0.5x, 0.85x, lxs 0.5x+30: 5 months Biomass n.s.
AOT40 = 0.1, 3.4,7.1,
19.7 ppm«h
AOT40 for 24 h for 1-3 years Total n.s.
1 to 3 years = 22 biomass
to 63 ppm»h
CF, Ix, 1.5x: 4 years Biomass 5.3 at 1 . 5x
AOT40 daylight for
4 years =1, 16, 79
ppm»h
CF, Ix, 1.5x, 2x : 4 years: Biomass No effect
total for 4 years = 98-124
211 to 569 ppm»h; days/year
daytime mean =
21-71
are indicated in bold.
Reference
Grulke et al.
(1996)
Kainulainen
et al. (2000)
Karlsson et al.
(2002)
Landolt et al.
(2000)
Braun and
Fluckiger
(1995)
Wallin et al.
(2002)
Laurence et al.
(1997)
Smoky Mountains National Park.
O
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6-
C/3
<-K Species
to l
ui Bean, cv. Lit
Table AX9-20.
Description
10-cm pots in OTCs in
Germany
Ethylene Diurea Effects on Vegetation Responses to Ozone
EDU Application
Soil drench 200 mL of
150 ppm solution per
plant every 14 days
Ozone Exposure Effects of EDU
CF, NF, CF-lx, CF-2x: O3 reduced pod, shoot, and
mean =1, 14, 15, 32 ppb root mass. EDU increased
root, leaf, and shoot mass, but
a significant interaction with
O3 occurred only for root
weight.
Reference
Brunschon-Harti
etal. (1995)
Bean, cv. BBL-290
2 expts in 5.5 L pots in
OTCs with 4 O3
treatments
Soil drench every 14
days, in expt 1 = 0.14, 28,
56, 120 mg/L potting
medium; expt 2 = 0, 8,
16, 32 mg/L
2 expts with CF, NF and
2 constant additions of
O3. 7-h mean O3 (ppb)
for Expt 1 = 34, 70, 95,
121; Expt 2 =19, 42. 74,
106
Visible injury and reduced
total biomass or yield, even in
CF treatment. Within an O3
treatment, sometimes
increased yield (Expt 2 only).
Miller et al. (1994)
vo
to
Bean, cv. Bush Blue
Lake 290, Bush Blue
Lake 274, lines SI56,
R123
Field-grown in fine sandy Foliar spray at 300 ppm
loam in Massachusetts, in every 7 days between
2 one-year experiments. full expansion of primary
leaves and pod
senescence.
Ambient, with 181 and
141 h> 40 ppb, 74 and
95 h> 60 ppb, and 23 h >
80 ppb in 2001 and 2002
respectively
EDU increased final above-
ground biomass in SI56 in
200 land 2002, but
significantly decreased above-
ground biomass in R123 in
both years and BBL 274 in
2002.
Elagoz and Manning
al. (2005a)
Bean
Pots with potting mix at Soil drench of 200 mL of AOT40 = 0.4-1.8 ppm-h 0 to 50% increase in pod
Ribas and Penuelas
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6
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Bean, cv. Lit
Bean, cv. Lit
3 locations in Spain
(2 years at 1 site)
Pots with potting mix at
4 sites in the Netherlands
Pots with potting mix at
1 site in Belgium
AOT40 = 0.64-0.98,
7-h mean = 49-55 ppb
increasing concentrations
of 100, 150,200,250
ppm every 14 days
(4-10 mg 1:1 soil)
Soil drench of 200 mL of
increasing concentrations
of 100, 150,200,250
ppm every 14 days
(4-10 mg 1:1 soil)
Soil drench of 200 mL of AOT40 = 0.81 ppm-h
increasing concentrations
of 100, 150,200,250
ppm every 14 days
(4-10 mg 1:1 soil)
mass, but did not restore yield (2000)
at sites with higher O3.
Average 20% yield increase at
all sites.
16% ield increase.
Tonneijck and Van
Dyk(1997)
Vandermeiren et al.
(1995)
-------
Table AX9-20 (cont'd). Ethylene Diurea Effects on Vegetation Responses to Ozone
S-
to
o
Species
Description
EDU Application
Ozone Exposure
Effects of EDU
Reference
Clover, subterranean
Plants in 10-cm pots at 4
rural sites in the
Netherlands for 3 years
100 ml of ISOppm
solution as soil drench
every 14 days for 2
months
AOT40 = 0-0.56 ppm-h
for 4-week periods
Injury, but not leaf biomass
was affected by EDU and O3
exposure.
Tonneijck and Van
Dijk (2002)
O
HH
H
W
Clover, white
15-cm pots in field, well
watered, 12 locations
throughout Europe, 3
years
100 mL of ISOppm
solution as soil drench
every 14 days for 3
months
AOT40 (28 days) = 0-20
ppm-h
Change in biomass ratio, weak
linear relationship (r2 = 0.16)
stronger relationship using
ANN and climatic factors
Ball etal. (1998)
Clover, white, cv. Menna
2 expts,10-cm pots in
field in Italy, see also
companion OTC expt
100 mL of ISOppm
solution as soil drench
every 14 days for 2
months
AOT40 = 15.5,
12.1 ppm«h;
7-h mean = 69, 60
Fumagalli et al.
(1997)
vo
to
oo
Clover, white, cv. Menna
Poplar, hybrid
2 expts,10-cm pots in
OTCs in Denmark
Stem injections, field,
cuttings, 1 or 2 years
Soil drench 100 mL of
150 ppm solution every
14 days
Approx. 125 or 250
mg/leaf (low, high EDU
treatments)
5 times every 14 days
CF,NF,NF+25,
NF+50 ppb,
O3 exposure not reported
1991:
7-h mean = 56,
AOT40 = 23;
1992:
7-h mean = 59,
AOT40 = 27
No effect of EDU despite
highly significant effect of O3
on above-ground biomass
No effect on biomass; 6%,
12% more severely O3
damaged leaves in high EDU
for 2 years
Mortensen and
Bastrup-Birk(1996)
Ainsworth et al.
(1996)
H
6
O
O
H
O
^w'
s
Pine, loblolly
Radish, cv. Cherry Belle
1 year old half-sib
seedlings in field in TX
for 3 years
Plants in pots in potting
mix exposed for 5 weeks
in southern Sweden.
150, 300, 450 ppm every
14 days
Soil drench containing 20
mg EDU applied 2 times,
14 days apart
1995, 1996, 1997,
no. h> 40 ppb =1723,
2297, 2052;
no. h > 60 ppb = 378,
584, 528;
peak =113, 102, 118
24-h mean = 31 ppb,
7-h mean = 36 ppb,
AOT40= 1.3 ppm-h.
For EDU 450 trt, above-
ground biomass increased
approx 46% (n.s. in other
treatments
24% increase in hypocotyl
mass, 18% increase in shoot
Manning et al.
(2003)
Pleijeletal. (1999b)
-------
1 mean indices should not be interpreted as a preference for them, but rather as a limitation in the
2 data reported in the literature. Additional information about O3 exposure for individual
3 experiments, including the number and type of O3 treatments (addition of a constant
4 concentration of O3 or an amount proportional to ambient levels), and duration, are reported in
5 Tables AX9-16 through AX9-19.
6 Since the 1996 O3 AQCD, the use of the AOT40 index has become quite common in
7 Europe for identifying and mapping areas of exceedance, but it has not been used much in the
8 United States. Thus, studies reporting O3 exposure only as AOT40 values are presented in tables
9 summarizing effects on annual, herbaceous perennial, and woody vegetation. However, such
10 studies are not as commonly cited in the text of this section, because AOT40 summary data
11 on O3 exposures in the United States are rarely available. This lack makes it difficult to compare
12 experimentally derived exposure-response data expressed as AOT40 to ambient U.S. O3
13 exposures. The development of critical levels in Europe has been based primarily on the AOT40
14 index, so this index is discussed in that context.
15 In addition to peak weighting, there is also evidence that the timing of exposure during
16 plant growth is important. For example, the greatest effects on grain yield are due to exposure
17 during grain filling, rather than earlier or later in the growing season (Lee et al., 1988; Pleijel
18 et al., 1998; Soja et al., 2000; U.S. Environmental Protection Agency, 1996; Younglove et al.,
19 1994). A recent study grew bush bean in OTCs with CF or above-ambient O3 using exposure
20 dynamics typical of the Midwestern United States for three time periods: (1) the entire season,
21 (2) the period prior to anthesis, (3) during pod filling and maturation (Tingey et al., 2002).
22 Ozone exposure prior to anthesis reduced growth by less than 1% per ppm-h (SUM06) while
23 exposure during pod filling and maturation reduced growth by 4 to 7%. A meta-analysis of
24 53 studies of O3 effects on soybean found that O3 had greater effects with increases in
25 developmental stage, with the greatest effect during seed filling (Morgan et al., 2003). The
26 importance of respite times was discussed in the previous criteria documents (U.S.
27 Environmental Protection Agency, 1978, 1986, 1996) but remains difficult to quantify (Section
28 AX9.4). Even when some of these aspects of O3 exposure can be elucidated, it is difficult to
29 apply this knowledge to developing exposure-response relationships based on data in the
30 scientific literature, because O3 exposure is often reported only in the form of a summary index
31 such as a 12- or 24-h mean, SUM06, or AOT40.
August 2005 AX9-259 DRAFT-DO NOT QUOTE OR CITE
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1 Table AX9-13 presents summaries of ambient O3 exposure patterns in the United States for
2 1982 to 1991 for several indices including the 7-h mean and SUM06. More recent summaries
3 for the entire United States for these indices are not available, but Table AX9-21 summarizes
4 more recent data for the central and eastern United States. As shown in Table AX9-21, from
5 1989 to 1995, mean 12-h 3-month SUM06 values (in ppm-h) at 41 rural sites in the Clean Air
6 Status and Trends Network were 31.5 for the Midwest, 18.9 for the Upper Midwest, 3 3.2 for the
7 Northeast, 13.2 for the Upper Northeast (New Hampshire, Maine), 34.5 for the South-Central,
8 and 19.2 for the Southern Peripheral subregions (Baumgardner and Edgerton, 1998). These
9 results are important because these sites were selected to represent rural areas, while many other
10 monitoring sites represent urban or suburban areas. For these same subregions, W126 values
11 ranged from 12.8 to 25.6 ppm-h. From 1989 to 1995, O3 concentrations decreased about 5% for
12 daily and 7% for weekly values for most of these sites, after adjusting for meteorological
13 conditions (Holland et al., 1999). These trends were statistically significant at about 50% of the
14 sites (p < 0.05). However, because the trend analysis was intended to examine the efficacy of O3
15 emissions controls, the trends were adjusted for meteorological conditions. Thus, they do not
16 reflect the actual trends in O3 exposure over time.
17
18 AX9.5.4 Effects of Ozone on Annual and Biennial Species
19 Much of the research on short-lived species during the last decade has been conducted in
20 Europe. Several European studies have focused on wheat with an emphasis on developing
21 critical levels as discussed in Section AX9.4.3 and reviewed briefly below in Section AX9.5.4.5.
22 An extensive search of the literature was performed using several electronic databases to
23 identify scientific articles containing quantitative information on both the amount of O3 exposure
24 and its effects on vegetation. Greater emphasis is placed on studies with longer duration with O3
25 exposure concentrations and environmental conditions that were as similar as possible to
26 ambient conditions. Many of the studies reviewed herein were conducted in OTCs. In the
27 United States, nearly all of such studies have used the type of OTC developed by Heagle et al.
28 (1973). For the few studies in the U.S. that used other types of OTCs, they are described briefly
29 in the text. In Europe, a wide variety of styles of OTCs have been used. See Section AX9.1 for
30 further information about the use of OTCs. The emphasis in this subsection is on quantifying
31 exposure-response relationships for annual plants, with a focus on the response of above-ground
August 2005 AX9-260 DRAFT-DO NOT QUOTE OR CITE
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1 biomass and yield of species grown as crops or occurring as native or naturalized species in the
2 United States. Emphasis is placed on studies not included in the 1996 AQCD (U.S.
3 Environmental Protection Agency, 1996), including a few studies published prior to 1996.
4 However, an attempt is made to compare the results of these more recent studies of individual
5 species to those reviewed in the 1996 AQCD.
6
7 AX9.5.4.1 Effects on Growth, Biomass, and Yield of Individual Species
8 Most research on the effects of O3 on herbaceous species has evaluated growth, biomass, or
9 yield of commercial portions of crop or forage species. It is well established that reproductive
10 organs such as seeds may be particularly sensitive to injury or biomass reductions due to O3, as
11 reviewed by (Black et al., 2000). As discussed in Section AX9.3, numerous analyses of
12 experiments conducted in OTCs and with naturally occurring gradients demonstrate that the
13 effects of O3 exposure vary depending on the growth stage of the plant. Plants grown for seed or
14 grain are often most sensitive to exposure during the seed or grain-filling period (Lee et al.,
15 1988; Pleijel et al., 1998; Soja et al., 2000; Younglove et al., 1994), whereas plants grown for
16 biomass production, such as alfalfa, may be sensitive throughout the growth period (Younglove
17 et al., 1994). However, because different species are sensitive during different periods of their
18 growth and, because planting or germination dates vary throughout large regions even for a
19 single species, no single phenological weighting scheme can appropriately and practically
20 represent all vegetation in all locations in the United States. For natural populations, reductions
21 in seed yield might be particularly important if subsequent seedling establishment is
22 compromised by O3.
23 Green beans (cv. Pros) were grown in pots in OTCs in the Netherlands for 62 days and
24 exposed to 6 treatments consisting of constant O3 additions to CF chambers (see Section AX9.1)
25 for 9 h/day (Tonneijck and Van Dijk, 1998). Bean yield response to O3 was nonlinear, with an
26 apparent threshold near the CF30 (charcoal filtered with a constant addition of 30 ppb O3)
27 treatment with a 9-h mean O3 concentration of 28 ppb and an AOT40 value of 0.1 ppm-h). Yield
28 was reduced by 29% at a 9-h mean value of 44 ppb corresponding to an AOT40 value of 3.6
29 ppm-h (Table AX9-16). Beans were grown in pots in OTCs for 3 months with the following O3
30 treatments: CF, nonfiltered (NF), CF with O3 added up to ambient, and CF with 2x-ambient O3
31 (Brunschon-Harti et al., 1995). Ozone reduced pod mass by 56% with a mean concentration of
August 2005 AX9-261 DRAFT-DO NOT QUOTE OR CITE
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1 32 ppb in the 2x ambient treatment as compared with 1 ppb in the CF treatment (daily averaging
2 time not reported). A second treatment factor in this experiment was addition of EDU, as
3 discussed below under the heading "Studies Using Ethylene Diurea as a Protectant." Bush beans
4 (cv Bush Blue Lake 290) were grown in pots in OTCs in Corvallis Oregon for 63 or 65 days in
5 two experiments, one from May to July and one from August to October (Tingey et al., 2002).
6 Plants were exposed to either CF air or CF air with above-ambient O3 with temporal frequency
7 and exposure dynamics typical of the Midwestern United States, with SUM06 values of 0.0 for
8 the CF treatment and 75.7 or 68.4 ppm-h for the two experiments. Ozone exposure reduced pod
9 dry weight by 51 and 57% in the two experiments. The sensitive cultivar S156 and the more
10 resistant cultivar Tenderette were grown in pots in OTCs in Raleigh, NC at either CF (12-h mean
11 of 23 ppb) or 1.4x (12-h mean of 72 ppb) (Heagle et al., 2002). At final harvest, the dry weight
12 of the sensitive cultivar was reduced 90% by the O3 treatment, but the more resistant cultivar
13 was not reduced. In two additional OTC experiments during 2000 and 2001 in Raleigh, NC, the
14 sensitive cultivar S156, the moderately sensitive cultivar Oregon-91, and the more resistant
15 cultivar R123 were grown in CF, NF, and ambient air treatments (Fiscus et al., 2005). For the NF
16 treatment of the two experiments, the yield of S156 was reduced 21% and 45%, that of Oregon-
17 91 by 27% only in 2001, while R123 was not significantly reduced. These yield reductions are
18 greater than those previously reported in four similar studies summarized in the 1996 AQCD
19 (Table 5-25 of U.S. Environmental Protection Agency, 1996). Greater sensitivity in the more
20 recent experiments may be due to cultivar differences or other differences in experimental
21 protocols.
22 In a study with OTCs on silty loam soil in Beltsville, MD, corn yield was reduced by 13%
23 with exposure to a 7-h mean concentration of 70 ppb O3 compared to a CF treatment with a 7-h
24 mean concentration of 20 ppb (Mulchi et al., 1995; Rudorff et al., 1996a;c). In this study,
25 different amounts of O3 were added above ambient levels for 5 days follows: 20, 30, 40, 50, 60
26 ppb, except that O3 was not added to exceed a total concentration of 120 ppb (Rudorff et al.,
27 1996a,c).
28 In two studies conducted in Raleigh, NC, cotton (cv. Deltapine 51) was grown in pots and
29 exposed to CF and 1.5* (nonfiltered, see Section AX9.1) O3 in one year, and CF, NF, and
30 1.5 x-ambient O3 in the second year, with ambient and elevated CO2 concentrations (Heagle
31 etal., 1999) (Table AX9-16). In the first year, yield decreased by 22% with 1.5 x -ambient O3
August 2005 AX9-262 DRAFT-DO NOT QUOTE OR CITE
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1 (12-h mean value of 71 ppb). In the second year, yield decreased by 21% and 49% with
2 exposure to ambient or 1.5x ambient O3 (12-h mean values of 51 and 78 ppb, Table AX9-16).
3 Increased CO2 levels prevented or reduced this yield suppression (Heagle et al., 1999). These
4 yield reductions are similar to those reported previously in four similar studies summarized in
5 the 1996 AQCD (Table 5-25 of U.S. Environmental Protection Agency, 1996).
6 In a study of oats in OTCs in southern Sweden, exposure to ambient (NF) O3 did not affect
7 grain yield (Pleijel et al., 1994a). Ambient O3 concentration expressed as a 7-h mean was
8 27 ppb, with only 1 h greater than 80 ppb and none above 90 ppb.
9 The interactive effects of elevated O3 and CO2 additions on potato yield (cv Bintje) were
10 studied in OTCs at 6 sites in northern Europe (Craigon et al., 2002). Ozone was added to a
11 target daily average value of 60 ppb, and AOT40 values across all years and experiments ranged
12 from ~6 to 27 ppm-h. The O3 treatment reduced total tuber yield an average of 4.8% with
13 elevated O3 treatment across all experiments (Craigon et al., 2002). This total effect was
14 statistically significant even though the effects of individual experiments generally were not
15 (Craigon et al., 2002), due to the increased statistical power of the pooled analysis. Several
16 publications report other aspects of the "CHIP" experiments or present results of individual
17 experiments (De Temmerman et al., 2002a,b; Donnelly et al., 2001a,b; Fangmeier et al., 2002;
18 Finnan et al., 2002; Hacour et al., 2002; Lawson et al., 2002; Pleijel et al., 2002; Vandermeiren
19 et al., 2002; Vorne et al., 2002). Resistant and susceptible cultivars of potato (Superior and Dark
20 Red Norland, respectively) were grown for one season in Raleigh, NC and exposed to CF, NF,
21 and 1.5x ambient O3 treatments with 12-h mean values of 15, 45, 80 (Heagle et al., 2003). Tuber
22 yield was decreased by 15 and 31% for Dark Red Norland in NF and 1.5x treatments, but by
23 only 11% in only the 1.5x treatment for Superior.
24 The effect of an intermittent constant addition of O3 using a free air exposure system in
25 Northumberland, UK was investigated with the oilseed rape cultivar Eurol (Ollerenshaw et al.,
26 1999). Ozone was added for 6 h/day for 17 days. The ambient treatment had a mean value of 30
27 ppb and the O3 addition treatment had a mean of 77 ppb. After overwintering, O3 was added for
28 32 days for 7 h/day between May and June (mean values of 31 and 80 ppb). Yield was reduced
29 by 14% despite the lack of any foliar symptoms.
30 Field fumigation chambers ventilated with fans on both ends were used to assess effects of
31 five O3 treatments on rice over 3 years in Japan (Kobayashi et al., 1994, 1995). All O3
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1 treatments used CF air, and O3 was added to the 0.5, 1.0, 1.5, 2.0, or 2.75x ambient
2 concentration for 7 h/day. Based on a linear regression for the 3 years, yield decreased by 3 to
3 10% at a 7-h mean concentration of 40 ppb (Table AX9-16). This decrease is greater than that
4 found for rice in earlier studies in California (Kats et al., 1985), although whether this difference
5 is due to differences in cultivars, experimental treatment, or environmental factors cannot be
6 determined.
7 During 3 years in Beltsville, MD, the soybean cultivars Essex and Forrest were exposed to
8 CF air and NF air in OTCs (Chernikova et al., 2000; Robinson and Britz, 2000) with O3 added as
9 described for experiments with corn and wheat at Beltsville (Mulchi et al., 1995; Rudorff et al.,
10 1996c). During 1994 and 1995, as previously found for these cultivars, Essex was less sensitive
11 than Forrest, with yield decreases of 10% (n.s.: p > 0.1) compared to 32% for Forrest (p < 0.01)
12 (Chernikova et al., 2000). There was no evidence of water stress in this experiment. In 1997,
13 the two O3 treatments were CF (7-h mean = 24 ppb) and NF with a constant addition of O3 (7-h
14 mean = 58 ppb) (Robinson and Britz, 2000). The yield of Essex was not significantly affected,
15 while the yield of Forrest was decreased by 21% (Table AX9-16).
16 In a study in Raleigh, NC, the soybean cultivar Essex was grown in pots and exposed to CF
17 and 1.5x ambient O3 concentrations during three growing seasons (Fiscus et al., 1997). Over the
18 3 years, exposure to an average 12-h mean O3 concentration of 82 ppb reduced soybean yield by
19 41% (Table AX9-16). In similar studies also in Raleigh, NC, Essex was exposed to CF, NF, and
20 1.5x ambient O3 for two seasons (Heagle et al., 1998). Yield decreased by 16% and 15% in the
21 2 years by ambient O3 (12-h mean values of 50 and 42 ppb), and decreased by 37 and 40% with
22 exposure to 1.5x ambient O3 (12-h mean values of 79 and 69 ppb, Table AX9-16). In this same
23 experiment in the second year, similar yield reductions were observed for the cultivar Holladay,
24 while the growth of cultivar NK-6955 was increased substantially by ambient O3 exposure.
25 All three cultivars were grown in the same chambers in this experiment, and the authors
26 suggested that NK-6955 plants may have shaded the other cultivars to some extent.
27 In a 2-year study using OTCs in Raleigh, NC, the soybean cultivars Coker 6955, Essex,
28 and S53-34 were exposed to CF, NF, and 1.5x ambient O3 treatments (Miller et al., 1994).
29 Seasonal mean 12-h O3 concentrations ranged from 14 to 83 ppb. As compared to the CF
30 treatment, ambient O3 exposure (NF treatment) reduced seed yield by 11 to 18% except for
31 Coker 6955 in the first year (1989), which showed a yield increase of 14%. The 1.5x ambient O3
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1 treatment reduced yield by 32 to 56% in all cultivars in both years. In a similar subsequent
2 experiment with the cultivar Essex, exposure to a 12-h mean ambient O3 concentration of 50 ppb
3 reduced yield by 11%, while exposure to 79 ppb reduced yield by 22% (Table AX9-16) (Miller
4 et al., 1998). In similar experiments with Essex during 1999 and 2000, plants were exposed to
5 1.5* ambient O3 in OTCs either in pots or planted in the ground (Booker et al., 2005). Exposure
6 to a 12-h mean ambient O3 concentration of 75 ppb reduced yield by 27% in pots and 24% in the
7 ground in 1999, and exposure to a 12-h mean ambient O3 concentration of 67 ppb reduced yield
8 by 41% in pots and 39% in the ground in 2000 (differences between results in pots and in the
9 ground were not statistically significant).
10 These yield reductions for soybean are generally similar to those reported previously in
11 13 similar studies summarized in the 1996 AQCD (Table 5-23 of U.S. Environmental Protection
12 Agency, 1996). A meta-analysis of 53 studies of O3 effects on soybean found that at an average
13 O3 exposure of 45 ppb, seed yield was decreased by 10% compared to the CF treatment, while at
14 70 ppb, seed yield was decreased by 24% (Morgan et al., 2003). The 95% confidence limits of
15 these responses based on a bootstrap method did not include a value of zero yield loss. These
16 results suggest that seasonal O3 concentration patterns that occur in some years throughout many
17 parts of the U.S. can reduce soybean seed yield.
18 A reanalysis of 7 years of data from OTC experiments with wheat in Ostad, Sweden
19 showed that relative yield linearly decreased with increasing O3, with a maximum yield loss of
20 23% at an AOT40 value of 15 ppm-h (Danielsson et al., 2003). A very similar response was
21 found using the flux (stomatal conductance) model of Emberson et al. (2000b) and a similar
22 amount of the variance was explained by the flux model (for AOT40 model, r2 = 0.34 and for the
23 Emberson flux model, r2 = 0.39). A modified flux model developed and calibrated for this site
24 also had a similar linear response equation, but explained much more of the variance (r2 = 0.90).
25 During the 1990s, a major European research program investigated the combined effects of
26 CO2, O3, and other physiological stresses on wheat (Bender et al., 1999; Hertstein et al., 1996,
27 1999; Jager et al., 1999). The ESPACE-wheat program included 13 experiments in OTCs at
28 eight sites in northern Europe over 3 years. Low- and high-O3 exposures in these experiments
29 had the following values: 12-h mean (SD) low = 26.3 ppb (12.2), 12-h mean (SD) high = 51.37
30 (18.3) ppb, AOT40 mean (SD) low = 6.2 (8,5) ppm-h, AOT40 mean (SD) high = 28.3 (23.0)
31 ppm-h, as calculated from data presented in Table 3 of Hertstein et al. (1999). An analysis of all
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1 13 experiments showed that high O3 at ambient CO2 reduced yield by 13% on average (Bender
2 et al., 1999). However, this reduction was not statistically significant based on an ANOVA, and
3 the authors concluded that the wheat cultivar Minaret may be relatively tolerant to O3 (Bender
4 et al., 1999). Results of some individual studies within this program have been reported
5 previously (Donnelly et al., 1999; Fangmeier et al., 1996, 1999; Mulholland et al., 1997,
6 1998a,b;Pleijeletal., 2000b).
7 In a study with OTCs on silty loam soil in Beltsville, MD, wheat yield was reduced by
8 20% on average over 2 years with 7-h mean concentrations of 61 and 65 ppb O3 compared with
9 CF treatment with a 7-h mean concentration of 20 ppb (Mulchi et al., 1995; Rudorff et al.,
10 1996a,c). In the above study, different amounts of O3 were added above ambient levels for 5
11 days as follows: 20, 30, 40, 50, 60 ppb, except that O3 was not added to exceed a total
12 concentration of 120 ppb (Rudorff et al., 1996c). Wheat grown in pots in OTCs was exposed to
13 elevated O3 and water stress in Germany, and yield was decreased by 35% in the 2x -ambient
14 treatment with a 7-h mean O3 concentration of 71 ppb, statistically significant effects were not
15 seen in the 1 x-ambient treatment (Fangmeier et al., 1994). In two studies conducted in Raleigh,
16 NC, soft red winter wheat was grown in pots and exposed to CF, NF, and 1.5x-ambient O3, with
17 ambient and elevated CO2 concentrations (Table AX9-16) (Heagle et al., 2000). In the first
18 experiment, eight cultivars were exposed to 12-h mean O3 concentrations of 27, 47, and 90 ppb,
19 and in the second experiment two of these cultivars were exposed to 22, 38, and 74 ppb. There
20 was a trend toward decreased yield in both experiments, but these trends were not statistically
21 significant. The wheat cultivar Drabant was exposed to CF, NF, and a constant addition of 35
22 ppb during 1992 and 1993 in Finland (Ojanpera et al., 1998) using the Heagle-type OTCs
23 (Heagle et al., 1973). The following 12-h mean O3 exposures were observed in 1992: 14, 30, 61
24 ppb. In 1993, the values were 9, 21, and 45 ppb, (see Table AX9-16 for AOT40 values and other
25 information). Yield was reduced 13% in each year by the added O3 treatment.
26 The effect of an intermittent constant addition of O3 using a free air exposure system was
27 investigated with the winter wheat cultivar Riband in Northumberland, UK (Ollerenshaw and
28 Lyons, 1999). Ozone exposures expressed as AOT40 values for September and October 1992
29 were 0.14 and 3.5 ppm-h; while for April to August 1993, values were 3.5 and 6.2 ppm-h. Yield
30 was reduced by 13%.
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1 These results provide an additional line of evidence supporting the OTC-studies that
2 demonstrated yield reductions in wheat due to O3 exposures that occur in the United States.
3 These yield reductions for wheat are generally similar to those reported previously in 22
4 comparable studies summarized in the 1996 AQCD (Table 5-25 of U.S. Environmental
5 Protection Agency, 1996).
6
7 AX9.5.4.2 Effects on Plant Quality
8 In addition to reductions in biomass or crop yield, O3 may also reduce the quality or
9 nutritive value of annual species. Many studies have shown effects of O3 on various measures of
10 plant organs that affect quality, with most studies focusing on characteristics important for food
11 or fodder (U.S. Environmental Protection Agency, 1996).
12 The effect of a continuous intermittent addition of O3 using a free air exposure system in
13 Northumberland, UK was investigated with the oilseed rape cultivar Eurol as discussed above
14 (Ollerenshaw et al., 1999). Ozone exposures expressed as AOT40 values for August to October
15 1991 were 0.2 and 3.8 ppm-h; for June 1992 they were 0.7 and 8.1 ppm-h. Yield quality
16 measured as crude protein and oil content was decreased significantly. Because the price of the
17 product is reduced in direct proportion to the oil content, such a decrease represents a substantial
18 loss to growers (Ollerenshaw et al., 1999).
19 Two wheat cultivars, Massey and Saluda, were each grown for one year each in Beltsville,
20 MD (Table AX9-16) and exposed to either CF or an addition of 40 ppb for 7 h/day for
21 5 days/week (Mulchi et al., 1995; Rudorff et al., 1996a,c). Milling and baking quality scores and
22 flour protein were not significantly affected by elevated O3 exposure, but the softness equivalent
23 was increased slightly (2.4%) in both experiments (Rudorff et al., 1996b). The authors
24 concluded that these changes, along with other slight changes due to an increased CO2 treatment,
25 suggested that O3 and CO2 had only minor effects on wheat grain quality. In wheat grown in
26 Sweden, the harvest index was significantly decreased and the protein content increased due to
27 exposure to a 12-h mean of 48 ppb (Gelang et al., 2000). In an analysis of 16 experiments
28 conducted with spring wheat and either O3 or CO2 exposures in four Nordic countries, a negative
29 linear relationship was found between grain yield and grain protein content (y = -0.38x +138.6,
30 expressed as percentages of the NF treatment (Pleijel et al., 1999a).
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1 For three soybean cultivars grown in Raleigh, NC, O3 significantly decreased oleic acid
2 content, although the authors stated that the reduction was not large enough to be economically
3 important (Heagle et al., 1998).
4 In a UK study, potato exposed during 1998 to an AOT40 value of 12.5 ppm-h in OTCs (in
5 Nottingham) resulted in the paste from tubers being more viscous (Donnelly et al., 200Ib). In
6 this study, an AOT40 exposure of 27.11 ppm-h in 1999 caused starch granules to be less resistant
7 to swelling, and total glycoalkaloid content was increased due to an increase in a-solanine
8 (Donnelly et al., 2001b). Such increases in glycoalkaloid content have been observed previously
9 in potato (Pell and Pearson, 1984) and may be important, because glycoalkaloids cause bitter
10 flavors and, at higher concentrations, toxicity. The authors indicated that levels found in this
11 study approached those that may cause bitterness, but not those of concern for toxicity (Donnelly
12 etal., 2001b).
13 In the CHIP program the effects of O3 were studied using OTCs at six sites in northern
14 Europe, and yield decreases were observed as described above. The reducing sugar and starch
15 content of tubers decreased linearly due to O3 exposure, while the ascorbic acid concentration
16 increased linearly (Vorne et al., 2002). Compared to the CF treatment, exposure to an AOT40
17 value of 14 ppm-h decreased starch concentrations by 2%, decreased reducing sugar
18 concentration by 30%, and increased ascorbic acid concentration by 20%. While the changes in
19 reducing sugars and ascorbic acid increase tuber quality, the reduction in starch concentration
20 decreases tuber quality.
21 In two 1-year studies using OTCs in commercial fields in Spain, the soluble solids content
22 of watermelon was decreased 4 to 8% due to seasonal O3 exposures as follows: AOT40 = 5.96
23 ppm-h and SUM06 = 0.295 ppm-h in one year; and AOT40 = 18.9, SUM06 = 4.95 in the second
24 year (Gimeno et al., 1999).
25
26 AX9.5.4.3 Effects on Foliar Symptoms
27 For most annual crop species, the most important effects of O3 are on yield of the
28 commercially important part of the crop, expressed as the mass of the harvested portion.
29 However, for some crops, foliar symptoms are important if they reduce the marketability of the
30 crop. This is why efforts have been made to identify O3 exposures associated with foliar
31 symptoms. In Europe, Level I critical levels have been determined for such effects based on
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1 observations from experiments conducted in 15 countries under the auspices of the United
2 Nations Economic Commission for Europe International Cooperative Programme on effects of
3 air pollution and other stresses on crops and non-woody plants (UN ECE ICP-Vegetation;
4 formerly ICP-Crops), as well as on observations of symptoms in commercial fields from 1993 to
5 1996 (Benton et al., 1995; Benton et al., 2000). Because the occurrence of symptoms increased
6 with greater humidity, these levels took into account the VPD. Two short-term critical levels
7 were derived from 1995 data: an AOT40 value of 0.2 ppm-h over 5 days when mean VPD is
8 below 1.5 KPa (0930 - 1630 h), and a value of 0.5 ppm-h when the mean VPD is above 1.5 Kpa
9 (Benton et al., 1996). The 1996 data supported the critical levels in 83% of observations,
10 although symptoms occurred on three occasions when the AOT40 was less than 0.05 ppm-h and
11 the VPD was very low — less than 0.6 Kpa. The authors concluded that these critical levels are
12 good indicators of the likelihood of foliar symptoms, but that further refinement may be
13 required, such as including factors that modify O3 uptake by stomata.
14 In a more recent study in Germany, 25 native herbaceous species were exposed to
15 several square-wave O3 exposures in CF OTCs (Bergmann et al., 1999). Six of the 25 species
16 showed O3-specific symptoms, and five species responded to single-day peaks. The most
17 sensitive species exhibiting O3-specific symptoms were Cirsium arvense and Sonchus asper,
18 which both responded to AOT40 values < 1.5 ppm-h (Bergmann et al., 1999).
19
20 AX9.5.4.4 Other Effects
21 Several studies during recent decades have demonstrated O3 effects on different stages of
22 reproduction. Effects of O3 have been observed on pollen germination, pollen tube growth,
23 fertilization, and abortion of reproductive structures, as reviewed by Black et al. (2000). This
24 issue is not addressed here, because reproductive effects will culminate for seed-bearing plants
25 in seed production, and the substantial body of evidence relating O3 exposure and reduced seed
26 production was discussed above. However, one example of a native species will be presented,
27 because of its implications for extrapolating exposure-response data to noncommercial species.
28 Spreading dogbane has been identified as a useful species for O3 biomonitoring, because
29 of O3-induced diagnostic symptoms (Kohut et al., 2000). A study in Massachusetts found that
30 exposure to O3 in NF OTCs or ambient plots for 103 days produced significantly fewer flowers
31 and that fewer of these flowers survived to produce mature fruits (Bergweiler and Manning,
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1 1999). Because foliar symptoms were not common, the authors concluded they are not required
2 for effects on reproduction to occur. Genotoxic effects and effects on population genetics were
3 discussed in Section AX9.3.
4
5 AX9.5.4.5 Scaling Experimental Data to Field Conditions
6 Substantial effort has been invested in the design of OTCs for assessing the effects of air
7 pollutants on vegetation under near-ambient conditions. The design, construction, and
8 performance of many types of chambers has been reviewed extensively (Hogsett et al., 1987a,b).
9 Despite such design efforts, the influence of experimental chambers on exposure-response
10 functions has been debated for many years (e.g., Manning and Krupa, 1992), because several
11 factors differ between OTC studies and actual fields. This issue was addressed in Sections
12 AX9.1.2.2 and AX9.1.2.4, and only a few comments about the implications of chamber artifacts
13 for interpreting exposure-response relationships are presented here.
14 While it is clear that chambers can alter some aspects of plant growth (for example,
15 (Bytnerowicz et al., 2004; Elagoz and Manning, 2005b), the more important issue is whether
16 they alter the response of plants to O3. A review of such chamber studies done in California
17 found that plants responded similarly to O3 whether OTCs, closed-top chambers, or air exclusion
18 systems were used; differences were found for fewer than 10% of growth parameters (Olszyk
19 et al., 1986). In another review of literature about Heagle-type OTCs (Heagle et al., 1988), the
20 authors concluded that "Although chamber effects on yield are common, there are no results
21 showing that this will result in a changed yield response to O3." A more recent study of chamber
22 effects examined the responses of tolerant and sensitive white clover clones to ambient O3 in
23 greenhouse, OTCs, and ambient plots (Heagle et al., 1996). For individual harvests, O3 reduced
24 the forage weight of the sensitive clone 7 to 23% more in the greenhouse than in OTCs.
25 However, the response in OTCs was the same as in ambient plots. A similar study with these
26 white clover clones near Naples, Italy also found no significant difference between O3 effects
27 measured in OTCs versus those measured by comparing the ratio of sensitive and resistant
28 clones in ambient air (Fagnano et al., 2004). Several studies have shown very similar yield
29 responses to O3 for plants grown in pots or in the ground, suggesting that even such a significant
30 change in environment does not alter the proportional response to O3, at least as long as the
31 plants are well- watered (Heagle, 1979; Heagle et al., 1983). As discussed in Section AX9.1,
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1 results from recent FACE studies are similar to those obtained from OTC studies, providing
2 another line of evidence that chamber effects in OTCs do not substantially alter O3 exposure-
3 response relationships.
4 Most experiments investigating O3 effects on annual vegetation provide adequate water to
5 avoid substantive drought stress. Because drought stress has generally been shown to reduce the
6 effect of O3 on annual vegetation, such experiments may tend to overestimate O3 effects on crops
7 and especially on unmanaged or seminatural vegetation.
8 As mentioned above, the use of O3 flux, rather than exposure, is theoretically more
9 realistic, and such an approach would also address the vertical gradient issue (Section AX9.1).
10 A number of investigators have suggested that modeling O3 flux can improve estimates of O3
11 effects on vegetation. Models of O3 flux can reduce the variation in the response to O3 that is
12 sometimes observed between years in an experiment (Emberson et al., 2000a, b; Fuhrer et al.,
13 1992; Griinhage et al., 1993; Griinhage and Haenel, 1997; Pleijel et al., 2000a). In a study of O3
14 deposition to an oat crop in OTCs, O3 flux in the chamber was estimated to be up to twice that in
15 an adjacent field based on a K-theory approach and measurements of stomatal conductance and
16 environmental conditions (Pleijel et al., 1994b). These measurements were made for 2 hours on
17 5 days when the canopy was physiologically active and wind speeds were moderate. However,
18 the O3 flux in a plant-less chamber was nearly as high as that in the open field. The authors
19 conclude that O3 uptake in the chamber was between 100 and 200% of that in the field. These
20 models of flux have a sound biological and meteorological basis and are useful for interpreting
21 experimental data. Flux models have been successfully applied at intensive study sites with
22 detailed site-specific data on stomatal conductance and micrometeorological conditions (e.g.,
23 Fredericksen et al., 1996; Griinhage et al., 1993, 1994)). Yet even at a single well-studied site,
24 different methods can provide different estimates of O3 flux. For example, at a site in a vineyard
25 in California, an evapotranspiration-based method overestimated the O3 flux as compared to an
26 eddy covariance approach by 20 to 26% (Massman and Grantz, 1995). At a site in a nearby
27 cotton field the evapotranspiration-based approach overestimated the eddy-covariance method
28 by 8 to 38%. Flux-response relationships have received substantial attention in Europe during
29 the past decade, as part of an attempt to move beyond the exposure-based Level 1 critical levels
30 to flux-based Level II critical levels (Section AX9.4). However, the database for flux-response
31 relationships is very limited (Griinhage et al., 2004), and these approaches do not always explain
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1 greater amounts of response variation than do exposure-based approaches (Karlsson et al., 2004).
2 The critical level approach is discussed briefly below, because it has been used to extrapolate
3 from field studies to landscapes, countries, and regions.
4 There has been criticism that the Level I critical level for crops overestimates O3 effects in
5 the Mediterranean countries, because it was developed based on studies in Northern Europe (De
6 Santis, 1999; De Santis, 2000). However, there is evidence of substantial crop loss due to O3 in
7 some southern European counties, such as the Po valley in Northern Italy (Fumagalli et al.,
8 2001). In these studies, Heagle-type OTCs were used. Losses in NF chambers as compared to
9 CF chambers over several years at two sites ranged from 11.2 to 22.8% for barley and wheat,
10 from 0.3 to 31.5% for other crop species, and from 4.1 to 19.8% for forage species (Fumagalli
11 et al., 2001). Surprisingly, the least effect was observed for soybean, despite AOT40 values of
12 9.32 ppm-h, 3 x the Level I critical level. Similarly, a review of studies in Northern Italy found
13 that ambient O3 episodes have been reported to cause foliar symptoms on 24 agricultural and
14 horticultural crops in commercial fields (Fumagalli et al., 2001). Ambient O3 has also been
15 reported to cause yield losses in several crop species, although no data on O3 exposure were
16 presented by the reviewers (Fumagalli et al., 2001).
17 The Level I approach has also been criticized for focusing only on a single annual species
18 (wheat) and a single woody perennial species (beech). However, this species focus is
19 appropriate, because the goal was to determine an exposure-response relationship for a sensitive
20 species based on available data. In support of standards in Germany, an effort was made to
21 combine data from different species, and consisted of a meta-analysis of studies conducted in
22 both closed and OTCs (Griinhage et al., 2001). In this study, experiments published between
23 1989 and 1999 were included and analyzed if they met the following conditions: (1) a
24 significant O3 effect was determined; (2) exposure conditions were well defined; (3) foliar
25 symptoms, growth, or yield was measured; and (4) plant species were relevant to Europe
26 (Griinhage et al., 2001). Despite the focus on European species, many of the species studies also
27 occur in the United States. Separate regressions for herbaceous plants and for tree species were
28 created as a function of duration of exposure at a given level of O3 exposure at the top of the
29 plant canopy. These regression equations, with confidence limits and with correction for the
30 vertical gradient in O3 from the top of the quasi-laminar boundary layer, can be used to define
31 whether effects are unlikely (below the lower confidence limit), probable (between the
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1 confidence limits), or highly likely (above the upper confidence limit) to occur near a given O3-
2 monitoring station.
3 A further concern about the Level 1 approach is that foliar symptoms, rather than biomass,
4 may be an important endpoint, because foliar symptoms may be more sensitive (VanderHeyden
5 et al., 2001). In an OTC study in southern Switzerland, it was shown that a number of tree
6 species show foliar symptoms at AOT40 values lower than the Level 1 value of 10 ppm-h
7 (VanderHeyden et al., 2001).
8 Exposure-response relationships developed primarily from OTC experiments, with
9 confirming evidence from other approaches such as resistant and sensitive clover clones exposed
10 in ambient air and FACE experiments, are useful for estimating the effects of ambient O3 on
11 vegetation in the U.S. However, despite the substantial number of experimental studies listed in
12 Table AX9-16 and in previous Air Quality Criteria Documents for O3 (for example, U.S.
13 Environmental Protection Agency, 1996), most studies have been conducted in only a few
14 locations with only a few species of economically important crops. While these studies provide
15 strong evidence that ambient O3 in the U.S. is likely reducing crop yields and plant growth
16 significantly in many regions in many years, it remains difficult to extrapolate to all landscapes
17 and all plant species and to determine whether exposure-response relationships based on existing
18 studies will protect all species in all locations from significant deleterious effects of O3.
19
20 AX9.5.4.6 Summary of Effects on Short-Lived Species
21 For annual vegetation, the data summarized in Table AX9-16 show a range of growth and
22 yield responses both within species and among species. Nearly all of these data were derived
23 from studies in OTCs, with only two studies using open-air systems in the UK (Ollerenshaw
24 et al., 1999; Ollerenshaw and Lyons, 1999). It is difficult to compare studies that report O3
25 exposure in different indices such as AOT40, SUM06, or 7-h or 12-h mean values. However,
26 when such comparisons can be made, the results of this more recent body of research confirm
27 the earlier results summarized in the 1996 O3 AQCD (U.S. Environmental Protection Agency,
28 1996). A summary of earlier literature concluded that a 7-h, 3-month mean of 49 ppb
29 corresponding to a SUM06 exposure of 24.4 ppm-h would cause 10% loss in 50% of 49
30 experimental cases (Tingey et al., 1991). A similar study using a 24-h, rather than 7-h,
31 averaging period found that a SUM06 exposure of 26.4 ppb would cause 10% loss in 50% of
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1 54 experimental cases (Lee et al., 1994a;b). Recent data summarized in Table AX9-16 support
2 this conclusion. These values represent ambient exposure patterns that occur in some years over
3 large portions of the United States. Some annual species such as soybean are more sensitive,
4 and greater losses may be expected (Table AX9-16). Thus, the recent scientific literature
5 supports the conclusions of the 1996 AQCD that ambient O3 concentrations are reducing the
6 yield of major crops in the United States.
7 Much research in Europe has used the AOT40 exposure statistic, and substantial effort has
8 gone into developing Level-1 values for vegetation. Based on regression analysis of 15 OTC
9 studies of spring wheat, including one study from the United States and 14 from locations
10 ranging from southern Sweden to Switzerland, an AOT40 value of 5.7 ppm-h was found to
11 correspond to a 10% yield loss, and a value of 2.8 ppm-h corresponded to a 5% yield loss
12 (Fuhrer et al., 1997). Because a 4 to 5% decrease could be detected with a 99% confidence
13 level, a critical level of an AOT40 value of 3 ppm-h was selected in 1996 (Karenlampi and
14 Skarby, 1996).
15 In addition to reductions in crop yield, O3 may also reduce the quality or nutritive value of
16 annual species. Many studies have shown effects of O3 on various measures of plant organs that
17 affect quality, with most studies focusing on characteristics important for food or fodder. These
18 studies indicate that there may be economically important effects of ambient O3 on the quality of
19 crop and forage species. Previous criteria documents have concluded that foliar symptoms on
20 marketable portions of crops and ornamental plants can occur with seasonal 7-h mean O3
21 exposures of 40 to 100 ppb (U.S. Environmental Protection Agency, 1978, 1986, 1996). The
22 recent scientific literature does not refute this conclusion.
23 The use of OTCs may reverse the usual vertical gradient in O3 that occurs within a few
24 meters above the ground surface (Section AX9.1). Such a reversal suggests that OTC studies
25 may overestimate, to some degree, the effects of an O3 concentration measured several meters
26 above the ground. However such considerations do not invalidate the conclusion of the
27 1996 AQCD (U.S. Environmental Protection Agency, 1996) that ambient (Tables AX9-13,
28 Table AX9-21) O3 concentrations are sufficient to reduce the yield of major crops in the
29 United States.
30
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Table AX9-21. Ozone Exposures at 35 Rural Sites in the Clean Air Status and Trends
Network in the Central and Eastern United States From 1989 to 1995
Subregion
Midwest
Upper
Midwest
Northeast
Upper
Northeast
South
Central
Southern
Periphery
SUM06
12-h, 3-Month
Mean
31.5
18.9
33.2
13.2
34.5
19.2
SUM06
12-h,
3-Month SD
10.2
8.5
11.9
8.6
16.6
7.6
W126
3-Month
Mean
25.1
16.0
26.6
12.8
25.6
15.2
W126
3-Month
SD
7.7
5.9
9.5
6.5
11.5
5.4
Max.
8h > 80 ppb(n)
Mean
13.8
5.6
15.8
3.3
7.1
1.9
Max.
8h > 80 ppb(n)
SD
10.6
5.6
12.2
5.5
10.0
1.6
Units for SUM06 and W126 are ppm-h.
Source: Baumgardner and Edgerton (1998).
1 AX9.5.5 Effects of Ozone on Long-Lived (Perennial) Species
2 Although there has been considerable research in Europe on annual species during the past
3 10 years, much research in the United States has focused on perennial species. In Europe, and in
4 a few studies in the United States, effects of O3 on mixtures of annual and perennial herbaceous
5 species have been investigated using growth chambers, greenhouses, and OTCs. Section
6 AX9.5.5.1 reviews such studies, with an emphasis on studies using OTCs.
7
8 AX9.5.5.1 Herbaceous Perennial Species
9 Two alfalfa cultivars were grown in pots and exposed to CF, NF, 1.5 x -ambient and
10 2x-ambient O3 concentrations in two 1-year studies in Quebec, Canada (Renaud et al., 1997).
11 One cultivar, Apica, is commonly grown in the region, and another, Team, is normally grown
12 farther south and is more tolerant to O3. For Apica in both years and for Team in 1991, O3
13 exposure caused a linear reduction in above-ground biomass. In the NF treatment, growth of
14 Apica was decreased by 31 and 21% in the 2 years, while the growth of Team was reduced by
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1 14% in 1991, but not reduced in 1992. The authors suggested that the differing effects on Team
2 could be due to different progenies and propagation methods in the 2 years or to more rapid
3 growth in 1991 along with higher O3 peak values in 1991. In 1991, O3 maxima exceeded 60 ppb
4 in 15 days, whereas in 1992 there were only three such days. At the end of the growing season,
5 total starch reserves in roots were decreased by O3, due primarily to a decrease in root mass, that
6 the authors suggested could accelerate decline in alfalfa yields. These yield reductions are
7 generally similar to those reported previously in five similar studies summarized in the 1996
8 AQCD (Table 9-25 of U.S. Environmental Protection Agency, 1996).
9 A study in Alabama exposed early- and late-season-planted bahia grass (cultivar
10 Pensacola) in OTCs to CF, NF, or 2x-ambient O3 treatments (Muntifering et al., 2000). Ozone
11 exposures expressed as 12-h mean values over the 24-week experiment were 22, 45, and 91 ppb,
12 and the highest ambient O3 concentrations were recorded in late June, late July, late August and
13 mid-September at approximately 90 ppb. Above-ground biomass growth was reduced by the NF
14 treatment for the first and second harvest by 34% and 29% for the early-season planting, but
15 statistically significant effects were not observed in the late-season planting (Table AX9-17).
16 The 2x -ambient treatment did not cause further significant reductions in biomass. The authors
17 suggested that the lack of a significant O3 effect in the late planting may have been due to the
18 shorter total O3 exposure time as well as to the lower O3-exposure concentrations during the
19 weeks immediately preceding harvest. These results are important, because this is an
20 economically important species and because previous studies have focused on grass species that
21 use the C3, rather than C4, metabolic pathway.
22 An investigation of the use of different O3 indices and averaging times on the correlation
23 with growth effects was undertaken with the North Carolina clover system (Heagle et al., 1995).
24 For 2 years of data at six sites in Massachusetts, Oregon, North Carolina, California (2 sites),
25 and Virginia, averaging time was found to be more important than the choice of the type of
26 index including mean, SUM06, and AOT40 (Heagle and Stefanski, 2000). The best correlation
27 between O3 exposure and the ratio of sensitive-to-tolerant clover types was found for the 6-h
28 period from 1000 to 1600 h. For this period, very similar r2 values (0.91 to 0.94) were found for
29 SUM06, W126, and AOT40 (Heagle and Stefanski, 2000). For the above indices, a linear
30 relationship was found, with no effect in Corvallis, OR with exposure to a SUM06 value of
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1 10.2 ppm-h and a ratio of 0.53 (sensitive/tolerant) at San Bernardino with a SUM06 exposure of
2 39.4 ppm-h (Heagle and Stefanski, 2000).
3 In a study of the biomass ratio of O3-sensitive versus O3-insensitive clover at 14 sites in
4 Europe during 1996 to 1998, a model was developed using ANN techniques (see Section AX9.1)
5 that had r2 values for the training data of 0.84 and for unseen validation data of 0.71 (Mills et al.,
6 2000). The predictive factors in the model were AOT40, 24-h mean O3, daylight mean
7 temperature, and 24-h mean temperature. This model was selected after a thorough investigation
8 of a number of models using many more or fewer parameters using both ANN and multiple
9 linear regression techniques. This model predicted that a 5% reduction in biomass ratio was
10 associated with AOT40 values in the range 0.9 to 1.7 ppm-h accumulated over 28 days, with
11 plants being most sensitive under conditions of low NOX, moderate temperature, and high 24-h
12 mean O3 concentration.
13 Exposure to a square-wave 8-h mean O3 concentration of 92 ppb for 62 days in an
14 experiment in OTCs the UK did not significantly reduce the total yield of strawberry fruits, but
15 did decrease the average size of the fruits by 14% (Drogoudi and Ashmore, 2000). This
16 contrasts with an increase in total yield (fruit weight) found in a previous study in California
17 (Takemoto et al., 1988).
18 When timothy was exposed in OTCs in Sweden to NF, CF, and CF+O3 treatments, there
19 was no effect of a 12-h mean O3 exposure of 68 ppb (NF treatment), but a 12-h mean exposure
20 of 152 ppb decreased yield by 58% (Danielsson et al., 1999). A similar lack of effect of
21 exposure to a 12-h mean O3 exposure of 62 ppb was found in a previous study in the United
22 States (Kohut et al., 1988).
23 Although most investigations of O3-response relationships focus on growth or yield of
24 marketable portions of plants, some studies also investigate effects on plant quality. In the study
25 of bahia grass in Alabama discussed above, in addition to the effects on yield, there were
26 significant effects on quality for ruminant nutrition (Muntifering et al., 2000). Concentrations of
27 neutral detergent fiber (NDF) were higher in primary-growth and regrowth forages from the
28 early-season planting when exposed to 2x-ambient O3 than when exposed to the NF treatment.
29 The concentration of acid detergent fiber was higher in the 2 x-ambient treatment than in NF
30 treatment regrowth, whereas acid detergent lignin concentration was higher in 2 x-ambient than
31 in NF primary-growth forage. Crude protein concentrations were lower in CF-exposed than in
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1 NF-exposed regrowth forage from the early planting and in CF- than in NF-exposed primary-
2 growth forage from the initial harvest of the late-season planting. No differences were observed
3 among treatments in concentrations of total phenolics in primary-growth or regrowth forages
4 from either planting, although concentrations of total phenolics tended to be higher in
5 CF-exposed than in NF-exposed primary-growth forage from the late-season planting. The
6 authors concluded that the alterations in quality of primary-growth and vegetative regrowth
7 forages were of sufficient magnitude to have nutritional and possibly economic implications to
8 their use for ruminant animal feed.
9 Sericea lespedeza and little bluestem were exposed to CF, NF, and 2x-ambient O3 in OTCs
10 in Alabama for 10 weeks (Powell et al., 2003). Ozone treatments expressed as 12-h mean
11 concentrations were 23, 40, and 83 ppb and expressed as seasonal SUM06 values were 0.2, 9.1,
12 and 61.0 ppm-h. Although there were few statistically significant effects of O3 on yield (the
13 yield of only the 2 x-ambient compared to NF for Sericea lespedeza in the last of six harvests),
14 plant quality as feed for ruminants was reduced. The nutritive quality of Sericea lespedeza was
15 decreased by 7% and that of little bluestem by 2% as a result of increased cell wall constituents
16 and decreased in vitro digestibility.
17 For some annual species, particularly crops, the endpoint for an assessment of the risk of
18 O3 exposure can be defined as yield or growth; e.g., production of grain. For plants grown in
19 mixtures such as hayfields, and natural or seminatural grasslands (including native
20 nonagricultural species), endpoints other than production of biomass may be important. Such
21 endpoints include biodiversity or species composition and measures of plant quality such as total
22 protein and effects may result from competitive interactions among plants in mixed-species
23 communities. Most of the available data on non-crop herbaceous species are for grasslands.
24 In a study of two perennial grasses (bent grass and red fescue) and two forbs (white clover,
25 Germander speedwell) grown in pots in OTCs, O3 effects differed among species and cutting
26 treatments (Ashmore and Ainsworth, 1995) (see also Table AX9-17). Fescue biomass increased
27 with higher O3 treatments both in pots that were not cut during the growing season (mid-June to
28 mid-September) and those that were cut every two weeks. However, bent grass biomass
29 decreased with higher O3 exposure in the uncut treatment and increased in the cut treatment.
30 White clover and Germander speedwell biomass decreased substantially with higher O3 exposure
31 with and without cutting, with greater decreases in the cut treatment. The authors cautioned that
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1 the experiment did not replicate field circumstances. The plants were all cut to 1 cm above the
2 ground, which does not simulate grazing, and there may have been effects due to growing the
3 plants in pots. However, two key results of this study likely apply to mixtures of species
4 growing in hay or forage fields or seminatural and natural communities. First, O3 exposure
5 increased the growth of O3-tolerant species, while exacerbating the growth decrease of O3
6 sensitive species. Second, the total biomass of the mixed-species community was unaffected by
7 O3 exposure due to the differential effects on O3-sensitive and O3-tolerant species.
8 In a 2-year study using OTCs placed over managed pasture in Switzerland, the above-
9 ground biomass of clover (red and white) was reduced linearly in response to increased O3
10 exposure (Table AX9-17) (Fuhrer et al., 1994). Exposure to a 12-h mean concentration of
11 39 ppb O3 reduced biomass by 24% as compared to the CF treatment with a 12-h mean
12 concentration of 21 ppb O3. There was a trend toward increased above-ground biomass of
13 grasses (primarily orchard grass), but this trend was not statistically significant. As often found
14 in other studies of mixtures of species, by O3 exposure did not significantly affect total above-
15 ground biomass O3 exposure.
16 A field-grown grass/clover mixture was exposed to CF, NF, and two O3 addition treatments
17 for two growing seasons in OTCs in southern Sweden (Pleijel et al., 1996). The mixture
18 consisted of 15% (by seed weight) red clover cv. Fanny, 60% timothy cv. Alexander, and 25%
19 fescue cv. Svalofs Sena. Ozone concentrations expressed as AOT40 ranged from 0 to
20 approximately 47 ppnrh and expressed as 7-h mean from 11 to 62 ppb. Over this range, a slight,
21 but statistically significant, linear decrease of 4% in total above-ground biomass was seen
22 growth over six harvests. No significant decrease was seen in the proportion of clover, and the
23 authors ascribed this lack of effect to the relatively higher O3 sensitivity of timothy and lower
24 sensitivity of this clover cultivar as compared to previously published results for other
25 grass/clover mixtures (e.g., Fuhrer et al., 1994).
26 A mixture of species in an old farm field in Alabama was exposed to O3 for two growing
27 seasons in large OTCs (4.8 m high and 4.5 m diameter); and a similar lack of effect of O3 was
28 found on total plant community growth measured as both canopy cover and vertical canopy
29 density (Barbo et al., 1998). Of the 40 species in the plots, O3 effects were examined only on the
30 most common species: blackberry, broomsedge bluestem, bahia grass, Panicum spp., and
31 winged sumac (second year only). Of these species, a 2x-ambient O3 treatment increased the
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1 percent canopy cover of blackberry over 2 years by 124%, while that of winged sumac was
2 decreased by 95% (Table AX9-17). Blackberry showed no significant effect on biomass, but
3 ripe fruit mass was decreased by 28% (Chappelka, 2002). However, there was a significant
4 chamber effect for this latter response. Biomass was not reported for other species in this study
5 due to a hurricane. Effects on loblolly pine grown in this experiment are discussed subsequently
6 in Section AX9.5.5.5.
7 In summary, results of studies on perennial herbaceous species conducted since the 1996
8 criteria document was prepared are presented in Table AX9-17. As for single-season
9 agricultural crops, yields of multiple-year forage crops are reduced at O3 exposures that occur in
10 some years over large areas of the United States (Tables AX9-13, AX9-21). This result confirms
11 that reported in the 1996 AQCD (U.S. Environmental Protection Agency, 1996). When species
12 are grown in mixtures, O3 exposure can increase the growth of O3-tolerant species while
13 exacerbating the growth decrease of O3-sensitive species (e.g., Ashmore and Ainsworth, 1995;
14 Fuhrer et al., 1994). Because of this competitive interaction, the total growth of the mixed-
15 species community may not be affected by O3 exposure (Ashmore and Ainsworth, 1995; Barbo
16 et al., 1998; Fuhrer et al., 1994). However, in some cases mixtures of grasses and clover species
17 have shown significant decreases in total biomass growth in response to O3 exposure in studies
18 in the United States (Heagle et al., 1989; Kohut et al., 1988) and in Sweden (Pleijel et al., 1996).
19 In Europe, a provisional critical level for perennials of an AOT40 value of 7 ppm-h over 6
20 months has been proposed to protect sensitive plant species from the adverse effects of O3.
21
22 AX9.5.5.2 Deciduous Woody Species
23 It is extremely difficult and costly to study entire mature trees under controlled conditions
24 such as those in OTCs, with the possible exception of some species managed for fruit or nut
25 production. For this reason, the great majority of investigations have been of seedlings in
26 growth chambers, greenhouses, or OTCs. A few investigations have been carried out on
27 saplings or more mature trees using free air exposure systems (Haeberle et al., 1999; Isebrands
28 et al., 2000, 2001; Werner and Fabian, 2002). Exposure-response functions based on 28
29 experimental cases of seedling response to O3 suggest that a SUM06 exposure for 3 months of
30 31.5 ppm-h would protect hardwoods from a 10% growth loss in 50% of the cases (Table
31 AX9-18). However, there is a substantial range in sensitivity among species. A risk analysis was
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1 undertaken to predict tree biomass growth reductions due to O3 based on exposure-response
2 equations for seedlings of individual species combined with the species' spatial distribution
3 across the eastern United States and interpolated O3 exposure expressed as SUM06 (Hogsett
4 et al., 1997). The growth of sensitive species such as aspen and black cherry was predicted to be
5 reduced by at least 20% across 50% of their ranges in a high O3 year and approximately 10% in a
6 lower-than-average O3 year (Hogsett et al., 1997).
7 A few investigations reported since the last criteria document was prepared have examined
8 saplings or mature trees, notably of oak species in the southern Appalachian Mountains and pine
9 species in California. Most of these studies have been of natural (uncontrolled) O3 exposures.
10 Additional studies have examined foliar symptoms on mature trees, and in recent years such
11 surveys have become more common and with greater attention to the standardization of methods
12 and the use of reliable indicator species (Campbell et al., 2000; Smith et al., 2003). Previous
13 criteria documents have noted the difficulty in relating foliar symptoms to effects on individual
14 tree growth, stand growth, or ecosystem characteristics (U.S. Environmental Protection Agency,
15 1996). This difficulty still remains to the present day.
16 Some investigators have suggested that a comprehensive risk assessment of the effects of
17 O3 on mature tree species might best be accomplished by extrapolating measured effects of O3
18 on seedlings to effects on forests using models based on tree physiology and forest stand
19 dynamics (Chappelka and Samuelson, 1998; Laurence et al., 2000, 2001). Several such efforts
20 are discussed in Sections AX9.3 and AX9.6.
21 In this subsection, emphasis will be placed on experimental evidence of O3 effects on the
22 growth of woody species under controlled conditions with some information from observational
23 studies under ambient conditions in forests. Experimental results are summarized for deciduous
24 species in Table AX9-18; the species are discussed below in the order in which they appear in
25 this table.
26 A series of studies in Michigan and Wisconsin during the 1990s on clones of trembling
27 aspen previously demonstrated that they differ in their O3 sensitivity (Coleman et al., 1995a,b,
28 1996; Dickson et al., 2001; Isebrands et al., 2000, 2001; Karnosky et al., 1996, 1998, 1999; King
29 et al., 2001). Several of those studies were undertaken with plants in pots or in the ground in
30 OTCs and additional studies were undertaken at three sites selected to differ primarily in O3
31 exposure (Karnosky et al., 1999). An ongoing study was undertaken using a FACE carbon
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1 dioxide and O3 enrichment facility in Rhinelander, WI (Isebrands et al., 2000, 2001). These
2 studies showed that O3-symptom expression was generally similar in OTCs, FACE, and gradient
3 sites, supporting the previously observed variation among aspen clones (Karnosky et al., 1999).
4 In the Michigan OTC study, plants were grown in pots and exposed to CF, 0.5x ambient, 1 x
5 ambient, 1.5* ambient, 2x-ambient O3 treatments for 98 days (Karnosky et al., 1996). Ozone
6 concentrations expressed as 3-month, 7-h mean values were 7 ppb (CF), 43 ppb (1 x) and 63 ppb
7 (2x). Ozone decreased total plant biomass between 2 and 22% for three clones previously
8 selected to represent high, intermediate and low O3 tolerance based on previous studies of larger
9 populations(clones 216, 271 and 259; Karnosky et al., 1996). Seedlings produced from 15 parent
10 trees responded similarly as did the 3 clones, with an average biomass reduction of 14% in the
11 1 x-ambient treatment for the seedlings compared to 16% for the clones. In a second experiment
12 using square wave exposures, biomass reduction for the clones was ranged from 23 to 39%
13 (mean = 31%) at a 7-h mean O3 concentration of only 45 ppb, which is similar to the response to
14 the 2 x-ambient treatment in the previous experiment at a 7-h mean O3 concentration of 66 ppb.
15 The FACE study evaluated the effects of multiple years of exposure to combinations of
16 elevated CO2 and 1.5* ambient O3 on growth responses in mixture of five trembling aspen
17 clones (Isebrands et al., 2000, 2001). Height, diameter, and stem volume (diameter2 x height)
18 were decreased by elevated O3. On average for all clones, stem volume was decreased by 20%
19 over the first 3 years in the elevated O3 treatment as compared with the 1 x-ambient treatment.
20 However, one clone showed increased growth in response to O3. Ozone concentrations were not
21 reported. Over the first 7 years of the study, average stem volume was decreased by 14% with
22 12-h mean O3 concentrations between 49 and 55 ppb as compared with effects at ambient O3
23 concentrations with 12-h mean values of 35 to 37 ppb (O3 exposure data are for the first 4 years,
24 as they have not been reported for subsequent years) (Karnosky et al., 2003, 2005). This FACE
25 facility study is important, because it confirmed responses reported previously with these clones
26 grown in pots or soil in OTCs, without the alterations of microclimate induced by chambers.
27 Currently, this is the only U.S. study using this technology to have examined the effects of O3
28 under these conditions. This study is also significant, because the elevated O3-exposure pattern
29 used was intended to reproduce the 6-year average pattern from Washtenaw County, Michigan
30 (Karnosky etal., 1999).
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1 Rooted cuttings of two aspen clones from Acadia National Park in Maine were exposed to
2 1 x-ambient, 1.7x-ambient, and 3x-ambient O3 concentrations in large OTCs in Ithaca, NY for
3 much of one growing season (15 June to 15 September) (Yun and Laurence, 1999). Both
4 circular (4.7 m diameter, 3.7 m height) and rectangular (7.4 m x 2.75 m x 3.7 m height)
5 chambers were used (Mandl et al., 1989). Exposure to 1.7x-ambient O3 (SUM06 = 20 ppm-h,
6 9-h mean = 74 ppb) reduced shoot growth by 14 and 25% compared to ambient O3 for the two
7 clones (Yun and Laurence, 1999). Total dry weight was reduced by 55 and 35% in the two
8 clones by the 3 x-ambient treatment (SUM06 = 62 ppm-h, 9-h mean =124 ppb) compared to the
9 ambient O3 treatment.
10 When black poplar cuttings in OTCs in Belgium were exposed to 8-h mean O3
11 concentrations of 5, 29, and 33 ppb, diameter growth decreased by 29% in the highest O3
12 treatment, but height growth was unaffected (Bortier et al., 2000b). A 2-month study of hybrid
13 poplar (Populus tremuloides x P. tremula) in a free air exposure system in Finland with 7-h
14 mean O3 concentrations of 30 and 38 ppb found a 6% decrease in height with no effect on
15 biomass (Oksanen et al., 2001). Eastern cottonwood cuttings in pots buried in the ground with
16 drip irrigation were exposed to ambient O3 at several sites in and near New York City in three
17 2-month experiments during three summers (Gregg et al., 2003). Ozone concentrations were
18 lower at urban sites than at rural sites within 100 km of the urban sites. Total biomass growth
19 was greater in urban than rural sites, with a strong linear decrease in biomass with increasing O3
20 across all sites and years (r2 = 93). Total biomass decreased 33% with 12-h mean O3 levels of 38
21 ppb compared to 23 ppb. Multiple regression analysis showed no significant temperature effect
22 on biomass. Therefore, the authors suggested that O3 exposures were the most likely explanation
23 for the reduced biomass in rural areas. The overall growth reductions and the variation among
24 genotypes seen on all of the above aspen and poplar studies is similar to those previously
25 reported in three OTCs studies summarized in the 1996 O3 AQCD (Table 9-26 of U.S.
26 Environmental Protection Agency, 1996).
27 For paper birch, over the first 7 years of the Wisconsin FACE study, average stem volume
28 was unaffected by 12-h mean O3 concentrations between 49 and 55 ppb as compared with effects
29 at ambient O3 concentrations with 12-h mean values of 35 to 37 ppb (O3 exposure data are for
30 the first 4 years, as they have not been reported for subsequent years (Karnosky et al., 2003,
31 2005). In contrast, significant effects were found in this study for aspen and sugar maple, so
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1 these results indicate that paper birch is relatively insensitive to O3 compared to these other
2 species.
3 Black cherry seedlings grown in pots were exposed in OTCs in the Great Smoky Mountain
4 National Park in Tennessee to O3 treatments ranging from CF to 2x-ambient in two experiments
5 during 1989 and 1992 (Neufeld et al., 1995). Ozone exposure, expressed as SUM06, ranged
6 from 0 to 40.6 ppm-h in 1989, and from 0 to 53.7 ppm-h in 1992. Corresponding AOT40 values
7 were 0.0 to 28.3 ppm-h in 1989, and 0 to 40.4 ppm-h in 1992. In 1989, total biomass was
8 decreased in the 1.5x-ambient treatment by 18% and in the 2x-ambient treatment by 38%.
9 In 1992, total biomass was decreased in the 1.5 x-ambient treatment by 27%, and in the
10 2x-ambient treatment by 59%. In this study, SUM06 and AOT40 provided better fits than did
11 SUMOO with Weibull regression analyses to log-transformed biomass data. Although a Weibull
12 model was used, responses to O3 expressed as SUM06 and AOT40 appeared to be linear. The O3
13 exposures in the 1.5 x-ambient and 2 x-ambient treatments were reported to be similar to those
14 for a site near Charlotte, NC in a high-O3 year (1988). In a 2-year experiment in OTCs in Ohio,
15 seedlings of black cherry, sugar maple, and yellow poplar were exposed to O3 treatments with
16 SUMOO values ranging from 16 to 107 ppm-h in 1990 and 31 to 197 ppm-h in 1991 (Rebbeck,
17 1996). After two seasons of exposure, only black cherry showed a growth decrease: total
18 biomass was reduced by 32% in the 2x-ambient O3 treatment compared to the CF treatment; root
19 biomass was decreased by 39%. These results contrast with those of a previous study with black
20 cherry seedlings in which significant biomass reductions with exposures up to 2 x-ambient were
21 not observed (7-h mean = 21 to 97 ppb), perhaps because of the small sample size (3 seedlings
22 per chamber (Samuelson, 1994) in the earlier study.
23 A multiyear study of effects of O3 on both seedling and mature (30-year-old) red oak trees
24 was conducted in Norris, TN in large OTCs with three replicates per O3 treatment. Trees were
25 exposed for 3 years to CF, 1 x-ambient, and 2 x-ambient treatments, with the following O3
26 exposures: SUM06 for 3 years = 0, 29, 326 ppm-h; SUMOO for 3 years = 147, 255, and 507
27 ppm-h. The net photosynthetic rate in mature trees was reduced by 25% in the ambient
28 treatment and by 50% in the 2x-ambient treatment (Samuelson and Edwards, 1993; Hanson
29 et al., 1994; Wullschleger et al., 1996). Despite these large decreases, no significant effects on
30 stem increment at the base, stem increment in the canopy, or leaf mass were observed for the
31 mature trees (Samuelson et al., 1996). Similarly, seedling biomass was not significantly reduced
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1 by O3 exposure. The difficulty in replicating experiments with mature trees makes it difficult to
2 detect changes in growth or biomass. However, the mean values of the stem increment at the
3 base and within the canopy in the ambient treatment were larger than those in the CF treatment,
4 although those in the 2x-ambient treatment were lower. Therefore, this study of mature trees
5 does not provide evidence that these ambient concentrations reduced above-ground tree growth,
6 even after 4-years exposure.
7 Sugar maple seedlings were exposed for 3 years to ambient, 1.7x-ambient, and 3 x-ambient
8 O3 treatments at both high-light (3 5% of ambient) and low-light levels (15% of ambient)
9 (Topa et al., 2001). This experiment was conducted in large OTCs near Ithaca, NY. Over the
10 3 years, O3 exposures expressed as SUMOO for the three treatments were 88, 126, and 185
11 ppm-h. After 3 years, total seedling biomass in the 3 x-ambient treatment was reduced by 64%
12 and 41% in the low- and high-light treatments, respectively (compared to the 1 x-ambient
13 treatment). The larger reduction of biomass under low-light conditions suggests that seedlings
14 growing under closed canopies may be substantially more sensitive to O3 than are seedlings
15 exposed to higher-light levels in gaps or clearings. These results differ from other studies in
16 which seedling biomass was unaffected by exposure to SUMOO values of 304 ppm-h over 2 years
17 (Rebbeck, 1996) or 591 ppm-h over 3 years (daytime mean of 40.7) (Laurence et al., 1996).
18 However the latter two studies used much higher light levels, which may have reduced the O3
19 effect, based on the results of Topa et al. (2001). Over the first 7 years of the Wisconsin FACE
20 study, average stem volume of sugar maple was decreased by 14% with 12-h mean O3
21 concentrations between 49 and 55 ppb as compared with effects at ambient O3 concentrations
22 with 12-h mean values of 35 to 37 ppb (O3 exposure data are for the first 4 years as they have not
23 been reported for subsequent years (Karnosky et al., 2003, 2005). These growth effects were not
24 statistically significant for the first 3 years, but became significant subsequently. These results
25 are important because they demonstrate that 3 years of exposure may not be long enough to
26 evaluate effects of O3 on the growth of tree species.
27 Although most studies demonstrate that O3 decreases biomass growth, occasional results
28 indicate that O3 can increase growth of some portions of woody perennials. When Casselman
29 plum trees near Fresno, CA were exposed to O3 in large, rectangular OTCs to three O3 treatments
30 (CF, 1 x-ambient, and an above-ambient O3 treatment) for 4 years (12-h mean = 31, 48, 91
31 ppm-h), stem increment increased 14% in the highest O3 treatment compared to the CF
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1 treatment; and this effect was statistically significant (Retzlaff et al., 1997). However, fruit yield
2 decreased in this treatment by 42% and also decreased by 16% in the 1 x-ambient-O3 treatment.
3 Root growth was not measured in this study. Hence, the increase in stem diameter may have
4 been at the expense of other organs. However, in a fifth year, all plants were exposed to
5 1 x-ambient O3, and there were no differences in fruit yield, suggesting that trees were able to
6 recover to some extent from the effects of O3 exposure in prior years.
7 When potted yellow poplar seedlings were exposed to O3 concentrations up to SUMOO
8 values of 107 ppm-h in one year and 197 ppm-h in a second year, no effects on biomass were
9 observed (Rebbeck, 1996). In a study at the same location with seedlings planted in the ground
10 and exposed to O3 concentrations with SUM06 values of 0.3, 228.7, and 661.8 ppm»h over 5
11 years, no effects on biomass were found (Rebbeck and Scherzer, 2002).
12 Many studies have demonstrated that root growth is more sensitive to O3 exposure than is
13 stem growth. For example, in a study with black cherry seedlings exposed in OTCs in
14 Tennessee in 1989, root biomass in a 2 x-ambient treatment was decreased by 42%, while stem
15 biomass was decreased by only 24%. However, in a second experiment in 1992, root and stem
16 growth reductions in the 2x-ambient treatment were similar (65% versus 60%) (Neufeld et al.,
17 1995). In Finland, reduced root growth was found for a number of clones of silver birch
18 (Oksanen and Saleem, 1999). After 5 years, root growth was decreased by 33%, but shoot
19 growth was not affected by O3 exposures of a 7-h mean of 15 ppm-h over 5 years in a FACE
20 system (Oksanen, 2001). When first-year poplar seedlings (P. tremuloides) were exposed in
21 OTCs to two O3 concentrations and six N concentrations, the root/shoot ratio was decreased soon
22 after exposure to O3 in all N treatments, even though O3 effects on total biomass were not
23 detected in the low-N and very high-N treatments (Pell et al., 1995). These results suggest that
24 effects on the root/shoot ratio occur before significant growth effects arise. In a series of OTC
25 experiments lasting 1 to 3 years at 3 different elevations in Switzerland, fine root growth in
26 European beech was found to be more sensitive to O3 than was shoot or total biomass (Braun and
27 Fluckiger, 1995). Although the estimated effect of O3 on fine root biomass was similar to that
28 for total biomass, fine root biomass was significantly decreased at AOT40 (24-h) values of only
29 3 ppm-h, while total biomass was not significantly decreased until AOT40 values reached 30 to
30 40 ppm-h.
31
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1 AX9.5.5.3 European Critical Levels
2 In Europe, a Level I critical level has been set for forest trees based on OTC studies of
3 saplings. This level is discussed here because it was based on a deciduous tree species. For
4 consistency with the approach used for crops, an AOT40 index value was selected. A few
5 studies have shown that O3 can be taken up by tree species at nighttime, e.g., young birch trees
6 (Matyssek et al., 1995). However, because most evidence suggests that O3 deposition at
7 nighttime is low (Coe et al., 1995; Rondon et al., 1993), a value for only daylight hours was
8 selected in Europe (Fuhrer et al., 1997; Karenlampi and Skarby, 1996). European beech was
9 selected for development of a Level I critical level, because data from several studies were
10 available for this species and because deciduous tree species were judged to be more sensitive to
11 O3 compared to evergreen tree species (Fuhrer et al., 1997; Karenlampi and Skarby, 1996).
12 A critical level was defined as an AOT40 value of 10 ppm-h for daylight hours for a 6-month
13 growing season (Karenlampi and Skarby, 1996). However, other studies have shown that other
14 species such as silver birch may be more sensitive to O3 than beech (Paakkonen et al., 1996).
15 Level I critical values are not designed for making quantitative estimates of the O3 effects on
16 vegetation at the regional scale, instead a so-called Level II critical value is required for this
17 purpose. For long-lived perennials, additional problems complicate extrapolation. As discussed
18 below (Section AX9.5.5.7), considerable scaling is required to extrapolate from experiments
19 conducted with tree seedlings to estimate effects on mature trees in forests. Because of these
20 scaling issues, there is greater uncertainty in estimating effects on forest trees than on annual
21 plants such as crops. While some information is available for addressing issues such as scaling
22 from seedlings to mature trees and estimating O3 uptake, this information may still be
23 insufficient for developing a Level II approach that can provide quantitative estimates of forest
24 growth losses due to O3 (Broadmeadow, 1998).
25
26 AX9.5.5.4 Summary of Effects on Deciduous Woody Species
27 Recent evidence from free air exposure systems and OTCs supports results observed
28 previously in OTC studies (Table AX9-15, Figure AX9-18). Specifically, a series of FACE
29 studies undertaken in Rhinelander, WI (Isebrands et al., 2000, 2001) showed that O3-symptom
30 expression was generally similar in OTCs, FACE, and ambient-O3 gradient sites, supporting the
31 previously observed variation among aspen clones using OTCs (Karnosky et al., 1999). This
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1 study also found no effects on sugar maple growth after 3 years, but in years 4 to 7 found
2 significant growth reduction due to O3 (Karnosky et al., 2005). These results are important,
3 because they indicate results obtained from OTCs are supported by results from free air exposure
4 systems and also that more than 3 years may be required to adequately investigate the effects of
5 O3 on the growth of tree species. Finally, this study found that competition may alter the effect
6 of O3, depending on environmental conditions and genotype (McDonald et al., 2002). New
7 evidence is also available comparing various aspects of O3 sensitivity between seedlings and
8 mature trees of some species, notably red oak. As has been observed in previous criteria
9 documents, root growth is often found to be the most sensitive indicator in terms of biomass
10 response to O3.
11 Results since 1996 support the conclusions of the 1996 AQCD (U.S. Environmental
12 Protection Agency, 1996) that individual deciduous trees are generally less sensitive to O3 than
13 are most annual plants, with the exception of a few genera such as Populus, which are highly
14 sensitive. However, the data presented in Table AX9-18 suggest that ambient exposures that
15 occur in different regions of the United States can sometimes reduce the growth of seedlings of
16 deciduous species. Results from multiple-year studies sometimes show a pattern of increasing
17 effects in subsequent years. Although, in some cases, growth decreases due to O3 become less
18 significant or even disappear over time. While some mature trees show greater O3 sensitivity in
19 physiological parameters such as net photosynthetic rate compared to seedlings, these effects
20 may not translate into measurable reductions in biomass growth. Because even multiple-year
21 experiments do not expose trees to O3 for more than a small fraction of their life span and
22 because competition may, in some cases, exacerbate the effects of O3 on individual species,
23 determining the effects on mature trees remains a significant challenge. Effects on mature trees
24 under natural conditions are discussed after the review of evergreen species below and more
25 fully in Section AX9.6, in the context of extrapolating from controlled studies to forest
26 ecosystems.
27
28 AX9.5.5.5 Evergreen Woody Species
29 Most investigations have shown evergreen tree species to be less sensitive to O3 compared
30 to deciduous species (U.S. Environmental Protection Agency, 1996). For example, exposure-
31 response functions based on 23 experimental cases of seedling response to O3, suggest that a
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1 SUM06 exposure for 3 months of 42.6 ppm-h would protect evergreen species from a 10%
2 growth loss in 50% of the cases (Table AX9-15). For deciduous species, the corresponding
3 SUM06 value was 31.5 ppm-h (Table AX9-15). As another example, experiments in the Great
4 Smoky Mountains National Park found black cherry seedlings to demonstrate substantial
5 decreases in biomass, as discussed above and shown in Table AX9-18 (Neufeld et al., 1995).
6 However, exposure for up to three growing seasons did not decrease the biomass of eastern
7 hemlock, Table Mountain pine, and Virginia pine seedlings exposed to O3 under similar
8 conditions in this location, as shown in Table AX9-19 (Neufeld et al., 2000).
9 As for deciduous species, there is a substantial range in sensitivity among evergreen
10 species. As discussed above for deciduous species, a risk analysis was undertaken to predict tree
11 biomass growth reductions due to O3 based on exposure-response equations for tree seedlings
12 combined with species distribution across the eastern United States and interpolated O3 exposure
13 (Hogsett et al., 1997). While some species such as Virginia pine were predicted to be affected
14 only slightly even in a high O3 year, the growth of sensitive evergreen species such white pine
15 was predicted to be reduced by 5% in a lower-than-average O3 year and 10% in a high O3 year
16 across 50% of its range (Andersen et al., 1997). The remainder of this section discusses
17 experimental results for evergreen species in the order shown in Table AX9-19.
18 Douglas fir seedlings were exposed to elevated O3 concentrations in a free air zonal air
19 pollution system in British Columbia, Canada for two growing seasons with 12-h mean values in
20 1988 of 18 to 41 ppb and in 1989 of 27 to 66 ppb (Runeckles and Wright, 1996). Although
21 substantial variation was seen in effects among the different treatments, there was a significant
22 decrease in the growth of the second flush weight in the second year, with reductions of 55% at
23 the highest O3 exposure, based on a linear regression. This result contrasts with the lack of
24 effect seen in a previous study with seedlings of this species grown in pots for 134 days and
25 exposed to 7-h mean O3 concentrations up to 71 ppb (Table 9-30 in U.S. Environmental
26 Protection Agency, 1996).
27 First-year loblolly pine seedlings of 53 open-pollinated families were exposed to
28 1 x-ambient O3 in OTCs for a single growing season, and average growth volume was decreased
29 by 14% compared to a CF treatment (McLaughlin et al., 1994). The 1 x-ambient O3 exposure in
30 this study expressed as 24-h SUMOO was 137 ppm-h, and the CF treatment reduced O3 by 47%.
31 In this study, the root-to-shoot ratio was decreased significantly in six of the nine families
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1 examined. Exposure to O3 with SUM06 values up to 162 ppm-h and 132 ppm-h in 2 successive
2 years in OTCs had no effect on seedlings grown in competition with various species of grasses
3 and forbs (Barbo et al., 2002). Exposure of 3-year-old seedlings to O3 exposures of up to
4 2.5x-ambient (12-h mean of 98 ppb) also had no significant effect (Anttonen et al., 1996).
5 Four-week-old loblolly pine seedlings were grown in large OTCs in Alabama and exposed to
6 CF, 1 x ambient, 2x ambient O3 treatments in 2 one year experiments, with seasonal 12-h
7 mean O3 concentrations of 13, 47, 98 ppm»h in 1998 and 12, 44, 97 ppm»h in 1999 (Estes et al.,
8 2004). Shoot biomass was decreased 15% in the 1x treatment and 22% in 2x in both years, while
9 root biomass was decreased by 26% in the 2x treatment in both years. Foliar symptoms were
10 significantly greater in the 1x treatment iin!999 and in the 2x treatment in both years.
11 Information summarized in the 1996 AQCD (U.S. Environmental Protection Agency, 1996),
12 indicated that significant effects on seedling growth were observed in several studies of
13 seedlings exposed to elevated O3 concentrations for one or more years. Several studies,
14 including that of McLaughlin et al. (1994), demonstrate considerable variation in O3 sensitivity
15 among different genotypes of loblolly pine.
16 For Ponderosa pine seedlings, the 1996 AQCD reviewed a number of studies with
17 exposures to elevated O3 concentrations for one to three growing seasons (U.S. Environmental
18 Protection Agency, 1996). More recent similar studies support the earlier results (Table
19 AX9-19); (Andersen et al., 2001; Takemoto et al., 1997). The 1996 criteria document also
20 discussed at some length the ongoing work examining effects of O3 across a naturally-occurring
21 O3 gradient in the San Bernardino Mountains in California. Since that time, much research on
22 ponderosa pine has focused on interactive effects of additional stresses such as nitrogen, and
23 effects of O3 on physiological parameters (Sections AX9.3, AX9.6). Effort has also focused on
24 the effects of O3 on root growth because such effects could alter sensitivity to drought or nutrient
25 stress. Ecosystem level effects of O3 are discussed further in Section AX9.6, but some
26 information relevant to exposure-response relationships is discussed below.
27 For several tree species, O3 has been shown in experimental studies with seedlings to
28 reduce root growth more than shoot growth (U.S. Environmental Protection Agency, 1996).
29 Ponderosa pine has been shown to be sensitive to O3, and studies with seedlings have shown
30 reduced root growth, decreases root-to-shoot ratios, and decreased allocation to roots (Andersen
31 et al., 1991, 1997; Andersen and Rygiewicz, 1995; Andersen and Scagel, 1997; Tingey et al.,
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1 1976). Data from a long-studied pollution gradient in the San Bernardino Mountains of southern
2 California suggests that O3 substantially reduces root growth in natural stands of ponderosa pine.
3 Root growth in mature trees was decreased at least 87% in a high pollution site as compared to a
4 low pollution site (Grulke et al., 1998), and a similar pattern was found in a separate study with
5 whole tree harvest along this gradient (Grulke and Balduman, 1999). Because other potential
6 influences on root growth, including shading by competing trees, soil temperature, soil moisture,
7 phenology, were not correlated with the observed pattern of reduced root growth, the authors
8 conclude that O3 was the cause of the observed decline in root growth. Further results of field
9 investigations with ponderosa pine and other pine species native to California are discussed
10 below under the heading "Scaling experimental data on long-lived species to field conditions" as
11 well as in Section AX9.6.
12 Table Mountain pine, Virginia pine, and eastern hemlock seedlings were exposed to
13 various levels of O3 (CF to 2* ambient) in OTCs for in a series of experiments two or three years
14 in Great Smoky Mountains National Park in Tennessee (Neufeld et al., 2000). There were no
15 statistically significant effects of O3 exposure on stem or root biomass, and only slight effects on
16 the biomass of the oldest needles in Table Mountain pine in the 2* ambient treatment.
17 As reviewed in the 1996 criteria document, studies of the response of red spruce to O3
18 exposures generally have not found effects on growth of seedlings or saplings, even after
19 exposure to high concentrations (12-h mean of 90 ppb) for up to 4 years. A report since that
20 time confirms that this slow-growing species is O3 insensitive for at least several years (Laurence
21 etal., 1997).
22 For perennial vegetation, cumulative effects over more than one growing season may be
23 important. For 3-year-old Norway spruce in Sweden, exposure to elevated O3 for three growing
24 seasons decreased total biomass by 18% and stem biomass by 28% (Karlsson et al., 1995).
25 However, after a fourth season of exposure, total biomass decreased significantly by only 8%
26 (Karlsson et al., 2002). In this experiment, the O3 exposures expressed as 12-h mean values
27 averaged over four growing seasons were 12 and 44 ppb for the CF and 1.5 x-ambient treatments,
28 respectively; and AOT40 values were 2 and 23 ppm-h, respectively. Despite 4 years of
29 exposure, this experiment did not demonstrate a consistent trend in the O3 effect on biomass that
30 would suggest a significant carry-over effect. However, a study of 3- to 7-year-old Norway
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1 spruce in OTCs in Finland found a 5.3% decrease in total plant biomass after 7 years, with an
2 elevated O3 AOT40 exposure value of 79 ppm»h (Ottosson et al., 2003; Wallin et al., 2002).
3
4 AX9.5.5.6 Summary of Effects on Evergreen Woody Species
5 In summary, the O3 sensitivity of different genotypes within species and between species
6 of evergreen vegetation varies widely. Based on studies with evergreen seedlings in OTCs,
7 major species in the United States are generally less sensitive than are most deciduous trees, and
8 slower-growing species are less sensitive than faster-growing ones. However, exposure to
9 ambient O3 may reduce the growth of seedlings of commonly occurring species. Because tree
10 species are long-lived, most experiments have only covered a very small portion of the life span
11 of a tree, making estimation of any effect on mature trees difficult. Considerations for scaling
12 the results of seedling studies to mature forest trees as well as additional information from field
13 surveys and studies of mature trees under natural conditions are discussed below and in
14 Section AX9.6.
15
16 AX9.5.5.7 Scaling Experimental Data to Mature Trees
17 As compared with annual crop species, it is much more difficult to define appropriate
18 exposure-response relationships for tree species. For annual species, an experiment may cover
19 the whole life span of the plant, but it is difficult and expensive to provide controlled-exposure
20 conditions for long-lived plants for any significant portion of their life spans, although a few
21 FACE studies have demonstrated that it is feasible. However, FACE studies cannot investigate
22 the effects of ambient O3 exposures, because lower-than-ambient O3 treatments cannot be
23 applied. Most studies have used small seedlings, because they are manageable under
24 experimental conditions; but seedlings and mature trees may have different sensitivities to O3.
25 For perennial species, effects of O3 may accumulate over more than 1 year, and may interact
26 with other stresses such as drought stress over multiple growing seasons. As for annual species
27 (Section AX9.3.2), substantial variability occurs among evergreen genotypes and this variation
28 may interact with other stress responses differently in different landscapes and regions. Despite
29 these difficulties, investigators have addressed some of these issues since the 1996 AQCD (U.S.
30 Environmental Protection Agency, 1996). New information is available on the response of
31 mature evergreen trees to O3 under field conditions, and models based on tree physiology and
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1 stand dynamics have been used to predict O3 effects on forest stands and regions. The following
2 issues are reviewed briefly below: (1) interaction of drought and O3 stress, (2) scaling data from
3 seedlings to mature-tree studies. Two additional scaling issues are addressed in Section AX9.6:
4 (1) scaling data to forest stands, and (2) scaling data to ecosystems and regions.
5
6 AX9.5.5.7.1 Interactive Effects of Drought and Ozone
1 Many interacting factors may influence the effect of O3 on vegetation. For crop plants,
8 environmental conditions are often managed such that nutrients and water are not strongly
9 limiting; but for native vegetation, including most perennial species, such factors are likely to
10 limit growth. The effects of interacting stresses on vegetation were reviewed in Section AX9.3.
11 However, because drought is common in many forests, and because there is substantial evidence
12 that it alters the response of trees to O3, it is discussed in this section in the context of
13 determining exposure-response relationships for trees.
14 Controlled experiments with seedlings provide direct evidence that drought can reduce the
15 impact of O3. For example, for 3-year-old Norway spruce in Sweden, exposure to elevated O3
16 for three growing seasons decreased total biomass by 18% and stem biomass by 28% (Karlsson
17 et al., 1995). However, for draughted trees, both total and stem biomass decreased only 5%,
18 with a statistically significant interaction with O3 for stem biomass. Yet after a fourth season of
19 exposure, there was no longer any interaction between drought and O3, while there was a
20 significant decrease of 8% in the biomass when both drought and well-watered data were
21 combined (Karlsson et al., 2002). In this study, seedlings were grown in sand in 120-L pots and
22 for the drought treatment, water was withheld for 4 weeks during the first year and for 7 to 8
23 weeks during each of the last 3 years. In this experiment, the O3 exposures expressed as 12-h
24 seasonal daylight mean averaged over four growing seasons were 12 and 44 ppb for the CF and
25 1.5x-ambient treatments, respectively. Over this period, the AOT40 values for the treatments
26 averaged 2 and 23 ppm-h respectively. Despite 4 years of exposures, this experiment did not
27 demonstrate a consistent trend in drought O3 interactions. The difference in effects seen between
28 the third and fourth season suggest that scaling drought-O3 interactions from seedlings to mature
29 trees may be difficult. However, evidence from biomonitoring surveys supports an interaction
30 between drought and O3 effects, at least for foliar symptoms. In systematic surveys of foliar
31 symptoms on species selected as biomonitors throughout much of the eastern United States,
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1 symptoms were more common and more severe in areas with high O3 concentrations (Smith
2 et al., 2003). However, in very dry years, such as 1999, the occurrence and severity of
3 symptoms was greatly reduced, even in areas with high ambient O3 concentrations.
4
5 AX9.5.5.7.2 Scaling from Seedlings to Mature Trees
6 Because most experiments are conducted with seedlings, various methods are required to
7 scale experimental data on seedlings to mature trees. An overview of physiological differences
8 between young and old plants, and the consequences of these differences for O3 sensitivity, was
9 provided in Section AX9.3.5.3. The discussion below focuses on information relevant to
10 developing exposure-response relationships for mature trees. Information from a few
11 experimental studies, as well as scaling efforts based on physiological characteristics
12 incorporated into models, are discussed in Section AX9.6.
13 Although most studies continue to examine the effects of O3 on seedlings, during the 1990s
14 some studies examined the effects of O3 on the response of mature trees. Studies of mature trees
15 demonstrate differences in some aspects of O3 sensitivity between seedlings and mature trees.
16 For some species, such as red oak, seedlings are less sensitive to O3 than are mature trees
17 (Hanson et al., 1994; Samuelson and Edwards, 1993; Wullschleger et al., 1996). Both red oak
18 seedlings and genetically related mature trees were exposed to CF, 1 x -ambient, or 2 x-ambient
19 O3 exposures in OTCs in Tennessee for two growing seasons (Hanson et al., 1994). Nine large
20 chambers (4.6 x 8.2 m) were used to enclose individual mature trees and standard EPA-style
21 OTCs were used for potted seedlings. Ozone exposures expressed as a 24-h SUMOO were 34,
22 79, and 147 ppm-h in 1992 and 37, 95, 188 ppm-h in 1993 for the sub-ambient, and 2x-ambient
23 treatments. Mature trees had a greater light-saturated net photo synthetic rate and stomatal
24 conductance compared to seedling foliage at physiological maturity. By the end of the growing
25 season, exposure to 1 x-ambient and 1 x-ambient O3 reduced the light-saturated net
26 photosynthetic rate and stomatal conductance of mature trees by 25 and 50%, respectively,
27 compared with the CF treatment (35 ppm-h). In seedlings, however, light-saturated net
28 photosynthetic rate and stomatal conductance were less affected by O3 exposure. The authors
29 concluded that extrapolations of the results of seedling-exposure studies to foliar responses of
30 mature forests without considering differences in foliar anatomy and stomatal response between
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1 juvenile and mature foliage may introduce large errors into projections of the O3 responses of
2 mature trees.
3 In a study of ponderosa pine in California, seedlings and branches of mature trees (in
4 branch chambers) were exposed to O3 concentrations of 0.5-, 1-, and 2x-ambient O3
5 concentrations (Momen et al., 1997). Net photosynthetic rate of 1-year-old, but not current-year,
6 foliage was reduced in mature trees but not significantly reduced in seedlings. This effect was
7 not due to alteration of stomatal conductance by O3. This result contrasts with those with earlier
8 studies of red spruce (Rebbeck et al., 1993).
9 In contrast to the findings for red oak and Ponderosa pine, giant sequoia seedlings had
10 higher rates of stomatal conductance, CO2-exchange rate, and dark respiration than did mature
11 trees (Grulke and Miller, 1994). As compared to older trees, stomatal conductance was more
12 than 7-fold greater in current-year, and 4-fold greater in 2-year-old, seedlings (Grulke and
13 Miller, 1994). The authors concluded that giant sequoia seedlings are sensitive to atmospheric
14 O3 until ~5 years of age. Low conductance, high water use efficiency, and compact mesophyll
15 all contribute to a natural O3 tolerance, or to O3 defense, or to both, in the foliage of older trees.
16 Similarly, lower stomatal conductance was found in mature Norway spruce in Austria
17 compared to seedlings grown with optimal water and nutrients in a growth chamber (Wieser,
18 1997). In this study, net photosynthetic rate was less sensitive to added O3 in mature trees
19 compared to seedlings. In a related study, the average rate of O3 uptake of 17-year-old trees
20 -0.6 nmol nT2 s"1, decreasing linearly in older trees, such that rates were only ~0.1 nT2 s"1 in
21 216-year-old trees (Wieser et al., 1999).
22 Based on a review of studies of stomatal conductance in both seedlings and mature trees,
23 Samuelson and Kelly (2001) concluded that O3 uptake in oak species, black cherry, sugar maple,
24 and American beech averaged 47% lower in potted seedlings than in mature trees. For evergreen
25 species, they concluded that O3 uptake in seedlings averaged 26% higher than in mature trees.
26 They also suggested that artifacts introduced by growth in pots confound these differences that
27 exposure-response functions derived from seedlings grown in situ are more applicable to mature
28 trees than are studies of seedlings grown in pots (Samuelson and Kelly, 2001).
29 As discussed above for annual vegetation, it has long been noted that internal O3 dose is
30 more appropriate than external O3 exposure for assessing the effects of O3 on vegetation, because
31 effects occur primarily via the uptake of O3 through the stomata (Section AX9.2.2). However,
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1 external O3 exposure sometimes has been shown to explain O3 effects as well or better than
2 calculated internal O3 dose. For ponderosa pine, Grulke and others (2002) found little difference
3 in the response of net photosynthetic rate and stomatal conductance to O3 exposure as compared
4 to calculated O3 uptake; and estimated O3 uptake by ponderosa pine and O3 exposure at several
5 sites were highly correlated (r2 = 0.92). For red oak, Hanson and others (Hanson et al., 1994)
6 found that SUMOO explained 83% of the variance in the response of light-saturated
7 photosynthetic rate to O3 levels, while estimated internal dose explained only 76% of the
8 variance. In this same study, SUM06 explained only 49% of the variance. Due to genetic
9 variation or other factors, individual mature trees will vary in their response to similar O3
10 exposures. For example, in 125-year-old giant sequoia trees exposed to -230 ppm-h of O3 in
11 branch chambers, O3 uptake in one individual was ~5 mmol nT2, while in another it was
12 -9.5 mmol nT2 (Grulke et al., 1996).
13 Based on these results, stomatal conductance, O3 uptake, and O3 effects cannot be assumed
14 to be equivalent in seedlings and mature trees. In general, mature deciduous trees are likely to
15 be more sensitive to O3 compared to seedlings, while mature evergreen trees are likely to be less
16 sensitive than seedlings. However, even when differences in physiological traits occur,
17 concomitant effects on stem growth may not be detected in the field. Additionally, complex
18 interactions may occur between environmental conditions and O3 responses; and artifacts may
19 occur for seedling studies, especially for seedlings grown in pots. Finally, competition between
20 species or genotypes within a species can either exacerbate or ameliorate the effects of O3. Such
21 effects are predicted by models of the growth of mixed species forests, as discussed in Section
22 AX9.6, and various interactions between competitive ability and O3 effects have been found for
23 aspen clones in the Wisconsin FACE study (McDonald et al., 2002). Section AX9.6 further
24 discusses issues that must be addressed when scaling data from individual mature trees to forests
25 and regions.
26
27 AX9.5.6 Studies with the Chemical EDU
28 The chemical EDU has been used with the goal of protecting plants from O3 effects
29 without controlling O3 exposure (Table AX9-20) (U.S. Environmental Protection Agency, 1986,
30 1996). As discussed in Section AX9.1.3.3, the use of EDU has the potential to be a low-cost,
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1 practical method of evaluating ambient O3 exposures on plants grown under natural conditions
2 without the limitations imposed by methodologies such as OTCs (Section AX9.1.3.3). However,
3 because EDU is phytotoxic, and may have effects on plants other than antioxidant protection, it
4 is crucial that the correct dosage for protection from O3 be determined without the direct effects
5 of EDU. For example, a study in Massachusetts applied EDU to foliage of field-grown bush
6 beans of two lines (SI 56, R123) and two cultivars (Bush Blue Lake 290, Bush Blue Lake 274)
7 (Elagoz and Manning, 2005a). EDU increased the above-ground biomass of one line but
8 decreased the above-ground biomass in the other line and in one of the cultivars. Other studies
9 have shown that EDU does not always have greater effects at higher O3 exposures (Ribas and
10 Penuelas, 2000; Tonneijck and Van Dijk, 1997). Such results suggest that it may be difficult to
11 quantify ambient O3 effects using EDU, because the amount of plant growth or yield expected at
12 a low (background) O3 concentration cannot be inferred from EDU-treated plants grown at
13 locations with higher O3 exposures. Unfortunately, although many studies with EDU have been
14 conducted in recent decades, very few have used multiple EDU application levels along with
15 multiple O3 exposures to characterize the EDU system for a given plant species. Therefore, the
16 text of this section focuses on how data from existing studies can be used for developing or
17 validating exposure-response relationships, rather than reviewing results of all individual studies.
18 Data from individual studies on O3 exposure, EDU application rates, and the effects of EDU are
19 presented in Table AX9-20. In addition to EDU, sodium erythorbate has been used in a few
20 studies as a protectant chemical. Since very few published studies have used sodium erythorbate
21 and attempts to establish appropriate doses for individual species are even more limited, the use
22 of this chemical is not reviewed here.
23 In summary, it is difficult to use data from existing EDU studies to develop exposure-
24 response relationships or to quantify the effects of ambient O3 exposure. Despite these
25 limitations, the EDU studies reviewed in previous criteria documents (U.S. Environmental
26 Protection Agency, 1986, 1996) and the more recent studies summarized in Table AX9-20
27 provide another line of evidence that ambient O3 exposures occurring in many regions of the
28 United States are reducing the growth of crops and trees.
29
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1 AX9.5.7 Summary
2 Data published during the last decade support the conclusions of previous criteria
3 documents that there is strong evidence that ambient concentrations of O3 cause injury and
4 damage to numerous common and economically valuable plant species. For annual vegetation,
5 the data summarized in Table AX9-16 show a range of growth and yield responses both within
6 and among species. Nearly all of these data were derived from studies in OTCs, with only two
7 studies using open-air systems in the UK (Ollerenshaw et al., 1999; Ollerenshaw and Lyons,
8 1999). It is difficult to compare studies that report O3 exposure using different indices, such as
9 AOT40, SUM06, or 7-h or 12-h mean values. However, when such comparisons can be made,
10 the results of more recent research confirm earlier results summarized in the 1996 O3 AQCD
11 (U.S. Environmental Protection Agency, 1996). A summary of earlier literature concluded that a
12 7-h, 3-month mean of 49 ppb corresponding to a SUM06 exposure of 26 ppm-h would cause
13 10% loss in 50% of 49 experimental cases (Tingey et al., 1991). More recent data summarized
14 in Table AX9-16 support this conclusion, and more generally indicate that ambient O3 exposures
15 can reduce the growth and yield of annual species. Some annual species such as soybean are
16 more sensitive, and greater losses may be expected (Table AX9-16). Thus, the more recent
17 scientific literature supports the conclusions of the 1996 O3 AQCD (U.S. Environmental
18 Protection Agency, 1996) that ambient O3 concentrations are probably reducing the yield of
19 major crops in the United States.
20 Much research in Europe has used the AOT40 exposure statistic, and substantial effort has
21 gone into developing Level-1 critical levels for vegetation using this index. Based on regression
22 analysis of 15 OTC studies of spring wheat, including one study from the United States and
23 14 from locations ranging from southern Sweden to Switzerland, an AOT40 value of 5.7 ppm-h
24 was found to correspond to a 10% yield loss, and a value of 2.8 ppm-h corresponded to a 5%
25 yield loss (Fuhrer et al., 1997). Because a 4 to 5% decrease could be detected with a 99%
26 confidence level, a critical level of an AOT40 value of 3 ppm-h was selected in 1996
27 (Karenlampi and Skarby, 1996).
28 In addition to likely reductions in crop yield, O3 may also reduce the quality or nutritive
29 value of annual species. Many studies have shown effects of O3 on various measures of plant
30 organs that affect quality, with most studies focusing on characteristics important for food or
31 fodder. These studies indicate that there may be economically important effects of ambient O3
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1 on the quality of crop and forage species. Previous criteria documents have concluded that
2 visible symptoms on marketable portions of crops and ornamental plants can occur with seasonal
3 7-h mean O3 exposures of 40 to 100 ppb (U.S. Environmental Protection Agency, 1978, 1986,
4 1996). The more recent scientific literature does not refute this conclusion.
5 The use of OTCs may reverse the usual vertical gradient in O3 that occurs within a few
6 meters above the ground surface (Section AX9.1). This reversal suggests that OTC studies may
7 somewhat overestimate the effects of an O3 concentration measured several meters above the
8 ground. However, such considerations do not invalidate the conclusion of the 1996 AQCD (U.S.
9 Environmental Protection Agency, 1996) that ambient O3 exposures (Tables AX9-13 and
10 AX9-21) are sufficient to reduce the yield of major crops in the United States.
11 As for single-season agricultural crops, yields of multiple-year forage crops are reduced
12 at O3 exposures that occur over large areas of the United States. This result is similar to that
13 reported in the 1996 O3 AQCD (U.S. Environmental Protection Agency, 1996). When species
14 are grown in mixtures, O3 exposure can increase the growth of O3-tolerant species and
15 exacerbate the growth decrease of O3-sensitive species (e.g., Ashmore and Ainsworth, 1995;
16 Fuhrer et al., 1994). Because of this competitive interaction, the total growth of the mixed-
17 species community may not be affected by O3 exposure (Ashmore and Ainsworth, 1995; Barbo
18 et al., 1998; Fuhrer et al., 1994). However, in some cases, mixtures of grasses and clover species
19 have shown significant decreases in total biomass growth in response to O3 exposure in studies
20 in the United States (Heagle et al., 1989; Kohut et al., 1988) and in Sweden (Pleijel et al., 1996).
21 In Europe, a provisional critical level for herbaceous perennials of an AOT40 value of 7 ppm-h
22 over 6 months has been proposed to protect sensitive plant species from adverse effects of O3.
23 For deciduous tree species, recent evidence from free air exposure systems and OTCs
24 supports results observed previously in OTC studies. For example, a series of FACE studies
25 undertaken in Rhinelander, WI (Isebrands et al., 2000, 2001) showed that O3-symptom
26 expression was generally similar in OTCs, FACE, and also at sites along an ambient O3 gradient,
27 supporting the previously observed variation among aspen clones using OTCs (Karnosky et al.,
28 1999). This study also found no effects on sugar maple growth after 3 years, but in years 4 to 7
29 found significant growth reduction due to O3 (Karnosky et al., 2005). These results are important
30 because, they indicate results obtained from OTCs are supported by results from free air
31 exposure systems and also that more than 3 years may be required to adequately investigate
August 2005 AX9-299 DRAFT-DO NOT QUOTE OR CITE
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1 effects of O3 on the growth of tree species. As has been observed in previous criteria documents,
2 root growth often is found to be the most sensitive biomass response indicator to O3.
3 Results reported since 1996 support the conclusion of the 1996 O3 AQCD (U.S.
4 Environmental Protection Agency, 1996) that deciduous trees are generally less sensitive to O3
5 than are most annual plants, with the exception of a few very sensitive genera such as Populus
6 and sensitive species such as black cherry. However, the data presented in Table AX9-18
7 suggest that ambient O3 exposures that occur in the United States can potentially reduce the
8 growth of seedlings of deciduous species. Results from multiple-year studies sometimes show a
9 pattern of increased effects in subsequent years. In some cases, however, growth decreases due
10 to O3 may become less significant or even disappear over time. While some mature trees show
11 greater O3 sensitivity in physiological parameters such as net photosynthetic rate than do
12 seedlings, these effects may not translate into measurable reductions in biomass growth.
13 However, because even multiple-year experiments do not expose trees to O3 for more than a
14 small fraction of their life span, and because competition may in some cases exacerbate the
15 effects of O3 on individual species, determining effects on mature trees remains a significant
16 challenge.
17 In Europe, a Level I critical level has been set for forest trees based on OTC studies of
18 European beech seedlings. A critical level was defined as an AOT40 value of 10 ppm-h for
19 daylight hours for a 6-month growing season (Karenlampi and Skarby, 1996). However, other
20 studies show that some species such as silver birch may be more sensitive to O3 compared to
21 beech (Paakkonen et al., 1996).
22 For evergreen tree species, as for other tree species, the O3 sensitivity of different
23 genotypes and different species varies widely. Based on studies with seedlings in OTCs, major
24 species in the United States are generally less sensitive than are most deciduous trees, and
25 slower- growing species are less sensitive than are faster-growing species. Interacting stresses
26 such as competition stress may increase the sensitivity of trees to O3. As for deciduous species,
27 most experiments with evergreen species have only covered a small portion of the life span of a
28 tree and have been conducted with seedlings, making estimating effects on mature trees difficult.
29 For all types of perennial vegetation, cumulative effects over more than one growing
30 season may be important; furthermore, studies for only a single season may underestimate
31 effects. Mature trees may be more or less sensitive to O3 than are seedlings, depending on the
August 2005 AX9-300 DRAFT-DO NOT QUOTE OR CITE
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1 species, but information on physiological traits may be used to predict some such differences. In
2 some cases, mature trees may be more sensitive to O3 than seedlings due to differences in their
3 gas exchange rates, growth rates, greater cumulative exposures, or due to the interaction of O3
4 stress with other stresses.
5
6
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53
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1 AX9.6 EFFECTS OF OZONE EXPOSURE ON NATURAL ECOSYSTEMS
2 AX9.6.1 Introduction
3 The preceding section on species-level responses (AX9.5) provides a lead-in to address the
4 response of ecosystems to O3. The conclusion of the 1996 O3 AQCD (U.S. Environmental
5 Protection Agency, 1996) was that aside from the results from the San Bernardino NF, there was
6 no direct evidence that O3 is altering natural ecosystems in the United States. This conclusion is
7 generally valid today, except that our understanding of the effects of O3 in the San Bernardino
8 NF has been tempered by additional understanding of the complicating role that N deposition
9 plays in this system. Despite the lack of any new, direct information linking O3 with ecosystem
10 changes, numerous publications since 1996 have highlighted ways in which O3 may affect
11 ecosystem structure and/or function. This section addresses new and (where appropriate) older
12 literature in order to illustrate possible shifts in energy or material flow through ecosystems as a
13 result of O3 exposure.
14 An ecosystem is defined as comprising all of the organisms in a given area interacting with
15 the physical environment, so that a flow of energy leads to a clearly defined trophic structure,
16 biotic diversity, and cycling of materials between living and nonliving parts (Odum, 1963).
17 Individuals within a species and populations of species are the building blocks from which
18 communities and ecosystems are constructed. Classes of natural ecosystems, e.g., tundra,
19 wetland, deciduous forest, and conifer forest, are distinguished by their dominant vegetation
20 forms. Ecosystems boundaries are delineated when an integral unit is formed by their physical
21 and biological parts. Defined pathways for material transport and cycling and for the flow of
22 energy are contained within a given integrated unit.
23 Each level of organization within an ecosystem has functional and structural
24 characteristics. At the ecosystem level, functional characteristics include, but are not limited, to
25 energy flow; nutrient, hydrologic, and biogeochemical cycling; and maintenance of food chains.
26 The sum of the functions carried out by ecosystem components provides many benefits to
27 mankind, as in the case of forest ecosystems (Smith, 1992). These include food, fiber
28 production, aesthetics, genetic diversity, and energy exchange.
29 Ecosystems are functionally highly integrated. Changes in one part of an ecosystem, such
30 as the primary producer component, may have consequences for connected parts, such as the
31 consumer and decomposer components. For example, when needles are shed prematurely as a
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1 result of O3 exposure, successional development of phyllosphere fungi inhabiting the surface of
2 Ponderosa pine needles may be truncated (Bruhn, 1980). In addition, decomposer populations in
3 the litter layer may be capable of higher rates of decomposition, due to the higher N content of
4 the younger age classes of needles falling from O3-damaged pines (Fenn and Dunn, 1989).
5 Because ecological systems integrate the effects of many influences, the results of O3 exposure
6 may depend on co-occurring influences that predispose an ecosystem to stress (Colls and
7 Unsworth, 1992). One important change in our thinking since the 1996 O3 AQCD is that, at the
8 high levels of O3 exposure that are known to result in detectable plant responses (>250 ppm h
9 accumulated over a growing season), N deposition must also be considered as a concurrent
10 stressor. Both O3 exposure and increased N deposition can cause changes in N cycling and
11 compartmentalization within ecosystems.
12 The vast majority of O3-effects literature addresses individual species responses (see
13 Section AX9.6.4.3), as was also true in 1996 (U.S. Environmental Protection Agency, 1996).
14 This section differs from the preceding one in that the physiological stress of individual species
15 is considered only within the context of its natural ecosystem. Changes in function at the
16 individual level propagate through the higher levels of organization, resulting in changes in
17 ecosystem structure and function. However, since ecosystem-level responses result from the
18 interaction of organisms with one another and with their physical environment, it takes longer
19 for a change to develop to a level of prominence at which it can be identified and measured. The
20 paucity of scientific literature on O3 effects at the ecosystem level is a result of both the
21 complexity of ecological systems, and long response times. In addition, "indirect" effects of O3
22 on plants (e.g., effects that alter the plants' ability to integrate environmental stresses) may be
23 more important than the direct effects on photosynthesis and respiration at the leaf level
24 (Johnson and Taylor, 1989).
25 A conceptual framework (see Table AX9-22) for discussing the effects of O3 on
26 ecosystems was developed by the EPA Science Advisory Board (Young and Sanzone, 2002).
27 Their six essential ecological attributes (EEAs) include landscape condition, biotic condition,
28 organism condition, ecological processes, hydrological and geomorphological processes, and
29 natural disturbance regimes (see Table AX9-22). The major ecological effects of O3, and gaps in
30 our knowledge of O3 exposure effects at the ecosystem level, are summarized at the end of this
31 chapter. While the main focus is O3 effects newly described since the 1996 O3 AQCD (U.S.
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Table AX9-22. Essential Ecological Attributes for Natural Ecosystems Affected by O3
Category
Species
Condition Measures
References
Landscape Condition
• Habitat Types
Biotic Condition
• Ecosystems and
Communities
Community Extent
and Composition
Trophic Interactions
Insects
Mixed conifer forest
Pinus ponderosa
Community composition,
Stand structure
Relative abundance
Grassland communities Species composition
Coastal sage scrub
Early successional plant
community
Populus tremuloides
and Betula papyrifera
Pinus ponderosa
Pinus taeda
Pinus ponderosa
Pinus ponderosa
Populus tremuloides
Populus tremuloides
Populus tremuloides
Species cover, richness,
equitability
Species richness, diversity,
evenness
Soil microbial community
Soil microbial community
Fungal morphotypes
Bark beetle severity
Bark beetle productivity and
predator/parasitoid density
Blotch leaf miner
performance
Aphid/natural enemy
abundance
Forest tent caterpillar/
paratisoid performance
Miller etal. (1989)
Miller and McBride (1999a)
Miller (1973);
Arbaugh et al. (2003)
Ashmoreetal. (1995);
Ashmore and Ainsworth (1995)
Westman( 1979, 1981)
Barbo etal. (1998)
Phillips et al. (2002)
Scagel and Andersen (1997)
Edwards and Kelly (1992);
Qui etal. (1993)
Cobb etal. (1968)
Dahlstenetal. (1997)
Kopper and Lindroth (2003a)
Percy et al. (2002)
Percy et al. (2002);
Holton et al. (2003)
Diseases
Populus hybrids
Populus hybrids
Populus tremuloides
Picea abies and
Picea sitchensis
Pinus ponderosa
Pinus taeda
Pinus sylvestrisl
mycorrhizae
Septoria occurrence
Rust occurrence
Rust occurrence
Needle fungi
Woodbury etal. (1994)
Beareetal. (1999a)
Karnosky et al. (2002)
Maganetal. (1995)
Root disease x O3 interactions Fenn et al. (1990)
Canker dimensions Carey and Kelley (1994)
Disease susceptibility Bonello et al. (1993)
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Table AX9-22 (cont'd). Essential Ecological Attributes for Natural Ecosystems
Affected by O3
Category Species
Community Dynamics Pinus ponderosa/Abies
concolor/Calocedrus
decurrens
Populus tremuloides
Pinus ponderosa/
Elymus glaucus
Pinus taeda/diverse
community
• Species and Populations
Population Size Pinus strobus
Pinus ponderosa
Genetic Diversity/ Lupinus bicolor
Population Structure
Populus tremuloides
Trifolium repens
Plantago major
Population Dynamics Trifolium repens
Plantago major
Condition Measures
Abundance
Competitive status
O3 sensitivity
Tree growth
Mortality
Mortality
% population sensitive
% population sensitive
% population sensitive
% population sensitive
Adaptation
Population changes over time
References
Minnichetal. (1995)
McDonald et al. (2002)
Andersen et al. (2001)
Barbo et al. (2002)
Kamosky(1981)
Carroll et al. (2003)
Dunn (1959)
Berrang etal. (1986, 1989, 1991)
Heagleetal. (1991)
Davison and Reiling (1995);
Reiling and Davison (1992b);
Lyons etal. (1997)
Heagleetal. (1991)
Davison and Reiling (1995)
Organism Condition
• Visible Symptoms
Pinus ponderosa
Foliar symptoms
Pinus Jeffreyi
Foliar symptoms
Prunus serotina
Foliar symptoms
Liriodendron tulipfera Foliar symptoms
Sassafras albidum
Foliar symptoms
Grulke and Lee (1997);
Arbaughetal. (1998);
Salardino and Carroll (1998);
Temple etal. (1992)
Grulke et al. (2003b)
Patterson and Rundel (1995);
Salardino and Carroll (1998);
Fredericksen et al. (1995, 1996);
Chappelka et al. (1997, 1999a,b)
Hildebrandetal. (1996);
Ghosh et al. (1998); Lee et al.
(1999); Ferdinand et al. (2000);
Schaub et al. (2003)
Yuska et al. (2003); Somers et al.
(1998); Hildebrand et al. (1996)
Chappelka et al. (1999a,b)
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Table AX9-22 (cont'd). Essential Ecological Attributes for Natural Ecosystems
Affected by O3
Category
Species
Condition Measures
References
Organism Condition (cont'd)
Visible Symptoms
(cont'd)
Physiological Status
Populus nigra,
Fraxinus excelsior and
Prunus avium
Fagus sylvatica
Fraxinus americana
Grassland species
Herbaceous species
Asclepias exaltata
Rudbeckia laciniata and
Verbesina occidentalis
Asclepias incarnata
Pinus halepensis
Populus tremuloides
Be tula pendula
Fagus sylvatica
Populus tremuloides
Pinus ponderosa
Festiva ovina
Betula pubescens
Populus tremuloides x
Populus tremula
Populus tremuloides
Pinus ponderosa
Betula pendula
Betula pendula
Acer saccharum
Foliar symptoms
Foliar symptoms
Foliar symptoms
Foliar symptoms
Foliar symptoms
Foliar symptoms
Foliar symptoms
Foliar symptoms
Allometry
Crown architecture
Crown architecture
Crown architecture
Root dry weight
Root/shoot ratio
Root/shoot ratio
Root/shoot ratio
Root/shoot ratio
Leaf area index
Carbon allocation to
mycorrhizae
Decreased winter bud
formation
Delayed bud break
Early bud break
Chappelkaetal. (1999a);
Novak et al. (2003)
Gerosa et al. (2003);
Vollenweider et al. (2003b)
Schaub et al. (2003)
Bungener et al. (1999a)
Bergmann et al. (1999)
Chappelkaetal. (1997)
Chappelka et al. (2003)
Orendovici et al. (2003)
Wellburn and Wellburn (1994)
Dicksonetal. (2001)
Kulletal. (2003)
Stribley and Ashmore (2002)
Colemanetal. (1996)
Grulkeetal. (1998a);
Grulke and Balduman (1999)
Warwick and Taylor (1995)
Mortensen(1998)
Landolt et al. (2000);
Paludan-Muller et al. (1999)
Oksanenetal. (2001)
Neufeld et al. (1995);
Wiltshire et al. (1996);
Andersen and Rygiewicz
(1995a,b)
Karnosky et al. (2003a)
Oksanen (2003a,b)
Prozherina et al. (2003);
Bertrand et al. (1999)
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Table AX9-22 (cont'd). Essential Ecological Attributes for Natural Ecosystems
Affected by O3
Category
Species
Condition Measures
References
Reproductive Status
Apocynun
androsaemifolium
Buddleia davidii
Rubus cuneifolius
Plantago major
Fragaria x ananassa
Plantago major
Understory herbs
Flowering time
Flowering time
Pollen germination
Pollen tube elongation
Fruit yield
Seed yield
Seed yield
Bergweiler and Manning (1999)
Findleyetal. (1997)
Chappelka (2002)
Stewart (1998)
Drogoudi and Ashmore
(2000,2001)
Lyons and Barnes (1998);
Pearson et al. (1996);
Reiling and Davison (1992a);
Whitfield et al. (1997)
Harward and Treshow (1975)
Ecological Processes
• Energy Flow
Primary Production Pinus ponderosa
Pinus ponderosa
Populus tremuloides
Photosynthesis
Needle retention
Photosynthesis
Be tula pendula
Betula pendula
Quercus rubra
Populus tremuloides
Pinus ponderosa
Quercus rubra
Photo sy nthe sis/conductance
Stem respiration and radial
growth
Root turnover
Soil respiration
Soil respiration
Carbon partitioning and
allocation
Populus ire muloides Carbon allocation
Miller et al. (1969); Clark et al.
(1995); Takemoto et al. (1997);
Grulke et al. (2002b)
Temple et al. (1993)
Colemanetal. (1995ab);
Noormets et al. (2001a,b);
Sharma et al. (2003);
Karnosky et al. (2003a);
Oksanen (2003a,b)
Matyssek et al. (2002)
Keltingetal. (1995)
Colemanetal. (1996)
King etal. (2001);
Andersen and Scagel (1997);
Colemanetal. (1995a)
Scagel and Andersen (1997);
Andersen (2000); Samuelson
and Kelly (1996)
Andersen et al. (1997);
Grulke etal. (1998a)
Grulke and Balduman (1999)
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Table AX9-22 (cont'd). Essential Ecological Attributes for Natural Ecosystems
Affected by O3
Category
Primary Production
(cont'd)
Net Ecosystem
Production
Growth Efficiency
Species
Pinus ponderosa
Betula pendula
Fragaria vesca
Pinus taeda
Lespedeza cuneata and
Schizacbyrium
scoparium
Liriodendron tulipfera
Prunus serotina
Pinus Jeffrey i
Pinus ponderosa
Pinus strobus
Pinus taeda
Fagus sylvatica
Picea abies
Populus tremuloides
Pinus ponderosa
Northern hardwoods
Northern hardwoods
Plantago major
Grassland species
Native herbs
Grasses and herbs
Populus tremuloides
Condition Measures
Carbon allocation
Carbon allocation
Carbon allocation
Root respiration
Yield
Radial growth
Radial growth
Radial growth
Radial growth (no effect)
Radial growth
Radial growth
Stem volume
Stem volume
Volume growth
Root growth
NPP estimates
Biomass estimates
Relative growth rate
Relative growth rate
Relative growth rate
Relative growth rate
Relative growth rate
References
Grulkeetal. (2001)
Karlsson et al. (2003);
Oksanen and Saleem (1999);
Saleemetal. (2001)
Manninen et al. (2003)
Edwards (1991)
Powell et al. (2003)
Somerset al. (1998);
Vollenweider et al. (2003a)
Somerset al. (1998)
Peterson et al. (1987)
Peterson et al. (1993)
Bartholomay et al. (1997)
McLaughlin and Downing
(1995; 1996)
Braunetal. (1999)
Wallm et al. (2002)
Isebrands et al. (2001)
Andersen et al. (1991)
Laurence et al. (2000)
Hogsettetal. (1997)
Davison and Reiling (1995);
Lyons et al. (1997); Reiling and
Davison (1992b); Davison and
Barnes (1998)
Bungener et al. (1999b)
Warwick and Taylor (1995)
Pleijel and Danielsson (1997)
Yun and Laurence (1999)
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Table AX9-22 (cont'd). Essential Ecological Attributes for Natural Ecosystems
Affected by O3
Category
Growth Efficiency
(Cont'd)
• Material Flow
Organic Carbon
Cycling
Nitrogen Cycling
Other Nutrient
Cycling
Species
Fagus sylvatica
Picea abies
Prunus serotina
Populus tremuloides
and Betula papyrifera
Andropogon virginicus
and Rubus cuneifolius
Liriodendron tulipera
Populus deltoides
Pinus ponderosa
Pinus sylvestris
Pinus ponderosa
Pinus ponderosa
Pinus taeda
Prunus serotina and
Liriodendron
tulip/era
Picea sitchensis and
Pinus sylvestris
Pinus ponderosa
Condition Measures
Relative growth rate
Relative growth rate
Relative growth rate
Altered foliar C:N ratio and
N resorption efficiency
Litter decomposition rate
Litter decomposition rate
Litter decomposition rate
Litter decomposition rate
Litter decomposition
(no effect)
Altered foliar N
Foliar N and O3 exposure
no effects
Altered foliar N metabolism
Altered foliar N
Foliar leaching no effect
Nutrient availability and O3
References
Bortier et al. (2000a)
Karlsson et al. (2002)
Lee et al. (2002)
Lindroth et al. (2001)
Kim etal. (1998)
Scherzer et al. (1998)
Findlay and Jones (1990)
Fenn and Dunn (1989)
Kainulainen et al. (2003)
Momen and Helms (1996)
Bytnerowicz et al. (1990)
Manderscheid et al. (1992)
Boerner and Rebbeck (1995)
Skeffington and Sutherland
(1995)
Bytnerowicz et al. (1990)
Hydrology and Geomorphology
• Water Budget
Picea rubens
Pinus armandi
Pinus jeffreyi
Picea abies
Fraxinus excelsior
Betula pendula
Populus hybrids
Water-use efficiency
no effect
Water-use efficiency
Canopy transpiration
Transpiration
(xylem sap flow)
Water stem flow
Water-use efficiency
Water-use efficiency
Laurence et al. (1997)
Shan etal. (1996)
Grulke et al. (2003a)
Maier-Maercker (1997)
Wiltshire et al. (1994)
Maurer and Matyssek (1997)
Reich and Lassoie (1984)
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Table AX9-22 (cont'd). Essential Ecological Attributes for Natural Ecosystems
Affected by O3
Category
Species
Condition Measures
References
Natural Disturbance Regimes
• Frequency Pinus ponderosa
Intensity
Extent
Duration
Pinus ponderosa
Picea sitchensis
Pinus halepensis
Picea rubens
Fagus sylvatica
Picea abies
Pinus ponderosa
Pinus ponderosa
Pinus ponderosa
Frequency of fire
Occurrence of bark beetle
outbreaks
Winter damage
Reduced winter damage
Freezing tolerance
Drought stress
Drought stress
Fire intensity
Extent of bark beetle attack
Duration of bark beetle attack
McBride and Laven (1976);
Minnichetal. (1995);
Miller and McBride (1999)
Pronosetal. (1999);
Dahlsten et al. (1997)
Lucas etal. (1988)
Wellburn and Wellburn (1994)
Waiteetal. (1994)
Pearson and Mansfield
(1993, 1994)
Maier-Maercker (1998);
Maier-Maercker and
Koch (1992)
Miller and McBride (1999a)
Minnichetal. (1995)
Minnichetal. (1995)
1
2
3
4
5
6
7
8
9
10
11
12
13
Environmental Protection Agency, 1996), many key historical papers are cited to demonstrate
ecosystem response, particularly where they remain the only examples in the literature.
Although the vast majority of published studies focus on individuals, six case studies (five
field examples and one FACE experiment) have measured several ecosystem components
simultaneously to better understand the overall ecosystem response to O3. We provide an
overview of these six studies up-front to provide a context for the subsequent discussion on
possible ecosystem effects on an EEA basis.
AX9.6.2 Case Studies
AX9.6.2.1 Valley of Mexico
The first evidence of air pollution impacts on vegetation in the Valley of Mexico (Mexico
City Air Basin) were observations of foliar injury symptoms in bioindicator plants attributed
to O3, PAN, SO2, and possibly other pollutants (De Bauer, 1972). Subsequently, O3 injury to
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1 foliage and crowns of pine trees were reported in forests to the south and southwest of Mexico
2 City (De Bauer and Hernandez-Tejeda, 1986; De Bauer and Krupa, 1990). Ozone is considered
3 to be the pollutant with the most severe impacts on vegetation within the Mexico City urban
4 zone and in forests downwind of the city. Pinus hartwegii is the most O3-sensitive pine species
5 and is severely impacted by high O3 exposures encountered to the south/southwest of the Mexico
6 City metropolitan area (Miller et al., 1994). The potential for O3 injury is particularly high in
7 this area, because O3 levels are high during the summer rainy season when soil moisture
8 availability and stomatal conductance are greatest; these factors enhance O3 uptake and injury.
9 Decline of Abies religiosa (oyamel) in the Desierto de los Leones NP is a well-known example
10 of dramatic dieback and mortality of entire forest stands due primarily to air pollution stress
11 (Alvarado et al., 1993). Other factors, such as a lack of stand thinning, also contribute to forest
12 decline. Lead in automobile gasoline was phased out in 1990, and foliar concentrations of heavy
13 metals in forest species are not now at phytotoxic levels (Fenn and De Bauer, 1999). Sulfur
14 dioxide concentrations decreased in the early 1990s as a result of regulatory mandates limiting
15 their emissions. Sensitive plants in the northeast and northwest sectors of the Mexico City urban
16 zone where concentrations are highest may still be impacted by exposure to ambient SO2 levels.
17 Deposition of ionic forms of N and S are high in forested areas southwest of Mexico City.
18 However, the effects of these chronic nutrient inputs to the forest are only beginning to be
19 investigated and understood.
20 The ecological perturbations caused by severe air pollution exposures in forests located
21 downwind of Mexico City are expected to continue for the near future (the next 5 to 10 years),
22 largely as a result of high O3 concentrations as well as N oxides emissions. The longer-term
23 response is more uncertain and depends largely on the effectiveness of regulatory emissions
24 control strategies. Currently, pollutant levels are declining. Forest responses to this trend will
25 depend on how long it takes to reduce levels sufficiently to allow sensitive species to recover.
26 Some of the change to the ecosystem is probably irreversible, such as the loss of lichen diversity
27 and the loss of other O3-sensitive species (Zambrano and Nash, 2000).
28
29 AX9.6.2.2 San Bernardino Mountains
30 The San Bernardino Mountains lie east of the Los Angeles Air Basin in California, and
31 significant levels of pollution have been transported into the mountain range, including into a
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1 Class I wilderness area. The effects of O3 exposure on the mixed conifer forest of the
2 San Bernardino Mountains is perhaps the longest and most thoroughly documented O3
3 ecological effects evaluation (Miller and McBride, 1999a). In this classic case study linking
4 tropospheric O3 exposure to damage to an entire forest ecosystem (U.S. Environmental
5 Protection Agency, 1996) (Table AX9-23), Miller et al. (1963) first identified the unique foliar
6 chlorotic mottle that was occurring on two of the dominant tree species, Pinus ponderosa and
7 P.jeffreyi. Levels of O3 averaging 100 to!20 ppb over 24 h with 1-h peaks well into the 200 ppb
8 range were common in the region in the 1960s and 1970s (Miller and McBride, 1999a). Single-
9 hour peak values have declined in recent years due to heavily regulated pollution control
10 (Arbaugh et al., 1998; Lee et al., 2003; Takemoto et al., 2001).
11 Since the 1996 O3 AQCD (U.S. Environmental Protection Agency, 1996) was written, the
12 concurrent role of N deposition in modifying ecosystem response to O3 exposure in the San
13 Bernardino Mountains has been further elucidated (Arbaugh et al., 2003; Bytnerowicz et al.,
14 1999; Bytnerowicz, 2002; Fenn et al., 1996; Takemoto et al., 2001). Both O3 exposure and N
15 deposition reduce foliar retention (Grulke and Balduman, 1999) and alter tissue chemistry of
16 both needles and litter (Poth and Fenn, 1998). In addition, confounding factors such as drought
17 and fire suppression add to the complexity of ecosystem response (Arbaugh et al., 2003;
18 Minnich et al., 1995; Takemoto et al., 2001). Extensive crown injury measurements have also
19 been made, linking ambient O3 exposure data to chlorotic mottle and fascicle retention (Arbaugh
20 et al., 1998). Ozone exposure and N deposition reduce carbon allocation to stems and roots
21 (Grulke et al., 1998a, 2001), further predisposing trees to drought stress, windthrow, root
22 diseases, and insect infestation (Takemoto et al., 2001). Increased mortality of susceptible tree
23 species (Ponderosa and Jeffrey pine) has shifted community composition toward white fir and
24 incense-cedar (Abies concolor, Calocedrus decurrens) and has altered forest stand structure
25 (Miller et al., 1989) (Table AX9-25). Ozone exposure is implicated in projected changes in
26 stand composition (McBride and Laven, 1999) toward a predominance of oaks, rather than
27 mixed conifer forests. Forest understory species have also been affected (Temple, 1999). These
28 individual species responses collectively have affected trophic structure and food web dynamics
29 (Dahlsten et al., 1997; Pronos et al., 1999), as well as C and N cycling (Arbaugh et al., 2003)
30 (Table AX9-24).
31
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Table AX9-23. Case Studies Demonstrating the Ecological Effects of O3
Study
Keystone
Species
Study Type
Period
Studied
Key Ecological Findings
Valley of
Mexico
Pinus hartwegii,
Abies religiosa
Field transects
San Bernardino Pinus ponderosa, Field transects
Mountains P. jeffreyi
35 yrs • Significant foliar injury
(De Bauer, 1972; De Bauer and
Hernandez-Tejeda, 1986; De Bauer and
Krupa, 1990)
• Community composition changes
(Alvaradoetal, 1993)
• Species richness changes (Zambrano and
Nash, 2000)
40 yrs • Community composition changes
(Arbaugh et al., 2003; Miller, 1973;
Minnich et al., 1995)
• Population changes
(McBride and Laven, 1999)
• O3-pine-bark beetle interaction
(Pronosetal., 1999)
• Altered C flows
(Arbaugh et al., 1999; Grulke et al.,
1998a, 2001, 2002b; Grulke and
Balduman, 1999)
• Interaction of O3, drought, N deposition
(Fennetal., 1996; Grulke, 1999;
Takemoto et al., 2001)
• Altered carbon cycling
(Arbaugh et al., 1999)
Sierra Nevada
Mountains
Pinus ponderosa,
P. jeffreyi
Appalachian
Mountains
Fraxinus
americana,
Liriodendron
tulip/era,
Pinus strobus,
Prunus serotina
Field 35 yrs • Wide-scale nature of effects
(Edinger et al., 1972; Miller and
Millecan, 1971)
• Link to decreased growth
(Peterson et al., 1987, 1991, 1995)
• Quantification of O3 flux
(Bauer et al., 2000; Goldstein et al., 2003;
Paneketal.,2002)
• Cumulative O3 effects
(Takemoto et al., 1997)
• Canopy level responses
(Grulke etal., 2003a,b)
• Population changes (Carroll et al., 2003)
Field 25 yrs • Link of visible symptoms to growth
decreases (McLaughlin et al., 1982;
Somersetal., 1998)
• Wide-scale nature of effects (Chappelka
et al., 1999a; Hildebrand et al., 1996)
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Table AX9-23 (cont'd). Case Studies Demonstrating the Ecological Effects of O3
Study
Keystone
Species
Study Type
Period
Studied
Key Ecological Findings
Aspen FACE
Plantago
Carpathian
Mountains
Acer sacchamm,
Betula
papyri/era,
Populus
tremuloides
Plantago major
Pinus sylvestris,
Picea abies
Open-air O3 6 yrs • Competitive interactions
exposure (McDonald et al., 2002)
• O3-aspen-rust interaction
(Karnoskyetal, 2002)
• Plant-insect interactions
• (Holton et al., 2003; Percy et al., 2002)
• C and N cycling
• (King et al., 2001; Lindroth et al., 2001)
• Moderation of CO2 responses by O3
(Isebrands et al., 2001; Karnosky et al.,
2003b; McDonald et al., 2002;
Wustman et al., 2001)
Field 20 yrs • Population structure
• (Davison and Reiling, 1995;
• Lyons etal., 1997)
• O3 resistance
• (Reiling and Davison, 1992c)
• Adaptation (Davison and Reiling, 1995)
Field 15 yrs • Significant foliar injury
• Community composition changes
• Species diversity changes
1
2
3
4
5
9
10
11
12
13
AX9.6.2.3 Sierra Nevada Mountains
Like the San Bernardino Mountains, the western slope of the Sierra Nevada Mountains in
central and southern California has also been exposed to elevated O3 for a long time, although
the effects have been much less.. Symptoms of O3 injury have been found on Ponderosa and
Jeffrey pines in all of the Sierra Nevada national forests and parks (Carroll et al., 2003). First
identified as a problem in the 1970s (Miller et al., 1972), elevated O3 with daytime means of
60-80 ppb are common (Bauer et al., 2000; Bohm et al., 1995; Bytnerowicz et al., 2002c; Panek
et al., 2002). The west-slope Sierra Nevada forests are also exposed to a wide range of
additional gaseous and paniculate pollutants, including various S and N compounds
(Bytnerowicz et al., 1999; Fenn et al., 2003b; Takemoto et al., 2001), but at levels much lower
than in the San Bernardino Mountains. Typical O3-induced visible foliar symptoms, including
chlorotic mottle, chlorophyll degradation, and premature senescence, are commonly found
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Table AX9-24. The Most Comprehensively Studied Effects of O3 on Natural Ecosystem
are for the San Bernardino Mountain Forest Ecosystem. Citations Focus on Research
Published Since U.S. EPA (U.S. Environmental Protection Agency, 1996).
Pollutant Occurrence
Reference
O3 exposure and N deposition
Cellular, Biochemical
Foliar pigments
Antioxidants
Foliar Responses
Foliar Symptoms
Gas Exchange
Photosynthesis and Conductance
O3flux
Foliar nutrients
Whole Organism
Growth/Biomass
• Above-ground
• Below-ground
• Root/shoot ratio
• Carbon allocation
• Crown vigor
Ecosystem
Community dynamics/succession
Simulations
Understory vegetation
Pest interactions
• Bark beetle/predators
• Disease occurrence
• Litter decomposition
Disturbance
• Bark beetle occurrence
• Fire frequency
Fenn et al. (1996; 2000); Grulke et al. (1998a,
2003a); Bytnerowicz et al. (1999)
Grulke and Balduman (1999); Grulke and Lee
(1997); Tausz et al. (1999a,b,c, 2001)
Arbaugh et al. (1998); Grulke and Lee (1997);
Miller and Rechel (1999)
Grulke (1999); Grulke et al. (2002a,b);
Grulke and Retzlaff (2001)
Grulke and Balduman (1999)
Grulke et al. (1998a); Grulke and Balduman
(1999); Grulke and Balduman (1999);
Grulke et al. (2001); Arbaugh et al. (1998);
Miller and Rechel (1999)
Arbaugh et al. (2003); Arbaugh et al. (1999);
McBride and Laven (1999)
Temple (1999)
Dahlsten et al. (1997); Pronos et al. (1999)
Miller and Rechel (1999); Pronos et al. (1999)
Miller and McBride (1999b); Minnich et al.
(1995); Minnich (1999)
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Table AX9-25. Effects of Ozone, Ozone and N Deposition, and Ozone and Drought
Stress on Pinusponderosa andPinus jeffreyi in the Sierra Nevada and the
San Bernardino Mountains, California. Citations are Focused on Research
Published since U.S. EPA (1996).
O3 O3 + N deposition
Foliar Biochemistry and
Tissue Chemistry
Total ascorbate d1
Dehydroascorbate i
Total glutathione d
Oxidized glutathione i
a Carotenoids i
Foliar nitrogen d
C:N ratio of foliage2 i
Starch n.d.
Chlorophyll content d
Gas Exchange
Amax lower canopy n.d.
A,^ whole canopy d
A^x seedlings di
Stomatal limitation n.d.
Stomatal conductance d
Foliar respiration n.s.
O3 flux d
d
n.d.
i
i
n.d.
i
ad.
d
id
i
ad.
ad.
ad.
di
i
n.s.
O3 + Drought References
i
d
d
d
d
d
d
i
d
d
d
ad.
i
d
d
d
Grulke et al. (2003b);
Tauszetal. (2001)
Grulke et al. (2003b)
Tauszetal. (2001)
Tauszetal. (2001)
Grulke et al. (2003b)
Grulke et al. (1998a); Grulke and
Lee (1997); Poth and Fenn (1998)
Poth and Fenn (1998)
Grulke et al. (2003b)
Grulke etal. (2001)
Grulke etal. (1998b, 2003b);
Grulke and Lee (1997);
Takemoto et al. (1997)(Grulke
(1999); Tauszetal. (2001)
Grulke (1999); Grulke etal.
(2002b); Grulke and Retzlaff
(2001); Panek (2004)
Grulke et al. (2003b);
Panek and Goldstein (2001)
Grulke and Retzlaff (2001)
Panek and Goldstein (2001)
Grulke (1999); Grulke et al.
(2003a); Panek (2004)
Grulke (1999);
Grulke et al. (2002a)
Panek et al. (2002, 2003);
Panek and Goldstein (2001)
Grulke et al. (2002a, 2004)
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Table AX9-25 (cont'd). Effects of Ozone, Ozone and N Deposition, and Ozone and
Drought Stress on Pinus ponderosa and Pinus jeffreyi in the Sierra Nevada and the
San Bernardino Mountains, California. Citations are Focused on Research
Published Since U.S. EPA (1996).
Growth and Productivity
Foliar biomass
Height growth
Bole diameter growth
Fine root biomass
03
ad.
ad.
d
d
O3 + N deposition
i
i
i
d
O3 + Drought
d
d
d
i
References
Grulke and Balduman (1999)
Grulke and Balduman (1999)
Grulke and Balduman (1999)
Grulke etal. (1998a)
Leaf Surfaces
Stomatalocclusion i n.d. n.d. Bytnerowiczetal. (1999);
Bytnerowicz and Turunen (1994)
Trophic Interactions
Bark beetle n.s. i i Pronos etal. (1999)
Ecosystem Level
Competitive indices n.d. d i Miller and Rechel (1999)
'Responses are shown as significant increases (i), significant decreases (d), both significant decreases and
increases reported (di), nonsignificant effects (n.s.), and no data (n.d.) compared to trees or seedlings at field sites
with lower O3, drought stress, or lack of significant N deposition (<10 kg ha"1 yr"1). Frequently n.d. was used
for lack of a control site without compounding high N deposition. Foliar analyses and leaf surface properties
were largely determined from previous year needles. Gas exchange data were generally from previous year
needles at peak growing season, prior to late summer drought (mid- to late July).
Abbreviations: C = carbon; N = nitrogen; A^ = maximum photosynthesis rate.
1 on O3-sensitive genotypes of Ponderosa pine (Arbaugh et al., 1998; Peterson et al., 1991; Staszak
2 et al., 2004) and Jeffery pine (Arbaugh et al., 1998; Grulke et al., 2003b; Patterson and Rundel,
3 1995; Peterson et al., 1987). Other important conifers in the region, such as giant sequoia,
4 appear to be relatively O3-tolerant (Grulke et al., 1996). The symptoms of foliar injury and
5 growth reductions have been verified on seedlings in O3 exposure chambers (Momen et al.,
6 2002; Momen and Helms, 1996; Temple, 1988).
7 Ozone foliar injury of dominant pine species in the Sierra Nevada Mountains is correlated
8 to decreased radial growth in both Ponderosa pine (Peterson et al., 1991) and Jeffrey pine
9 (Patterson and Rundel, 1995; Peterson et al., 1987). Because of the large amount of intraspecific
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1 variation in O3 sensitivity in these two species, O3 exposure may be a selective agent (Patterson
2 and Rundel, 1995), with differential mortality rates for sensitive individuals (Carroll et al.,
3 2003). The region's forests may also be experiencing subtle changes in species composition and
4 community structure (Patterson and Rundel, 1995; Takemoto et al., 2001).
5 Based on fire scar dating, reconstructions of stand age classes, historical records, and
6 present stand structure, fire has been largely excluded in western forests for the last 75 to 100
7 years (Minnich, 1999; Minnich and Padgett, 2003). Fire exclusion has resulted in fewer large
8 stand-replacing fires rather than a mosaic of smaller low-intensity fires. The change in fire
9 intensity may have selectively altered stand structure, fitness and competitiveness of component
10 species, along with their susceptibility to atmospheric pollutants and other stressors (Minnich,
11 1999). Short-lived (50 to 80 years) species such as knobcone and Coulter pine (Pinus coulteri D.
12 Don), which occur at the interface of the chaparral and the mixed conifer forest, may already
13 have been selected for O3 tolerance by seedling establishment (the most sensitive tree age class
14 in conifers) after large fires in the 1950s (Minnich, 1999). Strong measures to suppress fires
15 have largely kept chaparral fires from invading the mixed conifer forests, and stand densification
16 in the mixed conifer zone has increased. High stand density, in turn, may weaken the younger
17 cohorts and increase sensitivity to atmospheric pollution (Minnich, 1999).
18 Other disturbances that play a potential role in sensitivity to atmospheric pollution include
19 cycles of drought stress. Nearly every decade is marked by one or more years of very low
20 precipitation (Graumlich, 1993). During extended periods of drought, foliar injury is lower than
21 in subsequent years with higher average precipitation (Carroll et al., 2003). In the first several
22 years (1975 to!977) of a Sierran-wide assessment of O3 injury to pines, O3 injury increased,
23 because of greater water availability due to greater stomatal conductance and, presumably,
24 greater O3 uptake. Trees instrumented with monitors to directly measure canopy transpiration
25 had 20% greater stomatal conductance in mesic microsites (riparian areas, mid-slope seeps) than
26 trees in xeric microsites (rock outcrops) (Grulke et al., 2003a). Although the Sierra Nevada
27 experienced a prolonged drought between 1987 and 1993, it was less severe than other droughts
28 and O3 injury did not significantly decrease (Carroll et al., 2003). The same plots showed only a
29 slight increase in O3 injury between 1993 and 2000. While drought stress may make trees more
30 susceptible to insect and pathogen infestation, serious outbreaks of insect infestation are believed
31 to be indicators, not a cause, of existing stress in the forest (Wickman, 1992).
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1 AX9.6.2.4 Appalachian Mountains
2 The southern Appalachian Mountain region experiences some of the highest O3 exposures
3 of any natural areas in the eastern United States (Chappelka et al., 1997; Hildebrand et al., 1996;
4 Mueller, 1994; Samuelson and Kelly, 1997). Since the region is the home of the Shenandoah
5 and Great Smoky Mountains NPs, which have Class I air quality designations by the 1977
6 Clean Air Act, there has been considerable study of the region's dominant forest species to
7 determine O3 effects. Visible foliar symptoms of O3 have been found in natural ecosystems
8 consisting of sassafras (Sassafras albidum) (Chappelka et al., 1999a), black cherry (Primus
9 serotina) (Chappelka et al., 1997; 1999c; Hildebrand et al., 1996; Samuelson and Kelly, 1997),
10 yellow poplar and white ash (Fraxinus americand) (Chappelka et al., 1999a; Hildebrand et al.,
11 1996). Visible foliar symptoms induced by O3 have been recreated on the same species in
12 chamber studies (Chappelka et al., 1985; Chappelka and Chevone, 1986; Duchelle et al., 1982;
13 Fredericksen et al., 1995; Samuelson, 1994). No response to O3 exposure has been found for
14 other hardwood trees, nor for the three conifer species tested (Neufeld et al., 2000).
15 Long-term foliar injury symptoms have been correlated with decreased radial growth in
16 yellow poplar and black cherry (Somers et al., 1998) and with decreased biomass in cherry
17 (Neufeld et al., 1995). Although climatic conditions (drought) largely explained radial growth
18 reductions, O3 exposure may have also contributed (McLaughlin and Downing , 1996). Ozone
19 exposure may also be affecting the understory vegetation in the region (Chappelka et al., 1997;
20 2003; Davison et al., 2003; Duchelle et al., 1983; Duchelle and Skelly, 1981) and community
21 composition (Barbo et al., 1998), through impacts on both growth and reproduction (Chappelka,
22 2002). Foliar litter from trees exposed to elevated O3 have lower decomposition rates (Kim
23 et al., 1998). Other air pollutants are likely to be found in this ecosystem but not at the high
24 deposition values found in the California studies. A decline in forest health in the northern
25 Appalachians has been primarily attributed to the effects of acidic fog and rain on soil
26 acidification, lower Ca2+ availability, reduction in fine root biomass, and modification of
27 cuticular wax. However, fog- and O3-exposed red spruce forests in New England also show
28 winter injury (Percy et al., 1992).
29
30
31
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1 AX9.6.2.5 Plantago Studies in the United Kingdom
2 One of the most well-documented studies of population and community response to O3
3 effects are the long-term studies of sunflower (Plantago major} in native plant communities in
4 the United Kingdom (Davison and Reiling, 1995; Lyons et al., 1997; Reiling and Davison,
5 1992c). Sensitive populations of P. major had significant growth decreases in elevated O3
6 (Pearson et al., 1996; Reiling and Davison, 1992a,b; Whitfield et al., 1997) and reduced fitness
7 as determined by decreased reproductive success (Pearson et al., 1996; Reiling and Davison,
8 1992a). While spatial comparisons of population responses to O3 are complicated by other
9 environmental factors, rapid changes in O3 resistance were imposed by ambient levels and
10 variations in O3 exposure (Davison and Reiling, 1995). Molecular patterns of genetic variation
11 suggest that a change in O3 resistance over time probably resulted from natural selection in
12 genotypes already present in local populations, rather than through an influx of new P. major
13 germplasm (Wolff et al., 2000). At the site of sunflower seed collection the highest correlations
14 occurred between O3 resistance and ambient O3 concentrations occurred (Lyons et al., 1997),
15 rather than between O3 resistance and other climatic variables, as found for aspen (Berrang et al.,
16 1991).
17
18 AX9.6.2.6 Forest Health in the Carpathian Mountains
19 The Carpathian Mountains cross five countries (the Czech Republic, the Slovak Republic,
20 Poland, Romania, and the Ukraine) and contain many national parks and several biosphere
21 reserves. The forests were largely cleared in the 15th century and were replanted with Norway
22 spruce. As elevation increases, beech (Fagus sylvaticd) or beech-fir (Abies alba) forests grade
23 into Norway spruce or spruce-fir forests. Near the treeline, Norway spruce mixes with dwarf
24 mountain pine (Pinus mugo). Dwarf mountain pine forms an almost pure stand just below the
25 alpine vegetation.
26 The forests of the Carpathian Mountains have been subjected to anthropogenic stressors
27 (e.g., shepherding, metal mining, wood harvest for structures and paper) for hundreds of years,
28 as described for the Tatra Mountains in the southern Carpathians (Wezyk and Guzik, 2002). The
29 Carpathians have been subjected to regional air pollution stressors since industrialization. Most
30 of the effects of air pollution on forest health degradation were due to (1) heavy metal
31 deposition, (2) soil acidification by acid deposition, and (3) subsequent pest outbreaks, the
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1 combination of which led to the forest decline and dieback between 1970 and 1989 (Dunajski,
2 2002). Industrial pollutants such as SO2 and heavy metals have significantly declined since the
3 1980s, but O3 exposure has continued to increase (Bytnerowicz et al., 2002a; Bytnerowicz et al.,
4 2004). The increased ownership and use of private cars in Central Europe, as well as the
5 long-range transport of O3 from western Europe, are believed to be responsible for the continued
6 increase in photooxidants. In 1995, drought resulted in significant forest mortality, as well as an
7 epidemic of bark beetle infestation in subsequent years. A network of air quality monitoring
8 sites was installed across Europe in the late 1980s as part of the International Cooperative
9 Programme on Assessment and Monitoring of Air Pollutant Effects on Forests (ICP Forests).
10 Mean defoliation rates for six important forest species across Europe have increased or remained
11 unchanged from 1989 to 1999 (Percy et al., 2002). Ozone concentrations experienced in the
12 Tatra Mountains, especially along the southern slopes, occasionally reach 190 to 200 ppb as
13 2-week-long averages, with the highest values experienced in early summer at elevations of
14 1700 to 2300 m (Bytnerowicz et al., 2004). In other parts of the Carpathian Mountains, peak
15 2-week average O3 concentrations were lower, at 160 ppb (Bytnerowicz et al., 2002b). For all
16 trees inventoried, about 13% exhibited greater than 25% defoliation during 1997 to 2000. There
17 was no difference between extent of damage for broadleaves or conifers. Trees in Poland and
18 the Czech Republic were the most affected by air pollution, while the least damaged forests were
19 in Romania (Bytnerowicz et al., 2002b).
20 The extent to which O3 exposure affects forest health degradation, and slows forest
21 degradation, is still unknown in Europe. In many of the published studies, the response to a
22 known O3 gradient is largely confounded by other pollutants and/or climatic gradients (Szaro
23 et al., 2002; Widacki et al., 2002). Current levels of ambient O3 are believed to be high enough
24 to reduce bole radial growth (Percy et al., 2002). Although average O3 concentration alone was
25 not related to bole growth, the peak hourly O3 concentration was negatively correlated to bole
26 growth (Muzika et al., 2004). Recent evidence indicates that canopy health of European white
27 oak (Quercus robur), Norway spruce, maritime pine (Pinuspinaster), and beech has
28 significantly declined (Huttunen et al., 2001). However, the canopy health of Scots pine has
29 improved. The network of air quality monitoring stations and forest plots is extensive and
30 active. Subsequent correlative analyses including both meteorological and air quality attributes
August 2005 AX9-332 DRAFT-DO NOT QUOTE OR CITE
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1 throughout the European Union (EU) will help to determine the specific role of O3 exposure in
2 forest decline. Historical effects of anthropogenic disturbance may still be confounding.
3
4 AX9.6.2.7 Field Exposure System (FACE), Rhinelander, Wisconsin
5 The Aspen Free-Air CO2 Enrichment facility was designed to examine the effects of both
6 elevated CO2 and O3 on aspen (Populus tremuloides), birch (Betulapapyrifera), and sugar maple
7 in a simple reconstructed plantation characteristic of Great Lakes Aspen-dominated forests
8 (Karnosky et al., 2003b; Karnosky et al., 1999). Instead of using chambers to expose the plants
9 to desired gas concentrations, the gas is piped up vertical delivery tubes in the open air. The
10 vertical delivery pipes surround a 30-m diameter circular plot with five different aspen clones in
11 half of the plot, one quarter of the plot planted in aspen and birch, and one quarter in aspen and
12 maple. The O3 treatment for the first 5 years was 1.5x ambient, with ambient O3 exposures
13 averaging 35 to 37 ppb (12 h daytime average over the growing season) compared to elevated O3
14 rings averaging 49 to 55 ppb for the same time period (Karnosky et al., 2003b).
15 Elevated CO2, elevated O3, and elevated CO2 + O3 have had effects on most system
16 components being measured in the study (Table AX9-26) (Karnosky et al., 2003b). One
17 interesting finding of the project has been the nearly complete offset by elevated O3 of the
18 enhancements induced by elevated atmospheric CO2 for the pioneer keystone species aspen
19 (Isebrands et al., 2001) and birch (Percy et al., 2002) even though O3 exposure alone did not
20 always result in a significant response when compared to controls. They also found evidence
21 that the effects on above- and below-ground growth and physiological processes have cascaded
22 through the ecosystem, even affecting microbial communities (Larson et al., 2002; Phillips et al.,
23 2002). This study also confirmed earlier observations of changes in trophic interactions
24 involving keystone tree species, as well as important insect pests and their natural enemies
25 (Table AX9-26) (Awmack et al., 2003; Holton et al., 2003; Percy et al., 2002).
26
27 AX9.6.3 Landscape Condition
28 In the SAB framework (Figure AX9-20), landscape condition is assessed using the areal
29 extent, composition of component landscape ecosystems or habitat types, and the pattern or
30 structure of component ecosystems or habitat types (including biocorridors). To date, no
31 publications exist on the impacts of O3 exposure on landscape condition. The effects of O3
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Table AX9-26. Summary of Responses of Populus tremuloides to Elevated CO2
(+200 umol mol"*), O3 (1.5 x ambient), or CO2+O3 Compared with Control During
3 Years of Treatments at the Aspen FACE Project
(Modified from Karnosky et al. (2003b)
Foliar Gene Expression
and Biochemistry
Rubisco; RbcS2 transcripts
PAL transcripts
Ace oxidase, catalase
Ascorbate peroxidase
Glutathione reductase
Phenolic glycosides
Tannins
Foliar nitrogen
C:N ratio of foliage
Starch
Gas Exchange
Amax lower canopy
A^ whole canopy
Stomatal limitation
Stomatal conductance
Foliar respiration
Soil respiration
Microbial respiration
Stomatal density
Chlorophyll content
Chloroplast structure
O3flux
Growth and Productivity
Leaf thickness
Leaf size
Leaf area
C02
d1
d
d
d
d
I
n.s.
d
i
d
n.s.
ii
d
d
n.s.
ii
ii
n.s.
d
i
d
i
i
i
03
d
I
I
n.s.
I
d
i
n.s.
n.s.
d
dd
dd
n.s.
di
i
d
n.s.
n.s.
d
d
ii
n.s.
d
d
CO2 + O3
dd
d
d
d
d
n.s.
i
d
ii
n.s.
id
n.s.
d
d
n.s.
n.s.
n.s.
n.s.
d
d
i
n.s.
d
n.s.
Reference
Noormets et al. (200 la); Wustman et al. (2001)
Wustmanetal. (2001)
Wustman etal. (2001)
Wustmanetal. (2001)
Wustmanetal. (2001)
Kopper and Lindroth (2003a,b);
(2001)
Kopper and Lindroth (2003 a,b);
(2001)
Kopper and Lindroth (2003 a,b);
(2001)
Lindroth etal. (2001)
Wustmanetal. (2001)
Lindroth et al.
Lindroth et al.
Lindroth et al.
Noormets et al. (200 Ib); Takeuchi et al. (2001)
Noormets et al. (200 la); Sharma
Noormets etal. (200 Ib)
Noormets et al. (200 Ib)
et al. (2003)
Noormets et al. (200 la); Takeuchi et al. (2001)
King etal. (2001)
Phillips et al. (2002)
Percy et al. (2002)
Wustmanetal. (2001)
Oksanen et al. (2001); Takeuchi
Wustmanetal. (2001)
Noormets etal. (200 Ib)
Oksanen etal. (2001)
Wustmanetal. (2001)
Noormets et al. (200 la)
etal. (2001);
'Responses are shown as significant increases p <
p < 0.05, significant decreases p < 0.01 (dd), both
effects (n.s.), and no data (n.d.).
0.05 (I), significant increases p < 0.01 (ii), significant decreases
significant increases and decreases reported (id), nonsignificant
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Sources
Above-ground Processes
(-) Rubisco activity
(+) Lipid Peroxidation
(-) Stomatal Control
(-) Phloem Loading
(+) Senescence
Sinks
Above-ground Processes
(+) Antioxidant Synthesis
(+) Repair Processes
(+) Construction Costs
(+) Tissue Respiration
(-) Growth
(±) Reproduction
Storage Carbohydrates
(-) Pool Size
(-) Concentration
Root Storage
(-) Pool Size
(-) Concentration
Storage Carbohydrates
(-) Pool Size
(±) Concentration
Root Processes
(-) Growth
(±) Mycorrhizal
Colonization
(?) Turnover
Root Storage
(-) Pool Size
(-) Concentration
Figure AX9-20. A conceptual diagram of processes and storage pools in sources and sinks
that are affected by O3 exposure. A plus (+) denotes an increase in
process rate or pool size, a minus (-) denotes a decrease in process rate or
pool size, and a plus-minus (±) denotes that both increases and decreases
have been reported in response to O3. Primary effects in the shoots (1°)
are distinguished from secondary effects in roots (2°) since the primary
site of O3 action occurs in the leaves.
Source: Andersen (2003).
1 exposure have only been reported at the community or stand level (see Biotic Conditions,
2 below). The following is a description of current discussions by land stewards and of how
3 difficult it will be to quantitatively assess the effect of O3 exposure on landscape condition.
4 Landscapes are identified and preserved, such as national parks, Class I wilderness areas,
5 etc., so that they are protected from the effects of O3 exposure by law. Efforts to determine
6 whether landscapes have been affected by certain levels of exposure rely on valuation of
7 landscape and ecosystem components. Several different approaches of valuation have been
8 used, including pathological (visible symptoms), biomass and allocation, and biogeochemical.
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1 In the pathological approach, a "critical levels" concept is developed, with varying levels
2 of impact viewed as acceptable, interim targets, or as unacceptable. As an example, land
3 managers of Class I wilderness areas may consider a level acceptable if it resulted in no
4 visible O3 symptoms to sensitive species. In concrete terms, sensitive species may respond to
5 peak O3 exposures of 60 ppb (e.g., coneflower, in Great Smoky National Mountains NP;
6 [Davison et al., 2003]), and so the critical exposure level would be < 60 ppb for any hourly
7 valued during the growing season. An interim target would be that less than 5% of the sensitive
8 plants would have visible symptoms on <15% of the leaf surface. An unacceptable level of O3
9 exposure would be any result more pronounced than the interim target. The advantage of the
10 foliar injury approach is that large crews with relatively simple training can assess individual
11 species within the landscape and "see" the effect of the oxidant exposure. There are several
12 disadvantages, however. Some species (e.g., white fir) exhibit no foliar injury but do have shifts
13 in biomass allocation in response to oxidant exposure (Retzlaff et al., 2000). Other species have
14 shown significant decreases in foliar injury due to needle loss, with retranslocation of nutrients
15 to remaining foliage, and subsequent increased photosynthetic rate (Beyers et al., 1992).
16 In addition, the development of foliar symptoms within a species is related to sunlight and
17 microclimate (Davison et al., 2003).
18 In the biomass approach, O3 exposure resulting in a measurable decline in biomass (usually
19 of a target, sensitive species) is used to evaluate landscape condition. The bulk of the
20 information available is from seedling responses to controlled chamber exposures, reviewed in
21 the previous section. Some information exists for species in natural environments, but teasing
22 out concurrent stressors and finding adequate controls may be intractable. For example, in a
23 long-term gradient of O3 exposure, N deposition, and drought, the site with the highest O3
24 exposure had the greatest whole tree biomass (pole-sized trees) due to growth stimulation by N
25 deposition (Grulke and Balduman, 1999).
26 In the biogeochemical approach, changes in biogeochemical cycling are used to assess
27 landscape condition. O3-sensitive species have well known responses to O3 exposure, including
28 altered C allocation to below- and above-ground tissues, and altered rates of leaf production,
29 turnover, and decomposition. Changes in turnover rates of ephemeral tissues (leaves, fine roots)
30 also affect nutritional status of the remaining tissue. These shifts can affect overall C and N loss
31 from the ecosystem in terms of respired C, and leached aqueous dissolved organic and inorganic
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1 C and N. Instability in C and N pools and their dynamics can affect landscape-level nutrient
2 dynamics even without significant inputs of N deposition. The endpoint assessment is based on
3 changes in water quality from or in the landscape, correlated to a defined oxidant exposure level.
4 These approaches are linkable: visible injury at a particular level could be related to reduction in
5 photosynthate, which would reduce whole plant biomass (and carbon dynamics). If O3-sensitive
6 species dominate the landscape, then changes in C and N dynamics over time would be expected
7 to alter biogeochemical cycles. Examples of forest types that contain geographically extensive,
8 O3-sensitive species that could be used in assessing landscape-level changes include Ponderosa
9 pine in the western United States, yellow poplar or loblolly pine in the eastern United States
10 deciduous forests, and Norway spruce in the Carpathian Mountains of eastern Europe.
11 Water quantity may also be affected by O3 exposure at the landscape level. Moderately
12 high O3 exposure may affect the mechanism of stomatal opening (McAinsh et al., 2002),
13 resulting in sluggish stomatal opening and closing (Reich and Lassoie, 1984). During
14 moderately high O3 exposure in a drought year, canopy transpiration was greater for yellow
15 poplar than on adjacent days with lower O3 exposure, which could alter water use at the
16 landscape level. Oxidant exposure (O3 and NOX) may decrease the ability of exposed plants to
17 close stomata at night (Grulke et al., 2004), thus increasing water loss from the landscape.
18 Ecosystem models should aid in interpreting O3-exposure effects at the landscape level.
19
20 AX9.6.4 Biotic Condition
21 AX9.6.4.1 Ecosystems and Communities
22 The SAB framework described by Young and Sanzone (2002) identifies community extent,
23 community composition, trophic structure, community dynamics, and physical structure as EEAs
24 for assessing ecosystem health.
25
26 COMMUNITY EXTENT
27 Ecosystem function is dependent on areal extent, constituent species composition, trophic
28 structure and its dynamics, and community physical structure. Genetic variation within species,
29 and the dynamics of the interactions that exist among different species and their biotic and
30 abiotic environment, are also involved (Agrawal and Agrawal, 2000). There are no reports of O3
31 exposure altering community distribution or extent.
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1 COMMUNITY COMPOSITION
2 Significant changes in plant community composition resulting directly from O3 exposure
3 has been demonstrated in two forested areas: the mixed conifer forest of the San Bernardino
4 Mountains, CA and the mixed conifer forest of the Valley of Mexico near Mexico City. It is
5 also likely that community composition has changed in response to O3 exposure in the
6 coniferous forests of the Carpathian Mountains, but this has not yet been definitively shown.
7 The first forest communities shown to be affected by O3 were the Pinus ponderosa-
8 dominated stands of the San Bernardino Mountains in southern California (Miller, 1973). Miller
9 suggested that mixed forests of P. ponder osa, Pinus jeffreyi, and Abies concolor were changing
10 to predominantly A. concolor because of the greater O3 sensitivity of the pines. Significantly
11 greater mortality of young mature trees (50 to 99 years old) occurred in sites that also showed
12 higher foliar injury relative to sites that showed slight foliar injury (McBride and Laven, 1999).
13 For P. ponderosa, 33% of the trees in the high foliar injury sites died versus 7% of the trees in
14 the low foliar injury sites over the decade-long census. In contrast, 24% of Abies concolor died
15 in high foliar injury sites, whereas no trees died in slight injury sites. The authors suggested that
16 certain age classes were especially sensitive to O3 exposure, because they are emerging into the
17 canopy where higher O3 concentrations are encountered. Future projections based on past
18 changes in community composition have been conducted for 2024 and 2074 (McBride and
19 Laven, 1999). In their projections, the population of Ponderosa pine nearly disappears in all tree
20 age classes, and the community is dominated by California black oak (Quercus kelloggif) in all
21 tree age classes, followed by Incense-cedar (Calocedrus decurrens) and sugar pine {Pinus
22 lambertiana) by the year 2074. Their projections do not account for potential changes in genetic
23 structure of the more O3-sensitive species.
24 In the Valley of Mexico, the closed forest structure changed to a woodland from high
25 pollutant exposure (Fiscus et al., 2002). Cryptogamic community diversity also significantly
26 declined in response to prolonged, extreme O3 exposure (Zambrano and Nash, 2000). Together,
27 these two examples illustrate the potential for shifts in community composition in response to O3
28 stress.
29
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1 TROPHIC STRUCTURE
2 Above-Ground
3 One of the first reports of trophic level interactions in natural communities was
4 the O3-induced predisposition of Ponderosa pine to attack by bark beetles (Cobb et al.,
5 1968; Stark et al., 1968; Stark and Cobb, 1969). Trees exposed to oxidant injury had lower resin
6 production, flow, and exudation pressure. Also, several attributes associated with tree defense
7 against beetle attack including sapwood and phloem moisture content and phloem thickness
8 were compromised by oxidant exposure (Pronos et al., 1999). Another trophic level has been
9 implicated, in that O3-injured Ponderosa pine had the same rate of bark beetle infection, but
10 healthy trees had greater numbers of bark beetle predators and parasitoids (Dahlsten et al., 1997).
11 This suggests that O3 damage rendered the pines inhospitable for the natural enemies of the bark
12 beetles. Similar findings were presented by Percy et al. (2002) for aphids whose abundance was
13 increased in young Populus tremuloides stands exposed to elevated O3. In that study, the levels
14 of natural enemies of aphids (ladybirds, lacewings, spiders, and parasitoids) were significantly
15 decreased under elevated O3.
16
17 Below-Ground
18 Processing of plant-derived carbon compounds by soil organisms comprising the soil food
19 web is a fundamental property of a functional and stable below-ground ecosystem (De Ruiter
20 et al., 1998; Wolters, 1998). Soil food web organisms are responsible for recycling nutrients and
21 for development of soil properties such as porosity, aggregate structure, water-holding capacity,
22 and cation exchange capacity. A shift in food-web species diversity or functional complexity in
23 response to O3 stress may alter ecosystem processes.
24 Evidence that soil organisms are affected by O3 indicates the potential for changes in soil
25 food-web structure and function. Since O3 does not penetrate the soil beyond a few centimeters,
26 the proposed mechanism by which O3 alters soil biota is through a change in C input to soils
27 (Andersen, 2003). Ozone can alter C inputs to soil and hence soil processes through four
28 different pathways: (1) leaf-litter quality and quantity (see Material Cycling, below), (2) C
29 allocation to roots (see Physiological Status, below), (3) interactions among root symbionts, and
30 (4) rhizodeposition. The complex nature of the effects of O3 on trophic interactions and food
31 webs calls for additional basic research and modeling.
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1 There have been no comprehensive studies on the effects of O3 on structural components of
2 soil food webs; however, studies have shown that O3 affects free-living soil organisms of food
3 webs. In the few cases where soil microbial communities have been examined, O3 has led to
4 changes in bacterial and fungal biomass, and, in some cases, changes in soil enzyme activity.
5 Phillips et al. (2002) examined the effects of elevated CO2 and O3 on C flow through
6 heterotrophic microbial communities in soils collected from a FACE study in Wisconsin. Ozone
7 decreased abundance of fungal phospholipid fatty acids in aspen and birch-aspen plots but had
8 few other direct effects on measured soil parameters. The greatest effect of O3 was to eliminate
9 significant increases in microbial respiration resulting from elevated CO2, suggesting an
10 important role for O3 in altering C flow through soils. Shafer (1988) found that O3 tended to
11 increase the number of fungal propagules and bacteria exhibiting phosphatase activity in the
12 rhizosphere of sorghum. Ozone in combination with simulated acid rain stimulated soil
13 arylsufatase activity (Reddy et al., 1991). The response was observed at low concentrations, but
14 was reversed at high concentrations, suggesting a threshold level of O3, possibly involving
15 different mechanisms. Ozone significantly decreased soil microbial biomass in the fall after one
16 season of exposure in a wheat and soybean system (Islam et al., 2000). Other studies have
17 shown shifts in microbial and fungal biomass in response to O3 stress, but responses were
18 variable (Scagel and Andersen, 1997; Yoshida et al., 2001).
19 Decreased allocation to roots associated with O3 exposure alters N fixation in legumes and
20 actinorrhizal species. Ozone exposure was found to decrease nodulation in a number of species
21 (Manning et al., 1971; Tingey and Blum, 1973). In alder (Alnus sermlata), host root cells of
22 nodules showed cytoplasmic breakdown and lacked organelles when seedlings were exposed
23 to O3 for 27 days (Greitner and Winner, 1989).
24 Ozone has been shown to affect mycorrhizal colonization (Adams and O'Neill, 1991;
25 Edwards and Kelly, 1992; Ho and Trappe, 1984; McCool et al., 1982; Simmons and Kelly, 1989;
26 Smith and Read, 1997). Although short term in nature, several studies have found enhanced
27 mycorrhizal short-root formation under O3 stress. White pine (Pinus strobus) (Stroo et al.,
28 1988), Norway spruce (Rantanen et al., 1994), Northern red oak (Quercus rubrd) (Reich et al.,
29 1985), Douglas fir (Pseudotsuga menziesii) (Gorissen et al., 1991a), European silver fir (Abies
30 alba) (Wollmer and Kottke, 1990), and Scots pine (Kasurinen et al., 1999) all showed some
31 increase in mycorrhizal presence when exposed to O3. Others have shown minimal or no effects
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1 of O3 on mycorrhizas (Kainulainen et al., 2000; Mahoney et al., 1985; Meier et al., 1990). Stroo
2 et al. (1988) found that percent infection increased from 0.02 to 0.06 ppm O3, then decreased
3 from 0.06 to 0.14 ppm; the total number of short roots were unaffected, however. In cases where
4 stimulation was observed, the response was often noted shortly after initiation of exposure, often
5 at relatively low concentrations. Good examples of this transitory response can be found in
6 results with Norway spruce and Scots pine (Kasurinen et al., 1999; Rantanen et al., 1994). In
7 these studies, O3 increased mycorrhizal short roots initially but differences were not evident by
8 the end of the experiment.
9 Evidence suggests that decreased below-ground allocation associated with O3 stress alters
10 mycorrhizal host-symbiont compatibility. Edwards and Kelly (1992) found a shift in fungal
11 morphotypes present on loblolly pine roots, even though the number of mycorrhizal short roots
12 per gram fine root was not significantly affected by O3. Qui et al. (1993) found increased
13 numbers of morphotypes present in O3-sensitive loblolly pine seedlings exposed to O3. Roth and
14 Fahey (1998) found an interaction between O3 and acid precipitation treatments on the
15 composition of fungi forming ectomycorrhizae on red spruce saplings, possibly driven by
16 nutrient availability. Carbohydrate requirements vary among fungal species (Bidartondo et al.,
17 2001), and O3 may affect species composition by altering carbohydrate availability in roots.
18 A shift in species dominance could lead to a change in successional patterns of mycorrhizal
19 communities.
20 In the few studies that examined root exudation in response to O3 exposure, O3 was found
21 to alter the quantity and quality of root exudates. McCool and Menge (1983) found a significant
22 decrease in exudation of amino acids in tomato exposed to 300 ppb O3. McCrady and Andersen
23 (2000) observed increased root exudation in nonmycorrhizal wheat. No apparent change in root
24 exudation was found in labeling studies of ECM Ponderosa pine (Andersen and Rygiewicz,
25 1995a). Inconsistency in the literature probably results from species differences and
26 experimental protocols; however, these examples illustrate the potential effects of O3 on
27 rhizosphere C flux.
28 Decreased C allocation to roots of O3-exposed plants may reduce root longevity and
29 accelerate root turnover, increasing rhizodeposition of C and N. Fine root turnover decreased in
30 mature northern red oak exposed to elevated O3 (seasonal exposure ranging from 152 to
31 189 ppm-h), whereas seedlings did not show any reduction in turnover (Kelting et al., 1995).
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1 King et al. (2001) found a trend toward decreased live root biomass and increased dead root
2 biomass in aspen exposed to O3 in a FACE study, suggesting possible changes in both
3 production and longevity.
4 Other studies also suggest that O3 alters C flux to soils, resulting in changes in CO2 efflux
5 from soils. Both root respiration and soil CO2 efflux decreased from loblolly pine seedlings
6 exposed to O3 (Edwards, 1991). Soil CO2 efflux increased in response to O3 in Ponderosa pine
7 seedlings (Andersen and Scagel, 1997). No direct assessments of hyphal growth and turnover in
8 response to O3 stress have been conducted. Ozone decreased C allocation to extrametrical
9 hyphae of a Ponderosa pine mycorrhiza, which might be expected to decrease growth and
10 increase hyphal turnover (Andersen and Rygiewicz, 1995b).
11
12 COMMUNITY DYNAMICS, PHYSICAL STRUCTURE
13 One of the best-documented examples of change in long-term forest community dynamics,
14 of dominant overstory trees occurred in the San Bernardino Mountains between 1968 and 1974
15 (reported in Miller (1973) and Miller et al. (1989)). Plots were recently re-inventoried -25 years
16 after establishment (Arbaugh et al., 2003). Of the six codominant canopy species, white fir
17 showed the greatest change, increasing in both numbers and bole growth for a 286% change in
18 basal area/ha in the San Bernardino Mountains. Sugar pine basal area also increased
19 significantly (by 334%), but this species represents only a small portion (1%) of the total basal
20 area of the forest sampled. The most O3-sensitive species (Miller et al., 1982), Ponderosa and
21 Jeffrey pine, had the lowest increase in basal area/ha (76 and 62%, respectively). These two
22 species represented 72% of the basal area/ha of all stands inventoried. Ponderosa pine had the
23 greatest mortality rate of all canopy species inventoried (46%), followed by white fir and black
24 oak (35% and 33%), Jeffrey pine (29%) and Incense cedar and sugar pine (both 7%). In moist
25 sites (at the western end of the San Bernardino Mountains), there was significant recruitment of
26 Incense cedar, white fir, and sugar pine. Only one study directly attributed tree mortality to O3
27 exposure: it accounted for 7% of mortality in the Sierra and Sequoia NFs (Carroll et al., 2003).
28 Species diversity in the understory can be quite large, making studies of O3 effects on
29 understory community dynamics very challenging. However, there have been some attempts to
30 quantify understory responses, ranging from describing relative sensitivity to their visible
31 symptoms (Temple, 1999; Treshow and Stewart, 1973) to very complex measures of community
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1 structure and composition (Westman, 1979, 1981). The lowest percentage cover and lowest
2 species diversity in California coastal sage scrub was correlated with the highest O3 exposures as
3 estimated by extrapolation from the closest air monitoring stations (Westman, 1979). The
4 understory also has the potential to influence responses to O3 of dominant keystone species, as
5 has been shown in controlled experiments with both Ponderosa pine (Andersen et al., 2001) and
6 Loblloly pine (Barbo et al., 2002). Barbo et al. (1998) exposed an early successional forest
7 community to ambient air, charcoal-filtered air, non-filtered air, and 2* ambient in the
8 Shenandoah NP. They found changes in species performance, canopy structure, species
9 richness, and diversity index consistent with the view that O3 can induce a shift in vegetation
10 dominance and community structure.
11 There have been few studies evaluating the effect of O3 exposure on the physical structure
12 of natural ecosystems. Despite an extensive array of allometric equations for conifers in the
13 western United States (Ter-Mikaelian and Korzukhin, 1997), none appear to predict individual
14 tree shape in a site of moderate O3 exposure, suggesting that O3 may effect allometry (Grulke
15 et al., 2003a). Canopy structural changes are also implied by the measure of canopy
16 transparency used in the USD A Forest Service's Forest Health Monitoring (FHM) assessment.
17 The loss of epiphytic lichens within the canopy is a clear example of plant community structural
18 change occurring along an O3 gradient (Nash and Sigal, 1999; Zambrano and Nash, 2000).
19 As of yet, there have been no comprehensive studies on the effects of O3 on structural or
20 functional below-ground components (Andersen, 2003). Phillips et al. (2002) found evidence
21 for changes in the bacterial and fungal biomass below aspen and aspen/paper birch stands
22 exposed to elevated O3. Subsequent study showed that O3 exposure decreased cellobiohydrolase
23 activity in the soil microorganisms, driving the change in the microbial community (Larson
24 et al., 2002).
25
26 AX9.6.4.2 Species and Populations
27 Ozone can affect species and populations of species found in ecosystems through changes
28 in population size, genetic diversity, population structure and/or dynamics, and habitat suitability
29 (Young and Sanzone, 2002). For example, if individuals of a species are lost due to O3
30 exposure, population size declines. Often very young (e.g., conifer seedlings, see Section
31 AX9.6.4.3 below) and old individuals differ in their sensitivity, so that population structure also
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1 will be altered by O3 exposure. If resource allocation to reproductive output is altered by O3
2 exposure, population dynamics will be altered. Communities dominated by O3 sensitive species
3 in the canopy or understory may be altered sufficiently for the habitat to become unsuitable for
4 other species. Genetic selection acts on the individual plant, which represents a certain
5 proportion of the populations' genetic variation. If an O3- sensitive individual succumbs
6 through multiple stresses, including O3 stress, the genetic variation represented in the population
7 generally declines, unless sensitive individuals have low inherent genetic variability (e.g.,
8 Staszak et al., 2004).
9 While the concept of natural selection induced by O3 exposure and related changes in
10 natural plant communities has been around for a long time (Dunn, 1959; Miller et al., 1972), the
11 concept of evolution of O3 tolerance is still not widely accepted. The unequivocal demonstration
12 that considerable genetic variation in O3 resistance exists within and between plant populations,
13 and that ambient levels of O3 may differentially affect fitness-related traits (i.e., growth, survival,
14 and fecundity), suggests that O3 may potentially drive the natural selection for resistant
15 individuals. Dunn (1959) presented circumstantial evidence that ambient O3 in the Los Angeles
16 area was high enough to drive the selection of O3-resistant Lupinus bicolor genotypes. Since
17 Dunn's (Dunn, 1959) research on O3-induced population changes, researchers have
18 demonstrated differences in O3 tolerance among other plant populations. In the devastating
19 forest decline southeast of Mexico City, the remaining trees (primarily Abies religiosa in the
20 "cemetery forests") appear to be less affected by foliar injury than trees that were lost, despite
21 continued high-O3 exposures (Alvarado et al., 1993). However, even the most convincing work
22 in this field (Berrang et al., 1986, 1989, 1991), with Populus tremuloides, where a strong
23 correlation between visible foliar injury after O3 exposure and maximum O3 concentration at the
24 origin of the population was shown (Berrang et al., 1991), a change in gene frequency at any one
25 site over time has not yet been demonstrated (Bell et al., 1991; Reiling and Davison, 1992a).
26 Furthermore, the selection intensity of O3 has been questioned (Bell et al., 1991; Taylor et al.,
27 1994; Taylor and Pitelka, 1992) and the emergence of O3 exposure since the 1950s as an
28 environmental stressor may not have been long enough to affect tree populations with long
29 generation times (Barrett and Bush, 1991).
30 The loss of O3-sensitive individuals results in natural selection favoring O3-tolerant species
31 (Bradshaw and McNeilly, 1991). Increased levels of mortality of O3-sensitive individuals have
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1 occurred for Pinus jeffreyi and P. ponderosa exposed to ambient O3 along the western slope of
2 the Sierra Nevadas (Miller et al., 1998; Peterson et al., 1987), for Pinus strobus exposed to
3 ambient O3 in southern Wisconsin (Karnosky, 1981), and for P. ponderosa in the San Bernardino
4 Mountains (Carroll et al., 2003). In these examples, individuals that consistently had greater
5 foliar injury and lower needle retention were lost in repeated surveys. Ozone-induced loss of all
6 individuals except the most tolerant and breeding among the surviving individuals to yield more
7 more tolerant populations has not yet been demonstrated for plants exposed to O3, except for the
8 relatively short-term (2 years) adaptation exhibited in Trifolium repens (Heagle et al., 1991) and
9 Plantago major (Davison and Reiling, 1995). Heagle et al. (1991) were able to show the
10 adaptation of a Trifolium repens population to elevated O3 in just two growing seasons.
11 Similarly, Davison and Reiling (1995) compared the O3 resistance of P. major populations
12 grown from seed collected from the same sites over a period of increasing O3. The two
13 independent populations studied exhibited increased O3 resistance, consistent with the idea of
14 selection for O3 tolerance. Using random amplified polymorphic DNA primers, this team also
15 showed that the later populations are subsets of the earlier ones, consistent with in-situ evolution
16 rather than with catastrophic loss and replacement of the populations (Wolff et al., 2000). The
17 problem is determining whether spatial patterns in O3 resistance and changes in time are casually
18 related to O3, because there were very strong correlations with other factors (Davison and
19 Barnes, 1998; Reiling and Davison, 1992c). The potential for the evolution of O3 resistance has
20 been clearly demonstrated by Whitfield et al. (1997) in their study of O3 selection of common
21 plantain, where they showed that within a matter of a few generations, it was possible to
22 increase O3 resistance in an initially O3-sensitive population. Wild radish (Raphanus sativus)
23 developed O3 resistance after only one generation of exposure to O3 (Gillespie and Winner,
24 1989).
25 A third independent line of research suggesting O3 may be affecting the genetic diversity of
26 wild plant populations was presented by Paludan-Muller et al. (1999) who showed that northwest
27 European provenances of European beech were more sensitive to O3 than were southeast
28 European provenances that had experienced higher O3 levels. Recent research on the genetic
29 structure of 50-year-old Ponderosa pines in the San Bernardino Mountains suggests that distinct
30 differences in frequency of some alleles and genotypes occurred, with the O3-tolerant trees being
31 more heterozygous (Staszak et al., 2004). While both of these studies were only correlational,
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1 they are consistent with previous studies of this type, suggesting O3-induced population changes.
2 Again, other environmental stressors besides O3 exposure could have been involved in effecting
3 change within these populations.
4 Natural selection for O3 tolerance can also be facilitated by reductions in fitness related to
5 lower seed yields of O3-sensitive species or individuals. The impacts of O3 on reproductive
6 development, recently reviewed by Black et al. (2000) can occur by influencing (1) age of
7 flowering, particularly in long-lived trees that often have long juvenile periods of early growth
8 without flower and seed production; (2) flower bud initiation and development; (3) pollen
9 germination and pollen tube growth; and (4) seed, fruit, or cone yields and seed quality (Table
10 AX9-24). In addition, vegetatively propagated species can have lower numbers of propagules
11 under elevated O3 conditions (Table AX9-24).
12 Several studies suggest that reproductive structures are clearly sensitive to O3 and that O3
13 can affect fitness of plants by affecting either the sporophytic or gametic generations. Decreased
14 numbers of flower spikes and seed capsules per plant were found for plantain growing under
15 elevated O3 (Lyons and Barnes, 1998; Pearson et al., 1996; Reiling and Davison, 1992a).
16 Similar responses were seen for Brassica campestris plants exposed to a single dose of 100 ppb
17 O3 for 6 h. Stewart et al. (1996) and Bosac et al. (1998) reported an increase of flower bud
18 abortion for oilseed rape (Brassica napus L.) similarly exposed to a short duration of elevated
19 O3. Floral initiation period can be delayed in O3-sensitive plants, as was described for dogbane
20 (Apocynum androsaemifolium) grown under ambient O3 in the eastern United States. In one of
21 the few comparisons of whole plant O3 sensitivities with that of male gametophytes, Hormaza
22 et al. (1996) found a high correlation of relative O3 sensitivity of pollen tube elongation with that
23 of O3 effects on net photosynthesis and relative growth rates for 6 species of fruit trees.
24 Clearly, the concept of O3 -induced genetic change is an area that needs additional research
25 attention. Repeated collections over time from wild populations receiving high O3 exposures to
26 examine population responses and relative sensitivity changes, the sampling of genetic diversity
27 along known O3 gradients, and the use of modern biotechnological approaches to characterize
28 and quantify genetic diversity are useful approaches to test for O3-induced impacts on diversity
29 in natural ecosystems.
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1 AX9.6.5 Organism Condition
2 PHYSIOLOGICAL STATUS
3 The generalized effects of O3 exposure on plants are well known, and have been reviewed
4 from several viewpoints over the last decade (Darrall, 1989; De Kok and Tausz, 2001; Heath and
5 Taylor, 1997; Matyssek et al., 1995; Pell et al., 1997; Reich, 1987; Schraudner et al., 1997; U.S.
6 Environmental Protection Agency, 1996). The topic of individual species response and
7 modification of response by other factors has been addressed thoroughly in Sections AX9.6.3
8 and AX9.6.4 of this chapter. Here, this subsection will describe the physiological changes in
9 response to O3 that have been hypothesized to lead to changes in ecosystem structure or function.
10
11 Above-Ground Responses
12 The first critical step leading to O3 response is uptake of O3 by the leaves, leading to
13 changes in C and nutrient relations that are thought to alter plant growth and competitiveness
14 (see Section AX9.3). Ozone enters leaves through stomata, reacts with cell walls or membranes,
15 and starts a series of adverse reactions. Cuticular uptake of O3 is believed to be negligible (Coe
16 et al., 1995; Kerstiens and Lendzian, 1989). Once inside the leaf, O3 and its byproducts lead to
17 membrane disruption, chlorophyll breakdown, and decreased Rubisco levels (Schweizer and
18 Arndt, 1990). In turn, photosynthesis is decreased, as is stomatal conductance (Weber et al.,
19 1993). Also, O3 often leads to increased maintenance respiration, decreased foliar nutrient
20 content, and imbalances in tissue nutrient content and retention. When photosynthetic pigments
21 have been damaged, the pigment must be fully broken down (and/or new N and Mg must be
22 taken up and transported to the leaf) for the pigment to be regenerated (Bjorkman and Demmig-
23 Adams, 1995). Ozone exposure alters within-plant priorities for resources: less C is available
24 for allocation to roots and spring regrowth, and less foliar biomass is retained. At the whole-
25 organism level, O3 exposure decreases root mass (Grulke et al., 1998a) and radial bole growth
26 (Muzika et al., 2004; Peterson et al., 1991) with little impact on height growth. Visible
27 symptoms of O3 injury vary between species and genotypes but often include upper leaf surface
28 stipple, chlorotic mottle, or large bifacial blotches of necrotic tissue. Premature senescence is
29 typical of almost all O3-induced foliar damage. All of these changes can alter the plant's ability
30 to function in a broader ecosystem context.
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1 The underlying mechanisms of O3-injury response in conifers, broadleaf deciduous trees,
2 and herbaceous species are assumed to be similar. However, several differences in long-lived
3 species are important at the ecosystem level. Most of the research on O3 effects has been
4 conducted on herbaceous species (i.e., crops). Although a number of native herbaceous species
5 have been identified as O3-sensitive, there are no published physiological studies on the effect
6 of O3 exposure on herbaceous or shrub species in situ. In natural ecosystems, the majority of
7 species are not annuals, unless the system is highly disturbed. Nonetheless, response of crop
8 species to elevated O3 may be used as an analog for native annual response: phenological
9 staging is accelerated (soybeans; Booker et al. [2004]), thus "avoiding" additional O3 exposure.
10 Conifers have roughly half the stomatal conductance of deciduous broadleaf trees (Reich,
11 1987), leading to proportionally less O3 uptake at the same O3 exposure level. Yet, except for
12 species of larch, individual conifer needles are longer-lived and active over a greater portion of
13 the year. Therefore, needle longevity can also work against the tree by increasing cumulative O3
14 exposure and exposure to other stressors. Increased needle longevity is not always a
15 disadvantage, for example, conifers are physiologically active in early spring and late fall, during
16 times of lower oxidant concentrations. These periods can contribute significantly to a net
17 positive annual C balance and from the standpoint of nutrient storage, are important in reparation
18 responses to pollutants. Patterson and Rundel (1995) reported that Jeffrey pine had significant
19 stomatal opening (one third that of a typical summer day) in midwinter with snow on the ground.
20 At least pole-sized and larger trees can mitigate reductions in C acquisition due to oxidant
21 exposure in the summer with C assimilation on favorable days in the winter. The interaction of
22 environmental factors, plant phenology (the timing of growth events; birth and mortality of plant
23 parts), physiological status (nutritional or moisture status; dormant or active growth within the
24 year), and tree age (interannual differences in resource acquisition and allocation) all contribute
25 to the complexity of long-lived species (and hence ecosystem) response to O3 exposure.
26 One widely observed response to O3 exposure is premature leaf loss. As noted above,
27 premature leaf loss may reduce O3 uptake during high-O3 years, but it has several negative
28 consequences. Early leaf loss results in reduced C uptake through photosynthesis. Premature
29 needle loss also results in less N retranslocation compared to normally senescing leaves, which
30 reduces whole plant N balance (Fenn and Dunn, 1989) and carbohydrate availability for
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1 overwinter storage (Grulke et al., 2001). Because of such effects accumulated over several years
2 of O3 exposure, subsequent-year C and N reserves can be affected (Andersen et al., 1991).
3 Conversely, a series of drought years can decrease O3 uptake, as well as reduce C and
4 nutrient acquisition, altering resource allocation to defenses (e.g., antioxidants) (Grulke et al.,
5 2003b) (or resins) against insect infestation, rendering the tree more susceptible to O3 injury.
6 Conifers have thicker cuticles than either broadleaf deciduous or herbaceous species.
7 Continued O3 exposure may compromise cuticular integrity (Bytnerowicz and Turunen, 1994).
8 Once cuticular integrity is breached, individual leaves (needles) are likely to be excised, thus
9 contributing generally to defoliation and reduced C acquisition.
10 With the exception of the extensive research conducted on mature tree response to O3
11 exposure in California forests (Arbaugh et al., 1998; Grulke et al., 1996, 1998b, 2001, 2002b,
12 2003a,b, 2004; Grulke, 1999; Grulke and Balduman, 1999; Peterson et al., 1987, 1991, 1995;
13 Wieser et al., 2002), the vast majority of studies of O3 effects on forest trees have been
14 conducted on young seedlings (Chappelka and Samuelson, 1998) and little is known about
15 acclimation to O3 (Skarby et al., 1998). Chamber exposure studies can be used to document
16 foliar symptoms and develop response variables for the whole plant. These response variables
17 can then be field tested on mature trees using correlative analyses (e.g., Grulke and Lee [1997];
18 Grulke et al. [2003b]). Without the initial work in chamber exposure studies, field responses to
19 O3 exposure would be difficult to verify and distinguish from other concurrent stressors.
20 Predicting mature tree responses to O3 solely from seedling response studies is complex,
21 because seedlings or saplings do not necessarily respond to O3 in the same way as mature trees
22 (Karnosky et al., 2003a; Norby et al., 1999). Although species dependent, O3 has been found to
23 have stronger effects on leaf function in younger rather than older trees (Kolb and Matyssek,
24 2001). Each component physiological attribute "matures" at a different rate. Gas exchange
25 patterns differ between seedlings and mature trees. For example, leaf respiration of juvenile
26 Ponderosa pine was greater than that of mature trees (Momen et al., 1996). In the conifers
27 tested, the highest gas exchange rates (and by inference stomatal uptake of O3) are found in
28 seedlings (e.g., in scions of red spruce, Rebbeck et al. [1993]; giant sequoia, Grulke and Miller
29 [1994]; Ponderosa pine, Grulke and Retzlaff [2001]; and Norway spruce, Wieser et al. [2002]).
30 Patterns of biomass (Grulke and Balduman, 1999) and carbohydrate allocation (Grulke et al.,
31 2001) differ between immature and mature trees. Pole-sized trees had greater reduction in root,
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1 foliar, and bole carbohydrate concentrations than did old growth trees. Antioxidant defenses
2 vary with both tree age and needle age (Tegischer et al., 2002). Based on all attributes measured
3 in both Ponderosa pine and giant sequoia, the youngest tree age considered representative of
4 mature trees was 20 years old (Grulke et al., 1996; Grulke and Retzlaff, 2001). In some
5 broadleaf deciduous tree species, seedlings are more conservative, and mature trees have greater
6 gas exchange rates, as is the case for Quercus rubra (Edwards et al., 1994; Kelting et al., 1995;
7 Samuelson and Kelly, 1996, 1997) and Fagus sylvatica (Braun et al., 1999). In another
8 broadleaf deciduous tree species (black cherry), gas-exchange rates of seedlings were faster, but
9 total O3 flux to leaves of seedlings was lower than that of mature trees due to differences in leaf
10 ontogeny (Fredericksen et al., 1995, 1996).
11 Nitrogen deposition modifies the effects of oxidant exposure through several offsetting
12 physiological mechanisms (see Section AX9.4.4). Nitrogen deposition, in wet or dry particulate
13 form, ultimately increases site fertility, but increased soil N availability decreases C allocation to
14 roots, further exacerbating the effects of O3 exposure on roots (Grulke et al., 1998a). Increased
15 N availability also increases foliage turnover: fewer needle age classes are retained (Gower
16 et al., 1993). Therefore, the combination of both increased N and O3 exposure increases foliar
17 turnover. Finally, N deposition and increased plant N nutrition can increase stomatal
18 conductance, leading to increased O3 uptake. Alternatively, increased N counteracts the effect
19 of O3 on photosynthesis by increasing photosynthetic pigments and enzymes. Nitrogen
20 deposition may mitigate the degree of foliar injury from oxidant pollution via higher available
21 N for reparation of photosynthetic pigments. Nitrogen amendments also modify the antioxidant
22 defense system in complex ways (Polle, 1998).
23 Attributes of O3 injury to trees (foliar injury, needle retention, and canopy transparency),
24 as well as presence of pathogens and insect infestation, are routinely inventoried in established
25 sample plots distributed on Federal lands across the United States (Forest Health Protection,
26 USDA Forest Service). Foliar injury to several widespread, herbaceous species nationally
27 recognized as sensitive (bioindicators) is also assessed (U.S. National Park Service, 2003). This
28 assessment is part of a larger assessment of forest tree growth and dynamics (the Forest
29 Inventory and Analysis Program) (Smith et al., 2003; Smith, 2002)). Risk of O3 injury is then
30 estimated for the dominant forest tree species in the sample plots. For example, 12% of sampled
31 black cherry, 15% of loblolly pine, and 24% of sweetgum (Liquidambar styraciflud) were found
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1 to be in the highest risk category in the northeast and mid-Atlantic states (Coulston et al., 2003).
2 In the Carpathian Mountains, 12 to 13% of all trees (broadleaf and coniferous) have greater than
3 26% crown defoliation (Bytnerowicz et al., 2002b). In general, broadleaves (primarily beech)
4 trees were less affected (8 to 45%) than spruce (up to 37%) and fir (up to 50%) (Bytnerowicz
5 et al., 2002b). Ozone injury was directly correlated with cumulative O3 exposure in the Sierra
6 Nevada Mountains (Arbaugh et al., 1998); with the best correlation being found across sites
7 where >90% of the trees had O3 injury. Although direct links of visible foliar symptoms induced
8 by O3 to adverse effects on biomass are not always found, visible foliar symptoms have been
9 linked to decreased vegetative growth (Karnosky et al., 1996; Peterson et al., 1987; Somers
10 et al., 1998), as well as reproductive function (Black et al., 2000; Chappelka, 2002).
11 Foliar O3 injury has also been associated with adverse effects on competitive ability and
12 survival in forest communities (Karnosky, 1981; Karnosky et al., 2003a; McDonald et al., 2002).
13 Competition can alter organism condition and affect susceptibility to O3. Ponderosa pine
14 seedlings were more susceptible to O3, as determined by decreased plant biomass, when grown
15 in competition with blue wild-rye grass (Andersen et al., 2001). Similarly, the magnitude of O3
16 effects on height and diameter growth depended on the competitive status ofPopulus
17 tremuloides trees (McDonald et al., 2002). These studies show the importance of including
18 competition as a concurrent stressor in assessing whole plant responses to O3. The age of the
19 community ("time since disturbance") may also affect the ability of individuals to effectively
20 respond to O3. Unfortunately, the vast majority of O3 studies have been conducted on
21 open-grown plants, often grown in pots where competition is absent both above and below
22 ground.
23 Clearly, age-dependent O3 responsiveness and juvenile-mature correlations remain
24 important research questions in attempting to scale up to ecosystem level responses. Patterns of
25 allocation between root, stem, and leaf differ between immature and mature trees. Tree
26 architecture varies with tree age, and leaf area distribution in space and time may change in
27 response to elevated O3. All of these factors influence gas exchange in the canopy.
28 Furthermore, there may be few generalities that can be made from seedling to mature tree
29 response to O3 within a plant functional group (Karnosky, 2003; Norby et al., 1999).
30 Consequently, modeling ecosystem response is limited to either dealing with monospecific
31 plantations or assigning average responses to a mix of species.
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1 Below-Ground Responses
2 The effect of O3 on the soil ecosystem is thought to occur through physiological changes in
3 the root and interactions with soil organisms (Andersen, 2003). Comparatively little is known
4 about how changes in root growth and metabolism are translated through the soil food web,
5 resulting in changes in soil and, hence, ecosystem processes. An overview of physiological
6 changes likely to lead to changes at the ecosystem level is provided below.
7 Ozone stress decreases C allocation to roots (Cooley and Manning, 1987; Gorissen et al.,
8 1994; Gorissen and Van Veen, 1988; Manning et al., 1971; McCool and Menge, 1983;
9 McLaughlin and McConathy, 1983; Rennenberg et al., 1996; Spence et al., 1990; U.S.
10 Environmental Protection Agency, 1996). Since roots are often dependent on current
11 photosynthate for their structural development (Marshall and Waring, 1985; Ritchie and Dunlap,
12 1980; Van Den Driessche, 1978, 1991), C-limiting stressor such as O3 can have rapid and
13 significant effects on root growth. In many cases, decreased allocation to roots in response to O3
14 occurs quickly, with reductions in root growth occurring within one growing season (Andersen
15 and Rygiewicz, 1995a; Andersen and Rygiewicz, 1991; Gorissen et al., 1991b; Gorissen and
16 Van Veen, 1988; Spence et al., 1990; U.S. Environmental Protection Agency, 1996). Decreased
17 C allocation below ground is often associated with decreased root-shoot ratio, but observed
18 responses in root-shoot ratio are highly variable owing to several factors including intra- and
19 interspecies variation, culture conditions, and ontogenetic drift (Reich, 2002). Root-shoot ratio
20 is a point-in-time measurement that does not include C lost to exudation, respiration, or turnover.
21 Therefore, biomass and ratios of biomass (such as root-shoot ratio) do not necessarily reveal
22 physiological changes in response to O3 stress.
23 Decreased C acquisition leads to reduced carbohydrate levels and storage pools
24 in O3-exposed plants (Andersen and Scagel, 1997; Cooley and Manning, 1987; Gorissen et al.,
25 1994; Ito et al., 1985; McLaughlin et al., 1982; Rebbeck et al., 1988; Tingey et al., 1976).
26 Although it is difficult to quantify changes in the field, Grulke et al. (1998a) found decreased
27 medium and fine root biomass with increased pollutant load across an O3 gradient in southern
28 California. Coarse and fine root starch concentrations also were lowest in mature trees at the
29 most polluted site (Grulke et al., 2001). The effects of O3 could not be completely separated
30 from other known stresses across the pollutant gradient, but it appeared that O3 was an important
31 factor in the patterns observed.
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1 Decreased storage pools can lead to carry-over effects on root growth that are compounded
2 over time. Decreased carbohydrate storage pools were associated with decreased root growth
3 during the spring following exposure to O3, even in the absence of additional O3 exposure
4 (Andersen et al., 1991, 1997). Decreased spring root growth was attributed to decreased stored
5 C reserves as well as to premature loss of older foliage age classes during the previous fall.
6 Aside from the loss of photosynthetic surface area associated with premature senescence, early
7 loss of foliage in the fall occurs when allocation to roots is at a maximum in many species
8 (Kozlowski and Pallardy, 1997). Older needle age classes preferentially allocate photosynthate
9 basipetally to stems and roots (Gordon and Larson, 1970; Rangnekar et al., 1969); and the loss of
10 older needles in the fall during allocation to root growth and storage; and in the spring during
11 periods of root growth, preferentially impacts roots and root processes.
12 Ozone has also been shown to affect root metabolism as evidenced by changes in root
13 respiration. Edwards (1991) found decreased root and soil CO2 efflux during a 2-year exposure
14 of loblolly pine to O3. Fine root respiration increased in mature red oak exposed to O3, while
15 total soil CO2 efflux increased in the spring and decreased in the summer and fall (Kelting et al.,
16 1995). The authors attributed increased root respiration to increased nutrient uptake in support
17 of increased demands in the shoot. Ozone decreased root system respiration in aspen after
18 12 weeks of exposure, but the decrease was closely associated with decreased root biomass and
19 probably not metabolic processes (Coleman et al., 1996). Whether other metabolic shifts occur
20 in the roots of plants exposed to O3 needs to be examined.
21 Measurable effects on roots may occur before effects on shoots are observed because
22 shoots have immediate access to C for repair and compensation, whereas roots must compete
23 with shoots for C. Mortensen (1998) found decreased root, but not shoot, growth in Betula
24 pubescens at O3 exposures of 42 nMol mol-L (applied 12 h day"1), whereas both root and shoot
25 growth were reduced at higher exposures. Chromosomal aberrations were found in root tips of
26 Norway spruce exposed to O3, even in the absence of biochemical changes in needles (Wonisch
27 et al., 1998, 1999). Using relatively high O3 concentrations (0.15 ppm O3 6 h day"1), Hofstra
28 et al. (1981) found metabolic changes in Phaseolus vulgaris root tips prior to the development of
29 leaf injury. Morphological changes in root tips occurred within 2 to 3 days, and metabolism
30 declined within 4 to 5 days of initiation of O3 exposure.
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1 Feedback signals from roots can influence the degree of O3 response. Stolzy et al. (1964)
2 exposed tomato roots to periods of anaerobic conditions and tracked a change in leaf
3 susceptibility to O3. An exposure of roots to low oxygen conditions for 3 h did not alter
4 photosynthesis, but foliar damage was decreased when the roots were subsequently exposed
5 to O3. In this case, a signal originating in the root appeared to alter leaf sensitivity to O3, the
6 signal possibly being hydraulic in nature and leading to decreased O3 uptake.
7
8 SYMPTOMS OF DISEASE OR TRAUMA, SIGNS OF DISEASE
9 Although insects and diseases are dynamic components of forest ecosystems, trees can be
10 especially susceptible to outbreaks due to the presence of multiple stressors such as drought and
11 pollutant exposure. Ozone can have direct effects on insect or disease organisms, indirect effects
12 on the insect or pathogen through changes to the host, and direct or indirect effects on natural
13 enemies of the insect or pathogen (Pronos et al., 1999). A full discussion of O3 effects on insect
14 and pathogen interactions can be found in Section AX9.4.
15 Although the multitude of interacting factors makes it difficult to identify causative factors
16 in the field, some recent examples suggest a role for O3 in the timing or magnitude of disease
17 attacks in the field. After periods of drought stress (such as 1995 in central Europe), the
18 incidence of bark beetle (Ips spp.) appears to increase. In 1998 and 1999, the mean daily capture
19 of Ips was lowest in plots with low O3 exposure; the converse was also true (Grodzki et al.,
20 2004). Elevation confounded the relationships, but the differences in Ips frequency in relation to
21 O3 concentrations were highly significant at lower elevations. In the Valley of Mexico, a 1982
22 to 1983 drought was documented, but not described as precipitating a bark beetle attack in the
23 early 1980s. However, a link between air pollutant exposure and bark beetle attacks were
24 implicated, because attacked trees were already Distressed at the time of the bark beetle attack
25 (Alvarado et al., 1993).
26 An early study showed that oxidant exposure predisposed Ponderosa pine to the root
27 pathogen Fomes annosus (James et al., 1980). Both root diseases (Pronos et al., 1999) and O3
28 exposure (Grulke and Balduman, 1999) can each reduce root biomass, leading to increased
29 drought stress, insect attack, and subsequent windthrow or death. Trees may take several years
30 to die, and the patterns of precipitation and annual total rainfall interact to drive the level of
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1 drought stress experienced by the tree (Pronos et al., 1999). Additional research is necessary to
2 fully understand the complex interactions occurring between O3 stress and other biotic stresses.
3
4 AX9.6.6 Ecosystem, Chemical, and Physical Characteristics (water, soil)
5 AX9.6.6.1 Nutrient Concentrations, Trace Inorganic and Organic Chemicals
6 Ozone exposure reduces the nutritional content of tissues, as well as causing elemental
7 imbalances. Foliar nutrient content may be too high (toxic) or too low (deficient), but the
8 relative amounts and ratios among all nutrients can also result in imbalances.
9 Although N deposition and foliar N content increased with O3 exposure in the San
10 Bernardino Mountains, K, Mg, Fe, and Al were all also higher in Ponderosa pine at sites more
11 exposed to air pollution (Poth and Fenn, 1998). Trees with greater foliar injury (due to O3
12 exposure) had higher current-year needle concentrations of P, K, Zn, and Fe than trees at the
13 same site that were less injured. In drought-stressed Ponderosa pine with O3 exposure, foliar N
14 was also elevated and retained in the remaining needles (Temple et al., 1992). At a relatively
15 clean site in the eastern San Bernardino Mountains, N, P, and K were efficiently readsorbed, but
16 the amount of P remaining in the foliage was relatively high compared to defined thresholds.
17 The fact that other elements were modified besides the N being deposited emphasizes the degree
18 of chemical imbalance in the tissue. Foliar micronutrients were within the normal ranges
19 reported for Ponderosa pine (Poth and Fenn, 1998; Powers, 1981). Because both N deposition
20 and O3 exposure reduce root biomass, it was unlikely that the foliar nutrient content was higher
21 due to greater uptake. Instead, it appears that retranslocation from senescing tissue was
22 responsible. Across a pollution gradient in the Carpathian Mountains in eastern Europe, only
23 S/N (expected due to high S deposition) and Fe/Mn ratios were out of balance relative to
24 established norms. The S was relatively high due to SO2 deposition, and the Fe was relatively
25 high due to smelter plumes. No imbalances could be directly attributed to O3 exposure
26 (Mankovska et al., 2004).
27
28 AX9.6.7 Ecological Processes
29 AX9.6.7.1 Energy Flow
30 All green plants generate and use energy-containing C compounds through the processes of
31 photosynthesis and respiration. Whole-plant C uptake is dependent on photosynthesis rates, leaf
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1 area, and leaf phenology. The effects of O3 at the site of action in the leaf are discussed in
2 Section AX9.2. Here, the main focus is on whole-plant carbon dynamics resulting from changes
3 in C acquisition or use under O3 stress.
4 In natural ecosystems, O3 has been shown to depress photosynthesis in sensitive tree
5 species including Ponderosa pine (Grulke et al., 2002b; Miller et al., 1969; Takemoto et al.,
6 1997; Weber et al., 1993) and aspen (Coleman et al., 1995a; Noormets et al., 2001a,b; Sharma
7 et al., 2003; Yun and Laurence, 1999). In a study of mature Jeffrey pine, trees in mesic
8 microsites had greater O3 uptake over the growing season in comparison to trees in xeric
9 microsites (Grulke et al., 2003a) and greater O3 uptake was correlated with lower mid-canopy
10 needle retention, lower branch diameters, and lower foliar N content (Grulke et al., 2003a).
11 Chamber studies have also shown negative effects of O3 on tree seedling canopy structure
12 (Dickson et al., 2001) and leaf area (Neufeld et al., 1995; Wiltshire et al., 1994). It is well
13 known from chamber and field studies that O3 exposure is correlated with lower foliar retention
14 (Grulke and Lee, 1997; Karnosky et al., 1996; Miller et al., 1963, 1972; Pell et al., 1999; Topa
15 etal., 2001).
16 In contrast to the relatively consistent findings for photosynthesis, O3 effects on respiration
17 have been more variable. Stem respiration was unaffected by O3 exposure (Matyssek et al.,
18 2002), suggesting that construction costs of new stems are not affected by O3. However, the bole
19 represents a relatively large storage pool of carbohydrates in mature trees, and the timing of
20 phenological events among individual trees may help to confound the ability to statistically
21 detect differences in stem respiration across pollutant gradients (Grulke et al., 2001). Below-
22 ground respiration has been found to both increase and decrease in response to O3, depending on
23 the approach and timing of CO2 measures (Andersen and Scagel, 1997; Coleman et al., 1996;
24 King et al., 2001; Scagel and Andersen, 1997). The decreased soil respiration is thought to be
25 due to reduced root growth under O3 exposure, but could also be partially explained by
26 decreased microbial respiration in response to O3. Additional research is necessary to identify
27 the role of O3 in affecting root versus heterotrophic respiration, particularly over long time
28 intervals.
29 Carbohydrate availability and use influence the degree to which plants respond to O3
30 exposure. A model simulation of the effect of O3 exposure on bole growth of Ponderosa pine
31 showed a 15% reduction in mass (Weber and Grulke, 1995), largely influenced by differences in
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1 carbohydrates allocated and partitioned in repair processes elsewhere in the tree. Foliar
2 respiration is thought to increase under elevated O3 because maintenance costs (energy needs) of
3 leaves damaged by O3 are higher than normal (Grulke and Balduman, 1999; Noormets et al.,
4 200Ib). However, differences in foliar respiration are subtle and difficult to detect statistically.
5 Foliar carbohydrate studies also suggest that more C is used under O3 stress for repair processes
6 (Grulke et al., 2001; Topa et al., 2001) which would result in increased respiration. Ozone
7 exposure also reduced enzymatic activities of carbohydrate metabolism related to the breakdown
8 of sucrose (Einig et al., 1997). Changes in soil respiration in response to O3, even though O3
9 does not penetrate into the soil, illustrates the tight coupling of plant C balance and soil biota and
10 illustrates the potential role O3 plays in altering ecosystem C balances (Andersen, 2000).
11 Ozone can affect plant allometry through changes in energy use, potentially affecting net
12 primary production (NPP) at larger scales. The net effect of O3 impacts on photosynthesis and
13 respiration for sensitive components of natural ecosystems is that height growth (Isebrands et al.,
14 2001; Oksanen, 2003a) and radial growth (Isebrands et al., 2001; Oksanen, 2003b; Peterson
15 et al., 1987, 1991) can be negatively affected by O3. This has been extrapolated to decreased
16 NPP (Hogsett et al., 1997; Laurence et al., 2000).
17 Energy flow in plant communities can be altered by O3 through changes in C allocation.
18 It is well known that elevated O3 affects C allocation to roots (Andersen et al., 1997; Coleman
19 et al., 1995b; Grulke et al., 1998a, 2001; Grulke and Balduman, 1999) by decreasing or
20 inhibiting phloem loading of carbohydrates (Grantz and Farrar, 1999; Landolt et al., 1997), or of
21 carbohydrate metabolism (Einig et al., 1997). This leads to depressed root growth (Andersen
22 et al., 1991; Coleman et al., 1996; Grulke et al., 1998a) and the potential for plant communities
23 to have an increased susceptibility to drought through altered root-shoot balance. Furthermore, it
24 can negatively affect below-ground food webs (Phillips et al., 2002; Scagel and Andersen,
25 1997).
26 Another energetically costly response to O3 exposure is that the production of defense
27 compounds, such as antioxidants, tend to increase under elevated O3 conditions (Sheng et al.,
28 1997; Tausz et al., 1999c, 2002). Antioxidants help the plant scavenge free radicals before they
29 can cause damage to membranes or cell walls, but they demand C for production such that
30 growth can be adversely affected. In mature Jeffrey pine, stomatal uptake of O3 elicited one
31 complex of antioxidant defenses in mesic microsites, while endogenously generated free radicals
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1 in the chloroplast elicited a second complex of antioxidant defenses in xeric microsites (Grulke
2 et al., 2003b).
3
4 AX9.6.7.2 Material Flow
5 Plants as producers are responsible for using inorganic atmospheric C and reducing it into
6 organic forms used by consumers, thus driving nutrient processes in ecosystems. Ozone has the
7 potential to disrupt material flow through organic C cycling and changes in nutrient cycling,
8 particularly N and P cycling. Although there is indirect evidence that O3 is disrupting C and
9 nutrient cycling at the ecosystem level, there is little direct evidence that O3 alters nutrient
10 processing at ecosystem scales.
11 The greatest annual nutrient and C input to ecosystems is from foliar and root turnover.
12 Excision of plant parts and whole plant mortality are potentially much larger, but syncopated,
13 ecosystem inputs. Ozone exposure alters C cycling in the ecosystem by affecting the
14 within-plant C allocation and partitioning in dominant, O3-sensitive plants, and through chemical
15 composition and rate of decomposition of sloughed plant parts (roots, branches, leaves)
16 (Figure AX9-19).
17 In addition to O3-induced changes in the quantity of C and nutrient inputs into ecosystems,
18 O3 also can alter the nutrient quality of inputs. Ozone exposure alters nutrient levels in the
19 foliage (Boerner and Rebbeck, 1995; Fenn and Poth, 1998; Lindroth et al., 2001; Momen et al.,
20 2002) and affects the C:N ratio (Andersen et al., 2001; Grulke et al., 2003b; Grulke and Lee,
21 1997; Lindroth et al., 2001). Concentrations of compounds such as tannins, lignin, and
22 phenolics (Baumgarten et al., 2000; Findlay et al., 1996; Kim et al., 1998; Saleem et al., 2001)
23 are also affected by O3 exposure, which in turn alters decomposability (Fenn and Dunn, 1989)
24 and litter buildup in the ecosystem.
25 There are several possible pathways by which O3 may affect litter quality and, hence, litter
26 decomposition, thus altering nutrient flow in ecosystems. These include altered C quality,
27 altered nutrient quality, and alteration of leaf surface organisms important in decomposition
28 pathways. For example, yellow poplar and black cherry litter exposed to O3 showed greater N
29 loss during decomposition than charcoal-filtered controls, although mass loss did not vary
30 among O3 treatments (Boerner and Rebbeck, 1995). Subsequent studies showed that although
31 foliar N was not affected by O3 exposure in yellow poplar leaves, foliage decomposed more
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1 slowly (Scherzer et al., 1998). Other studies have also shown a change in foliar N concentration
2 in response to O3 treatment, affecting the C:N ratio and possible litter quality (Andersen et al.,
3 2001).
4 In some cases, it appears that N remobilization from foliage into the plant is not complete
5 at the time of foliage abscission in O3-exposed plants (Findlay and Jones, 1990; Matyssek et al.,
6 1993; Patterson and Rundel, 1995; Stow et al., 1992). Greater N content of senesced litter could
7 increase rates of decomposition. When O3-exposed cottonwood (Populus deltoides) leaves
8 abscissed at the same time as control leaves, they decomposed at similar rates; however,
9 prematurely senesced foliage from O3-exposed cottonwood decomposed more slowly than
10 controls despite their higher N content (Findlay et al., 1991; Findlay and Jones, 1990). Higher N
11 in senesced leaves appeared to be related to organic complexes formed by bound phenolics
12 in O3-exposed leaves, making the litter less palatable to decomposers, and slowing
13 decomposition rates (Findlay et al., 1996; Jones et al., 1994). Increased phenolics also have
14 been found in European silver birch (Betulapenduld) exposed to O3 (Saleem et al., 2001).
15 Carbon quality in leaf litter also changes in O3-exposed foliage. Compositional changes in
16 leaf structural characteristics, such as lignin content, would be expected to alter rates of litter
17 decomposition (Fogel and Cromack, 1977; Kim et al., 1998; Meentemeyer, 1978). Blackberry
18 litter exposed to elevated O3 had greater permanganate lignin than the control treatment, a
19 difference that was inversely related to mass-loss rates in decomposition studies (Kim et al.,
20 1998).
21 Ozone may affect early stages of decomposition by altering populations of leaf surface
22 organisms before or after senescence. Magan et al. (1995) found a shift in phyllosphere fungi on
23 Scots pine, Sitka spruce (Picea sitchensis), and Norway spruce exposed to O3, but the potential
24 effect of these changes on subsequent litter decomposition was uncertain. The slowest
25 decomposition rates of preexposed blackberry leaves were found when senesced foliage was
26 exposed to O3 during decomposition, suggesting a possible direct effect of O3 on microorganisms
27 in decomposing litter (Kim et al., 1998). Whether O3 concentrations at the soil surface influence
28 initial stages of litter decomposition remains to be addressed.
29 Ozone exposure also reduces nutritional content of foliage because of the degradation of
30 chlorophyll. Reconstruction of chlorophyll may be limited by nutritionally poor soils or low soil
31 moisture, as well as alteration of root uptake by O3 exposure and other stressors. Foliar exposure
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1 to O3 may also increase leaching of nutrients (Kerstiens and Lendzian, 1989). Ozone exposure
2 promotes early senescence of foliage (Heath and Taylor, 1997; Miller and Elderman, 1977), with
3 higher nutrient content than if excised later in the growing season (Poth and Fenn, 1998).
4 Since O3 can slow decomposition through changes in leaf quality and quantity, leaf litter
5 can accumulate (Fenn and Dunn, 1989). The accumulation of soil organic matter from increased
6 leaf litter, even in the absence of acidic deposition, can lower soil pH (Binkley, 1992). Lower
7 soil pH can promote loss of nutrients, particularly cations, from the system, further reducing
8 nutrient availability to the plant. Complex organic compounds in decomposing litter may also
9 tie up nutrients, rendering them less available to plants.
10 Other factors such as nutrient deposition also affect the degree to which O3 influences C
11 and nutrient flow through ecosystems. In high-pollution sites, the effect of N deposition is
12 difficult to separate from O3 exposure, and reviews of the effect of acidic (and N) deposition on
13 ecosystem nutrient dynamics are important to consider (see Binkley [1992]; Fenn et al. [1998,
14 2003b]). For example, at moderately high pollution sites, foliar content of N is higher than that
15 at lower pollution sites, but so are P, Mg, and Fe contents (Poth and Fenn, 1998). Although
16 significant changes in foliar tissue chemistry have occurred in response to long-term pollutant
17 deposition in the Carpathians (Fenn et al., 2002; Mankovska et al., 2004), much of this response
18 is correlated to heavy metal and N and S deposition. At this point, the contribution of O3
19 exposure alone cannot be isolated without careful between-site comparisons of plant response
20 and understanding the deposit!onal velocities of constituent atmospheric species. The significant
21 effects of plant response along known pollution gradients are important to consider, because
22 heavy metal and N and S deposition has significantly declined over the last decade, while O3
23 exposures remain high and declines in forest health have been sustained.
24 Models provide a means to track material flows through ecosystems. Two biogeochemical
25 models were parameterized to capture long-term effects of O3 exposure, N deposition, and
26 climate on a Ponderosa pine-dominated site in the eastern San Bernardino Mountains.
27 Simulated O3 exposure resulted in faster production and turnover of foliage and a shift in C from
28 the canopy (15% reduction) to the forest floor (increase of 50 to 60%) (Arbaugh et al., 1999).
29 When O3 exposure was combined with that of N deposition, litter mass increased exponentially.
30 The direct effect of O3 exposure on below-ground nutrient dynamics and ecosystem
31 material flow is poorly understood. Additional research will be necessary to understand spatial
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1 and temporal dynamics of nutrient and C flow in ecosystems and to separate the effects of O3
2 from those attributable to N deposition.
3
4 AX9.6.8 Hydrological and Geomorphological
5 At present, there are no publications on the effects of O3 exposure that are carried through
6 at the ecosystem level to changes in mass water flow, channel morphology, riparian habitat
7 complexity, or sediment movement. It is possible that processes occurring at smaller scales are
8 affecting geomorphological processes in ecosystems; however, difficulty in scaling these
9 responses spatially and temporally have made it difficult to show experimentally. It is possible
10 that O3 exposure affects water quality through changes in energy and material flows, as
11 discussed previously.
12
13 AX9.6.9 Natural Disturbance Regimes
14 There has been little research on how natural disturbances interact with O3 to affect
15 performance of plants, communities, and ecosystems. The frequency, intensity, extent, and
16 duration of natural disturbances are variable and unpredictable. However, there have been
17 enough ecophysiological studies to suggest that O3 could predispose plant communities to
18 certain natural stresses, e.g., drought stress or extreme low-temperature stress during the winter.
19 While several studies have shown that drought stress reduces O3 uptake through stomatal
20 closure, evidence also suggests that O3 can alter plant water use and susceptibility to drought. In
21 controlled studies, Reich and Lassoie (1984) showed that relatively low O3 concentrations could
22 diminish stomatal control and alter water use efficiency. Ash trees (Fraxinus excelsior) exposed
23 to elevated O3 had greater water use early in the growing season, but less water use late in the
24 growing season when exposed to elevated O3 (Wiltshire et al., 1994). Under moderate drought
25 stress, Norway spruce trees grown under elevated O3 consumed water faster and showed higher
26 stomatal conductances than controls (Karlsson et al., 1995). Pearson and Mansfield (1993)
27 showed that successive O3 episodes disrupted stomatal function, making beech seedlings more
28 susceptible to drought. Previous year O3 exposure was shown to have a carry-over effect in the
29 following growing season for beech (Pearson and Mansfield, 1994).
30 Few studies showing the effects of O3 on water relations of field-grown trees are found in
31 the literature. However, Grulke et al. (2003a) examined the effects of O3 on canopy transpiration
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1 of Jeffrey pine from mesic and xeric microsites and found that trees from mesic sites had 20%
2 more O3 uptake than those in the xeric sites. The authors also concluded that the mesic trees had
3 greater O3 injury as evidenced by lower needle retention, whereas trees in xeric microsites had
4 greater chlorotic mottle. Chlorotic mottle induced by stomatal uptake of O3 is indistinguishable
5 from that of endogenously produced oxidants resulting from partially closed stomata, a reduction
6 of CO2 inside the leaf, and production of strong oxidizers within the chloroplast when excited
7 electrons are passed to O2 instead of CO2 under high light levels.
8 Trees living near the limits of their freezing-tolerance range may be especially susceptible
9 to predisposition of freezing injury by O3 (Sheppard et al., 1989). However, Aleppo pine
10 exposed to elevated O3 had enhanced winter hardiness (Wellburn and Wellburn, 1994). As with
11 the seasonal carryover of drought susceptibility, the influence of elevated O3 on freezing
12 tolerance is carried over from summer to winter. Such effects have been demonstrated for Sitka
13 spruce (Lucas et al., 1988) and for red spruce (Waite et al., 1994). Sorting out the role of
14 elevated O3 in contributing to frost or low-temperature damage in forests remains difficult due to
15 the presence of other factors that may affect senescence.
16
17 AX9.6.10 Scaling to Ecosystem Levels
18 The vast majority of literature describing O3 effects comes from short-duration herbaceous
19 plant or tree seedling studies under controlled conditions. Scaling results from these studies
20 requires extrapolation over both space and time in order to understand the full extent of changes
21 in ecosystems. In addition to spatial, temporal, and age-related complexities, ecosystems are
22 composed of organisms whose lifetimes range from hours to centuries (Laurence and Andersen,
23 2003). Forested ecosystems are affected by environmental conditions such as water and nutrient
24 availability, as well as by intra- and interspecies competition. Therefore, direct experimentation
25 to determine the response of forested ecosystems is not simply a matter of determining the effect
26 of O3 on individual mature trees. In addition, even if an experiment can be conducted,
27 extrapolation of the results across landscapes and regions remains challenging. Nonetheless,
28 models provide a means to explore possible long-term changes and to identify important
29 research uncertainties.
30 Approaches to scaling fall roughly into two categories: (1) process-based modeling to
31 extrapolate physiological responses to O3 based on seedling studies and (2) field assessments
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1 using surveys and growth correlations, often in association with stand-level models to address
2 ecosystem complexity. Comparatively good information is available on process level effects
3 of O3 in seedlings and, therefore, some models offer the opportunity to use this information to
4 scale O3 effects at the stand and regional scales (Chappelka and Samuelson, 1998; Fuhrer et al.,
5 1997; Hogsett et al., 1997; Laurence et al., 2000). Field assessments offer the opportunity to
6 examine larger plot sizes, older trees, and trees growing under realistic competition. These two
7 approaches are discussed in more detail below.
8
9 AX9.6.10.1 Scaling from Seedlings to Mature Trees
10 A number of investigators have used simulation models based on physiological processes
11 to integrate available data and predict the effects of O3 on mature trees. Such models predict tree
12 growth by simulating fundamental mechanisms, rather than through a statistical analysis of
13 empirical data. For instance, the process of photosynthesis is simulated based on environmental
14 conditions and physiological characteristics, and then the fixed C is allocated to plant growth
15 using principles of plant physiology. Models based on mechanisms should be applicable across
16 wide areas if the important functional relationships are represented accurately in the models and
17 if the environmental conditions are accurately identified. The ability of six models (TREGRO,
18 CARBON, ECOPHYS, PGSM, TREE-BGC, and W91) to simulate the effects of climatic
19 change and O3 have been reviewed by Constable and Friend (2000). Of these models, only
20 PGSM and TREGRO explicitly simulated the effects of O3 on foliar processes.
21 The TREGRO model was used to simulate C allocation and tissue growth in seedlings and
22 mature red oak trees based on the experimental data discussed above (Weinstein et al., 1998).
23 For seedlings at 2x-ambient O3, only the total nonstructural carbohydrate (TNC) storage pool
24 was predicted to be affected. For mature trees, large decreases were predicted for TNC, leaves,
25 stem, branch, and both fine and coarse roots. Most predicted effects in mature trees were
26 consistent with observations in the field, but the simulations overestimated the effect of
27 2x-ambient O3 on root TNC and growth. The authors suggested that this discrepancy may have
28 been due to trees reducing respiration in response to O3 stress, a response not simulated in the
29 model.
30 For Abies concolor, TREGRO was parameterized and simulated growth of a mature tree
31 for 3 years to test for effects of O3 exposure and drought stress (Retzlaff et al., 2000).
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1 Reductions in O3 exposure-mediated carbon assimilation were translated to losses in whole tree
2 biomass that probably would not be detectable in the field. However, TNC levels in branch
3 tissue were simulated to be lowered by over 50%, and branch growth was reduced in a
4 moderately polluted site relative to a clean site. Low O3 exposure (sufficient to decrease C
5 assimilation by 2.5%) and drought stress (25% reduction in annual precipitation, which is
6 common on a decadal scale) acted synergistically to reduce C gain of whole tree biomass of
7 A. concolor. Simulated results of the tests were comparable to effects found in OTCs for
8 seedlings and pole-sized trees in clean and moderately polluted sites.
9 Models such as TREGRO are usually parameterized from many different sources of data,
10 including chamber experiments and plantations, from seedlings to mature trees, making it
11 difficult to validate that they reproduce changes that occur as trees develop from seedlings to
12 maturity. To address this issue, physiological and growth data were collected from a natural
13 stand of P. ponder osa and used to parameterize the TREGRO model (Grulke and Retzlaff,
14 2001). Representative trees of each of five tree age classes were selected based on population
15 means of morphological, physiological, and nearest neighbor attributes. Seedlings were
16 observed to differ significantly from pole-sized and older trees in most physiological traits. The
17 changes in biomass with tree age predicted from the model closely matched those of trees in the
18 natural stand.
19 The PGSM model was used to simulate Pinus ponderosa seedling growth responses to O3
20 exposure and drought stress (Chen et al., 1994). Drought stress was predicted to reduce the
21 effect of O3 on growth, as was observed in the experimental data. The TREGRO model was
22 used to simulate responses of Pacific Coast and interior varieties of P. ponder osa to five
23 simulated O3 exposures between subambient and 3x-ambient (Constable and Taylor, 1997).
24 Simulated growth of var. ponderosa was reduced more than that in var. scopulorum with all O3
25 exposures. Drought was protective of O3 exposure. Similar results were also found in a
26 relatively moist Ponderosa pine plantation (Panek and Goldstein, 2001), whereas drought was
27 synergistically deleterious with cumulative O3 exposure in a natural stand (Grulke et al., 2002b).
28 For Pinus taeda, the Plant Growth Stress Model (PGSM) was calibrated with seedling data
29 and then used to simulate the growth of mature trees over a 55 year period in the Duke Forest,
30 NC, using estimates of historical O3 concentrations (Chen et al., 1998). Simulated stem diameter
31 and tree height were comparable to observed values. In another simulation using TREGRO,
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1 loblolly pine was more sensitive (greatest reduction in C gain) to a peak O3 episode in July
2 (Constable and Retzlaff, 1997), whereas mature yellow poplar (Liriodendron tulipiferd) was
3 more sensitive to a peak O3 episode in August.
4 For Populus tremuloides, the ECOPHYS model was used to simulate the relative above-
5 ground growth response of an O3-sensitive clone (259) exposed to square-wave variation in O3
6 concentration (Martin et al., 2001). The model adequately simulated several effects of O3,
7 including a greater effect on stem diameter than on stem height, earlier leaf abscission, and
8 reduced stem and leaf dry matter production at the end of the growing season. For Acer
9 sacchamm, the TREGRO model was use to predict effects of a 10-year O3 exposure on root and
10 stem growth of a simulated 160-year-old tree (Retzlaff et al., 1996). Twice-ambient O3 exposure
11 (for Ithaca, NY) was predicted to deplete the TNC pools and reduce fine root production.
12
13 AX9.6.10.2 Surveys, Growth Correlations and Stand-Level Modeling
14 Stand-level studies have included surveys of O3 symptoms, correlations of radial growth
15 with O3 and other environmental factors, and regional-scale modeling. In addition, open air O3
16 exposure systems, such as those being used on Populus tremuloides-mixed stands in northern
17 Wisconsin (Karnosky et al., 2003b) and on Fagus sylvatica and Picea abies in Germany (Nunn
18 et al., 2002) offer an opportunity to examine larger plot sizes, older trees, and trees growing
19 under realistic competition. Plots along natural O3 gradients, as have been used very effectively
20 in southern California forest studies (Miller and McBride, 1999b) and with P. tremuloides stands
21 in the Great Lakes region (Karnosky et al., 1999), offer additional insights into ecosystem level
22 responses. Undoubtedly, however, simulation modeling will have to become an integral
23 component of research in order to predict adequately ecosystem responses to O3 (Laurence and
24 Andersen, 2003). Results of these approaches are discussed below, organized into three U.S.
25 regions: (1) northern states (including the upper Midwest and the Northeast), (2) southeastern
26 states, and (3) western states (primarily California). A fourth section contains selected
27 information from Europe.
28
29 Northern and Midwestern United States. In recent years, the USD A Forest Service has
30 conducted systematic O3 biomonitoring surveys in most north-central and northeastern states
31 (Coulston et al., 2003; Smith et al., 2003). Plots are located on a systematic grid, and trained
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1 field crews evaluate up to 30 plants of up to six species that have foliar injury symptoms
2 diagnostic of O3 damage. For the United States as a whole, injury has been found more often in
3 eastern than in interior or west-coast states. As expected, O3 injury is more common and more
4 severe in areas with higher O3 concentrations. Of sampled Prunus serotina plots, -12% were
5 estimated to be at high risk for injury based on a injury index derived from the survey data
6 (Coulston et al., 2003). P. serotina was estimated to be at risk for injury on the Allegheny
7 Plateau and the Allegheny Mountains (in Pennsylvania, West Virginia, and Maryland), as well as
8 in the coastal plain of Maryland and Virginia.
9 Ozone concentration, foliar injury, and physiological traits were measured on P. serotina
10 trees of different sizes in Pennsylvania (Fredericksen et al., 1995). The proportion of foliage
11 injured was 46% for seedlings, 15% for saplings, and 20% for canopy trees. Cumulative O3 flux
12 was the most useful O3 metric for predicting injury. Injury was negatively correlated (r2 = 0.82)
13 with net photosynthetic rates, but was not related to stomatal conductance. P. serotina is
14 discussed further below, because detailed surveys have been conducted in the Shenandoah and
15 Great Smoky Mountains NPs in the southeastern United States.
16 Over the past several decades, some surveys of white pine (Pinus strobus) have reported
17 significant associations between foliar injury and reduced growth (Anderson et al., 1988; Benoit
18 et al., 1982). However, a review of 93 surveys conducted from 1900 through the late 1980s
19 concluded that methodological problems were pervasive, including such issues as proximity to
20 roads, lack of peer review, lack of random sampling, small sample sizes, and lack of quantitative
21 methods to estimate severity (Bennett et al., 1994). Because of these problems, along with
22 evidence of adequate growth rates for P. strobus regionally and contradictory evidence from
23 numerous studies of symptom production in response to controlled O3 exposure, these authors
24 concluded that there was no clear evidence of decline in P. strobus (Bennett et al., 1994).
25 A more recent study in Acadia National Park (ANP) in Maine found no association between O3
26 exposure in OTCs and symptom development in P. strobus, calling into question whether
27 symptoms previously ascribed to O3 may be caused by some other stress (Kohut et al., 2000).
28 However, another ANP study found significant correlations between O3 exposure and the radial
29 growth of trees during 10 years in 7 of 8 stands examined (102 trees total; (Bartholomay et al.,
30 1997). Taken together, these results suggest that there may not an association between growth of
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1 P. strobus trees and putative O3 symptoms, but there may be an association between O3 exposure
2 and radial growth of mature trees in the field.
3 The study of O3 effects was undertaken from 1990 to 1993 in the ANP in Maine, because
4 this location experiences elevated O3 exposures due to transport from urban areas located upwind
5 (Kohut et al., 2000). Thirty-two species of plants found in the park were propagated and
6 exposed to O3 in OTCs. In addition, ambient O3 concentrations were monitored at the study site
7 at 15 m above sea level and near the top of Cadillac Mountain at 470 m above sea level. At the
8 study site, the maximum 1-h O3 concentration was 140 ppb, which occurred in both 1990 and
9 1991. Daytime 12-h O3 concentrations were 35, 41, 36, and 37 ppb during the four years; and O3
10 concentrations were consistently higher at the high-elevation site. Species showing foliar injury
11 at ambient O3 concentrations included Prunus serotina, Populus tremuloides, Fraxinus
12 americana, Pinus banksiana, big-leaf aster, and spreading dogbane. Species showing foliar
13 injury at 1.5 x ambient O3 concentrations included Betulapopulifolia, small sundrops, and
14 bunchberry. Species remaining uninjured at 2 x ambient O3 concentrations included Betula
15 papyri/era., Pinus strobus., Pinus rigida, Picea rubens, Thuja occidentalis, Quercus robur,
16 Canada bluejoint grass, wild radish, and Canada mayflower. Because of their O3 sensitivity and
17 diagnostic symptoms, big-leaf aster, spreading dogbane, Populus tremuloides, Fraxinus
18 americana, and Prunus serotina were recommended as bioindicators for the ANP.
19 The PnET-II model was applied to 64 locations across the northeastern United States to
20 simulate the effects of ambient O3 on mature hardwood forests (Ollinger et al., 1997). In this
21 model, O3 effects on each of several layers of the forest canopy were represented by a single
22 linear equation relating predicted O3 uptake to decreased net photosynthetic rate. Wood growth
23 was predicted to decrease between 3 to 22%, with greatest reductions in southern portions of the
24 region where O3 levels were highest and on soils with high water-holding capacity where
25 drought stress was absent. Little variation was predicted among years, because high O3 often
26 coincided with hot, dry weather conditions that reduced predicted stomatal conductance and O3
27 uptake.
28 In order to estimate the impact of O3 on forests, effects must be evaluated not only on
29 individuals, but also on mixtures of species and the composition of forest stands. The PnET
30 model described above evaluated the effects of O3 on broad forest types (an evergreen/deciduous
31 mix), but did not address specific forest species composition. In order to address competition
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1 among species, the TREGRO model was linked to the ZELIG forest stand growth model and a
2 geographic information system was created to predict the effects of O3 across the north-central
3 and northeastern United States (Laurence et al., 2000). ZELIG is a gap-succession model used
4 to simulate succession in mixed stands typical of eastern and northern forests. Ambient O3
5 generally caused a reduction of 2 to 4% in the growth ofQuercus rubra across the region during
6 the 100-year simulation. The response followed the pattern of O3 exposure, with little effect in
7 the northwest part of the region, but with greater effect in southern locations. The O3 response of
8 Acer saccharum to O3 varied widely, but the overall growth response was always positive,
9 indicating that the evergreen/deciduous mix was able to take advantage of the decrease in the
10 growth of Q. robur and other species caused by O3. In the northernmost part of the region, A.
11 saccharum growth increased by up to 3%, but in the southern part of the region, its growth
12 increased by up to 12%. The authors ascribed this enhanced growth to a combination of warmer
13 temperatures and reduction in the growth ofPmnus serotina, a minor component of the
14 simulated stand that was very sensitive to O3.
15
16 Southeast United States. In a survey of the Great Smoky Mountains NP, foliar injury
17 attributed to O3 was found on 47% of the more than 1,600 plants examined (Chappelka et al.,
18 1997). In subsequent surveys of injury in the park, injury was found on mature trees of the
19 following species: Sassafras albidum, Prunus serotina, and Liriodendron tulip/era (Chappelka
20 et al., 1999b,c). In a similar study in Shenandoah National Park, injury was found on Fraxinus
21 americana, P. serotina, andZ. tulip/era (Hildebrand et al., 1996).
22 For Prunus serotina seedlings grown in soil in OTCs and exposed to relatively low
23 ambient levels of O3 in Pennsylvania, there was no correspondence between visible foliar stipple,
24 leaf gas exchange, and seedling growth between two families previously shown to differ in O3
25 symptoms (Kouterick et al., 2000). However, significant exposure-response relationships were
26 found for foliar injury in the Great Smoky Mountains and Shenandoah National Parks. In each
27 park, foliar injury was evaluated on mature P. serotina trees on three plots at different elevations
28 near O3 monitors during 1991 to 1993 (Chappelka et al., 1999a,b; Hildebrand et al., 1996). In
29 1991, incidence was 60% and 45% for the two parks and 33% in both parks during 1992 and
30 1993. Symptoms were greater at the highest elevations where O3 concentrations were highest.
31 In another study, radial growth rates were measured in 44 P. serotina trees ranging in age from
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1 19 to 56 years old with and without O3 symptoms, at three sites in the Great Smoky Mountains
2 National Park. Trees with O3 symptoms were compared to similar-sized trees with few
3 symptoms. There was no evidence that trees with O3 symptoms had lower growth rates (p = 0.6)
4 (Somersetal., 1998).
5 In the Great Smoky Mountains National Park, radial growth rates were measured for 44 L.
6 tulip/era trees ranging in age from 30 to 58 years old, with and without O3 symptoms, at three
7 sites at different elevations (Somers et al., 1998). Trees with O3 symptoms averaged 30% lower
8 growth rates over ten years (p = 0.0005). Seedlings of Liriodendron tulip/era were exposed for
9 two seasons to 2x-ambient O3 exposures in OTCs in Delaware, OH (seasonal SUMOO exposures
10 of 107 and 197 ppmh) (Rebbeck, 1996). Foliar O3 symptoms were observed, but growth was not
11 reduced.
12 In order to evaluate the influence of interspecies competition on O3 effects, the linked
13 TREGRO and ZELIG modeling system was used to predict the effects of O3 over 100 years on
14 the basal area of species in a Liriodendron tulipfera-dominated forest in the Great Smoky
15 Mountains NP (Weinstein et al., 2001). Ambient O3 was predicted to reduce the basal area of L.
16 tulip/era by 10%, whereas a 1.5 x -ambient exposure was predicted to cause a 30% reduction.
17 Basal area of Acer rubrum and Prunus serotina was predicted to increase for some years, but
18 then decrease by up to 30%, with few changes in the total basal area of all species by the end of
19 the simulation.
20 In order to evaluate the influence of interspecies competition on O3 effects, the linked
21 TREGRO and ZELIG modeling system was used to predict the effects of O3 on the basal area of
22 Pinus taeda and Liriodendron tulip/era growth throughout their ranges (Laurence et al., 2003).
23 The models were parameterized using biological and meteorological data from three sites in the
24 southeastern United States (in Alabama, Louisiana, and North Carolina). Forest stand response
25 to five O3-exposure regimes with annual SUM06 values ranging from 0 to 100 ppmh per year
26 was simulated for 100 years. The simulated basal area of the two species was generated within
27 the context of four other tree species common in southeastern forests. Basal area of P. taeda was
28 highly responsive to precipitation and O3 exposure, with the greatest increases under high-
29 precipitation, lowO3-exposure scenarios and the greatest decreases under low-precipitation, high
30 O3-exposure scenarios. The basal area of L. tulipifera did not significantly differ (+10%) from
31 simulations using a "base case" (ambient O3, average precipitation).
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1 Systematic biomonitoring surveys found that approximately 24% of sampled sweetgum
2 and 15% of sampled Pinus taeda plots were estimated to be at high risk for foliar injury on the
3 coastal plain of Maryland and Virginia (Coulston et al., 2003; Smith et al., 2003). In a study in
4 Tennessee, the effect of ambient (uncontrolled) O3 on 28 mature canopy-dominant 50 to
5 90-year-old P. taeda trees in five stands was measured over a 5-year period (McLaughlin and
6 Downing, 1995, 1996). Of many O3 metrics, a 3-day average of hourly O3 values > 40 ppb
7 (AOT40) was found to best explain short-term variation in stem expansion as measured with
8 dendrometer bands. Interactions between O3, temperature, and drought stress (as indicated by
9 the weekly moisture stress index) accounted for 63% of the short-term variation in stem growth
10 rates. Because there are interactions among O3, drought stress, and temperature that may differ
11 with the averaging time (days to years), this type of study cannot provide conclusive proof of
12 cause and effect (Reams et al., 1995). However, the results do suggest that the effects of O3
13 measured on loblolly seedlings may also be occurring in mature trees in both wet and dry sites.
14 The magnitude of effects of O3 on growth, including interactions with other variables in this
15 study, ranged from 0 to 15% over 5 years, with an average of 5.5%.
16
17 Western United States. The USD A Forest Service conducted O3 biomonitoring surveys in
18 Washington, Oregon, and California during one year (1998), and in the Sierra Nevada and
19 Sequoia NFs every other year for several decades (Campbell et al., 2000). Overall, only one plot
20 showed any symptoms of O3 injury outside of the Sierra Nevada and Sequoia National Forests.
21 In the Sierra Nevada National Forest, between 30 and 40% of trees showed injury from 1989
22 through 1997. In the Sequoia National Forest, between 40 and 50% of the trees surveyed
23 showed injury from 1990 through 1998.
24 For Pinus ponder'osa along a well-studied gradient of O3 exposure in the San Bernardino
25 mountains, chlorotic mottle was highest on foliage at the most polluted site, as has been found
26 previously (Grulke and Balduman, 1999). Based on whole-tree harvests, root biomass was
27 lowest at the most polluted sites, confirming previous studies with seedlings under controlled
28 conditions, as discussed above. Ozone responses in highly polluted environments such as
29 Southern California may not be predicted adequately by extrapolating effects from single-factor
30 experiments. Instead, combined approaches utilizing field experiments and modeling efforts
31 may be required to properly account for a combination of stressors including O3, N deposition,
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1 and drought. Furthermore, the available studies underscore the lack of correlation between O3
2 symptoms and mature tree effects. If a field survey fails to find a correlation between mature
3 tree growth and O3, this result may be due to the dominant effect of another factor such as N
4 deposition and may not be evidence that O3 does not reduce the growth of mature trees.
5 The TREGRO model was used to evaluate how projected future temperature and CO2
6 concentrations might affect the response of individual Ponderosa pine to O3 at seven sites in
7 California, Oregon, and Washington (Tingey et al., 2001). As expected, growth decreased with
8 increasing O3 exposure. Differences in O3 response among sites appeared to be due primarily to
9 differences in precipitation.
10 Often air quality standards do not translate directly into measurable improvements in tree
11 growth or productivity. To evaluate whether past improvements in air quality have improved
12 Ponderosa pine growth, TREGRO was used to simulate growth at sites in the San Bernardino
13 Mountains in California (Tingey et al., 2004). Ozone and meteorological data from the past
14 37 years was used to run the simulations. Despite variation in precipitation and temperature, O3
15 was found to reduce simulated tree growth. The authors were able to simulate growth
16 improvements as air quality improved during the 1980s and 1990s, suggesting that
17 improvements in emission control strategies benefitted Ponderosa pine. The model simulations
18 were qualitatively consistent with improvements in canopy condition that were observed at sites
19 where O3 reductions were the greatest.
20
21 Studies in Europe. In a 4-year study ofFagus sylvatica in Switzerland at 57 forest sites
22 ranging in age from 65 to 173 years, stem increment was found to decrease with increasing
23 maximum O3 exposure (Braun et al., 1999). In this study, O3 concentration was estimated by
24 interpolation among monitoring stations, and other site conditions such as soil water status and
25 temperature were interpolated from weather stations. Other factors such as N deposition, tree
26 diameter, and canopy dominance were also found to be significantly associated with stem
27 increment. The maximum annual O3 dose (expressed as AOT40) was found to be more strongly
28 associated with decreased stem increment than was the average O3 dose over the 4 years.
29 A growth reduction of 22.5% (confidence interval 14.3 to 28.6%) was associated with each
30 10 ppmh increment of O3 (expressed as AOT40). This decrease was steeper than the 6.1%
31 growth reduction summarized previously from several OTC studies with F. sylvatica seedlings
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1 (Fuhrer et al., 1997). However, the authors suggest that this difference may be explained largely
2 by the 4 years of exposure in their forest survey study as compared to the 1-year exposures for
3 seedlings. As with any forest survey, these results must be interpreted with caution because O3
4 exposure was correlated with other variables, such as tree age and the deposition of NO2
5 and SO2.
6
7 AX9.6.11 Summary of Ecological Effects of Ozone Exposure on Natural
8 Ecosystems
9 In this chapter, an effort has been made to discuss the adverse effects of O3 on natural
10 ecosystems within the context of the SAB framework for assessing and reporting ecological
11 conditions (Young and Sanzone, 2002) (Figure AX9-21). Using this framework, there is
12 evidence that tropospheric O3 is an important stressor of natural ecosystems, with
13 well-documented impacts on the biotic condition, ecological processes, and chemical/physical
14 nature of natural ecosystems. In turn, the effects of O3 on individual plants and processes are
15 scaled up through the ecosystem affecting processes such as energy and material flow, intra- and
16 interspecies competition, and NPP. Thus, effects on individual keystone species and their
17 associated microflora and fauna may cascade through the ecosystem to the landscape level. This
18 suggests that by affecting water balance, cold hardiness, tolerance to wind, and by predisposing
19 plants to insect and disease pests, O3 may even influence the occurrence and impact of natural
20 disturbance. Despite the probable occurrence of such effects, however, there are essentially no
21 instances where highly integrated ecosystem-level studies have conclusively shown that O3 is
22 indeed altering ecosystem structure and/or function.
23 Systematic injury surveys demonstrate that foliar injury occurs on O3-sensitive species in
24 many regions of the United States. However, the frequent lack of correspondence between foliar
25 symptoms and growth effects means that other methods must be used to estimate the regional
26 effects of O3 on tree growth rates. Investigations of the radial growth of mature trees, in
27 combination with data from many controlled studies with seedlings, as well as a few studies with
28 mature trees suggest that ambient O3 is reducing the growth of mature trees in some locations.
29 Studies using models based on tree physiology and forest stand dynamics suggest that modest
30 effects of O3 on growth may accumulate over time and may interact with other stresses. For
31 mixed-species stands, such models predict that overall stand growth rate is generally not likely to
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Hydrolie Alteration
Habitat Conversion
Habitat Fragmentation
Climate Change
Invasive Non-native Species
Turbidity/Sedimentation
Pesticides
Disease/Pest Outbreaks
Nutrient Pulses
Metals
Dissolved Oxygen Depletion
Ozone (Tropospheric)
Nitrogen Oxides
Nitrates
Hydroiic Alteration
Habitat Conversion
Habitat Fragmentation
Climate Change
Over-Harvesting Vegitation
Large-Scale Invasive
Species Introduction
Large-Scale Disease/Pest
Outbreaks
Hydroiic Alteration
Habitat Conversion
Climate Change
Over-Harvesting Vegitation
Disease/Pest Outbreaks
Altered Fire Regime
Altered Flood Regime
Hydroiic Alteration
Habitat Conversion
Climate Change
Turbidity/Sedimentation
Pesticides
Nutrient Pulses
Metals
Dissolved Oxygen Depletion
Ozone (Tropospheric)
Nitrogen Oxides
Nitrates
Sulfates
Salinity
Acidic Deoosition
t/J
£
+•>
w
Hydroiic Alteration
Habitat Conversion
Climate Change
Pesticides
Disease/Pest Outbreaks
Nutrient Pulses
Dissolved Oxygen Depletion
Nitrogen Oxides
Nitrates
Sylfates
Hydroiic Alteration
Habitat Conversion
Habitat Fragmentation
Climate Change
Turbidity/Sedimentation
Figure AX9-21. Common anthropogenic stressors and the essential ecological attributes
they affect.
Source: Modified from Young and Sanzone (2002).
1
2
3
4
be affected. However, competitive interactions among species may change as a result of growth
reductions of sensitive species. These results suggest that O3 exposure over decades may be
altering the species composition of forests in some regions.
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1 RESEARCH NEEDS
2 The knowledge base for examining the range of ecological effects of O3 on natural
3 ecosystems is growing, but significant uncertainties remain regarding O3 effects at the ecosystem
4 level. For example, there is a need for information on the following ecosystem-level responses:
5 • Ecosystem Processes. Little is known about the effects of O3 on water, C, and nutrient
cycling, particularly at the stand and community levels. Effects on belowground
ecosystem processes in response to O3 exposure alone and in combination with other
stressors are critical to projections at the watershed and landscape scales. Little is yet
known about the effects of O3 on structural or functional components of soil food webs, or
how these impacts could affect plant species diversity (Andersen, 2003).
6 • Biodiversity and Genetic Diversity. The study of genetic aspects of O3 impacts on natural
ecosystems has been largely correlational in nature and it remains to be shown
conclusively whether O3 affects biodiversity or genetic diversity (Davison and Barnes,
1998; Pitelka, 1988; Winner et al., 1991). Studies of competitive interactions under
elevated O3 levels are needed (Laurence and Andersen, 2003), and reexamination via new
sampling of population studies to bring in a time component to previous studies showing
spatial variability in population responses to O3 are needed. These studies could be
strengthened by modern molecular methods to quantify impacts on diversity.
7 • Natural Ecosystem Interactions with the Atmosphere. Little is known about feedbacks
between O3 and climate change on volatile organic compound (VOC) production, which in
turn, could affect O3 production (Fuentes et al., 2001). At moderate-to-high O3 exposure
sites, aberrations in stomatal behavior could significantly affect individual tree water
balance of sensitive trees, and if the sensitive tree species is dominant, hydrologic balance
at the watershed and landscape level could be affected. This has not been addressed in any
model because O3 exposure effects, if included in the modeling effort have assumed a
linear relationship between assimilation and stomatal conductance.
8 • Below-Ground Inter actions. While the negative effects of O3 on below ground growth are
well characterized, interactions of roots with the soil or microorganisms are not.
9 • Other Interactive Effects. Interaction studies with other components of global change
(e.g.,warming, increasing atmospheric CO2, N deposition, etc.) or with various biotic
stressors are needed to better predict complex interactions likely in the future (Laurence
and Andersen, 2003). Whether O3 will negate the positive effects of an elevated CO2
environment on plant carbon and water balances is not yet known; nor is it known if these
effects will scale up through the ecosystem. How might O3 affect the progress of pest
epidemics and insect outbreaks as concentrations increase is unclear (Ball et al., 1998).
10 • Reproduction Effects. Information concerning the impact of O3 on reproductive processes
and reproductive development under realistic field or forest conditions are needed as well
as examination of reproductive effects under interacting pollutants (Black et al., 2000).
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1 • Comparative Extrapolation. The vast majority of O3 studies of trees have been conducted
with young, immature trees and in trees that have not yet formed a closed canopy.
Questions remain as to the comparability of O3 effects on juvenile and mature trees and on
trees grown in the open versus those in a closed forest canopy in a competitive
environment (Chappelka and Samuelson, 1998; Kolb and Matyssek, 2001; Samuelson and
Kelly, 2001).
2 • Scaling-Up Issues. Scaling the effects of O3 from the responses of single or a few plants
to effects on communities and ecosystems is a complicated matter that will require a
combination of manipulative experiments with model ecosystems, community and
ecosystem studies along natural O3 gradients, and extensive modeling efforts to project
landscape-level, regional, national and international impacts of O3. Linking these various
studies via impacts on common research quantification across various scales using
measures of such factors as leaf area index or spectral reflective data, which could
eventually be remotely sensed (Kraft et al., 1996; Panek et al., 2003), would provide
powerful new tools for ecologists.
3 • Comparative Risk Assessment Methodologies. Methodologies to determine the important
values of services and benefits derived from natural ecosystems such that these could be
used in comprehensive risk assessment for O3 effects on natural ecosystems (Heck et al.,
1998).
4
5
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1 AX9.7 ECONOMIC EVALUATION OF OZONE EFFECTS ON
2 AGRICULTURE, FORESTRY AND NATURAL ECOSYSTEMS
3 AX9.7.1 Introduction
4 The adverse consequences of ambient air pollutant exposures on vegetation, ecosystems,
5 and components of the material environment have been documented since the beginning of the
6 industrial revolution. Attempts to quantify the monetary damage and injury resulting from
7 tropospheric O3 exposures to managed agriculture, forests, and natural ecosystems date back at
8 least to the 1950s.
9 Both methodological and data problems plagued early efforts to assess the monetary
10 damages of air pollution to crops and natural vegetation. Adams and Crocker (1989) discussed
11 the methodological issues, e.g., a lack of reliable data on effects from air pollutants on crop
12 yields or the failure to develop and apply appropriate economic models. Some of these problems
13 were remedied by the EPA's National Crop Loss Assessment Network (NCLAN) in the 1980s.
14 The EPA's NCLAN facilitated the performance of economic assessments by providing O3-crop
15 yield data with which to estimate O3-crop yield response functions (see Heagle [1988] for a
16 review of NCLAN procedures and findings). NCLAN also funded a series of economic
17 assessments that, along with subsequent economic assessments, documented substantial
18 economic damages to agriculture. (See Spash [1997] for a detailed review of economic
19 assessments, many of which used NCLAN data.)
20 Since the completion of the NCLAN program in the late 1980s, the number of economic
21 assessments of air pollution studies focusing on terrestrial ecosystems in general, and agriculture
22 in particular, has declined. For example, for the period of 1980 to 1990, 33 economic studies
23 of O3 and other air pollutant effects on U.S. crops were published in peer-reviewed journal
24 outlets (Spash, 1997). However, in preparing this section of the current criteria document, only
25 four peer-reviewed economic assessments were found for the decade of 1991 to 2000 that
26 addressed vegetation in the United States. In addition, one peer-reviewed article (Kuik et al.,
27 2000) was found dealing with agriculture in the Netherlands. Recent interest in global climate
28 change and the potential effects of global warming on O3 and other photochemical oxidants, has
29 renewed interest in the effects of air pollution on both managed and unmanaged terrestrial
30 ecosystems (Adams et al., 1998). In addition, concern is growing regarding the effects of air
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1 pollutants on natural ecosystems and on the services they provide (Daily, 1997). Unfortunately,
2 this interest has not yet translated into additional peer-reviewed publications addressing O3 or
3 other air pollutant effects on ecosystems.
4 This section of the current criteria document first discusses the availability of economic
5 information and its usefulness in forming environmental policy. Next, economic assessments of
6 air pollution effects and findings from the 1996 AQCD (U.S. Environmental Protection Agency,
7 1996) are discussed, followed by a synthesis of the limited literature available since the 1996
8 AQCD with respect to O3 effects on agriculture, forestry, and ecosystems. Finally, limitations
9 and continuing uncertainties are reviewed. The most fundamental of these is the lack of
10 measurements of the economic effects of air pollution on natural ecosystems. Other issues
11 include the variability of performance in both managed and natural ecosystems under increased
12 climatic and air pollution variability as well as the challenges related to spatial and temporal
13 scales used in performing economic assessments. To date, this set of effects has been sparsely
14 addressed.
15
16 AX9.7.2 The Measurement of Economic Information
17 Economic science is an exercise in deductive logic in which testable hypotheses about the
18 behavior of economic agents (i.e., farmers, consumers, resource owners) and markets are
19 deduced from a body of theory. That body of theory is based on a series of premises proposed
20 by economists and philosophers dating back over two centuries to Adam Smith. These premises
21 gradually evolved into a theoretical foundation primarily dealing with microeconomics and
22 culminating in structural relationships that define the operation of markets. This foundation was
23 first laid out in a comprehensive and rigorous fashion by Alfred Marshall in 1920. Samuelson
24 (1948) formalized these theoretical relationships, resulting in what is sometimes referred to as
25 modern, or neoclassical, economics.
26 The insights gained from the theoretical foundation of economics helped shape the nature
27 of applied economic, or policy-relevant research. An example of such applied research is when
28 economists seek to measure the economic consequences of air pollution on agriculture. Such an
29 application is described in Adams and Horst (2003), who provided a graphical representation of
30 the measurement of the effects of air pollution on the well-being of producers and consumers.
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1 Economic theory is applied to real-world problems when the methods of economics and the need
2 for data from other disciplines come into play. When measuring the economic effects of an
3 environmental change, economists need an economic model or method that is theoretically
4 consistent, i.e, defensible, as well as data with which to estimate both economic and
5 environmental science relationships for use in the model. Among accepted economic assessment
6 methods, the actual choice is frequently determined by the nature of the problem to be addressed.
7 It should be noted that the choice of assessment method can affect the type of economic
8 information that is obtained. Even with a given assessment method, results are sensitive to
9 specific data treatment or assumptions (Adams, 1999). For example, some methods only
10 measure effects on a particular group e.g., farmers. Other methods may measure effects across
11 several groups. Thus, one should not expect the magnitudes of damage or benefits to be
12 identical across economic assessments. One should, however, expect that the direction of the
13 effects will be similar.
14 Once it is established that an assessment meets basic economic criteria, e.g., including
15 human behavioral responses, the selection of the specific economic assessment method is often a
16 relatively minor issue in terms of estimating benefits of air pollution control (or disadvantages of
17 increases in air pollution). Although results differ across approaches, the differences are largely
18 attributable to specific features of the assessment (e.g., whether the natural science data include a
19 particular effect or relationship, whether effects on consumers are measured, and so forth). The
20 nature and quality of the air quality forecasts used in the assessments can greatly influence the
21 sensitivity of the assessments (Adams and Crocker, 1989). This is particularly noticeable when
22 dealing with forecasts of seasonal air pollution changes (Adams et al., 1988). From the
23 standpoint of providing policy guidance, the differences in economic estimates attributable to the
24 assessment methods are often swamped by uncertainty in the natural and physical science
25 forecasts. This has also been noted in recent economic assessments of climate change (Adams
26 et al., 1998). In many settings, the quality of economic assessments of air pollution is likely to
27 be improved more by refining the physical and natural science data used in the assessments than
28 by intensive efforts to fine-tune the assessment techniques (Adams, 1999).
29
30
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1 AX9.7.3 Understanding of Air Pollutants Effects on the Economic Valuation
2 of Agriculture and Other Vegetation in the 1996 Criteria Document
3 Evidence from the plant science literature cited in the 1996 O3 AQCD (U.S. Environmental
4 Protection Agency, 1996) is unambiguous with respect to the adverse effects of tropospheric O3
5 on some types of vegetation. For example, the 1996 AQCD noted that findings from the EPA
6 multiyear NCLAN program in the 1980s provided rigorous corroboration of at least two decades
7 of previous research and a century of anecdotal observations that showed that O3 at ambient
8 levels caused physical damage to plants in general and to important crop species in particular.
9 Specifically, NCLAN established that ambient O3 levels resulted in statistically significant
10 reductions in yields for some crop species (Heagle et al., 1988). The 1996 AQCD also assessed
11 the results of studies regarding O3 effects on crops, forests, and natural vegetation in more detail.
12 More recent reviews, such as the comprehensive survey of the economic literature on
13 agricultural effects by Spash (1997), corroborate the synthesis of results reported in the 1996
14 AQCD.
15 The number and quality of assessments of the economic consequences of O3 exposures on
16 vegetation reported in the 1996 AQCD are primarily a function of the state of evidence obtained
17 from scientific studies in each vegetation category. For example, the plant science evidence
18 reviewed in the 1996 AQCD concerning effects of O3 exposures on agricultural crops was
19 reported to be more valid than for individual tree species or plant communities (ecosystems).
20 As a result, most economic assessments discussed in the 1996 AQCD focused on the data
21 obtained from studies of agricultural crops. The economic literature dealing with O3 effects on
22 forest productivity in the 1996 AQCD is sparse. The few economic assessments of tree or forest
23 effects reported in the 1996 AQCD were confined to evaluations of assumed or hypothetical
24 changes in output, such as board feet of lumber (e.g., Haynes and Adams, 1992). As noted in the
25 1996 AQCD, O3 effects on ecosystems and their services had not been measured in any
26 systematic fashion and no peer-reviewed economic assessments were yet reported.
27 This section first briefly reviews economic assessments drawn from the review in the 1996
28 criteria document. This review is the benchmark against which recent articles are then discussed
29 in the subsequent section. As was the case in 1996, the discussion of economic valuation of
30 ecosystem effects is generally limited to conceptual and methodological issues, given the
31 continued lack of empirical analyses in this category.
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1 AX9.7.3.1 Agriculture
2 In view of the importance of U.S. agriculture for both domestic and world consumption of
3 food and fiber, reductions in U.S. crop yields could adversely affect human welfare. The
4 plausibility of this premise has resulted in numerous attempts to assess, in monetary terms, the
5 losses from ambient O3 exposures, or the benefits of O3 control, to agriculture. Twenty-three
6 assessments of the economic effects of O3 exposures on agriculture were reviewed in the 1996
7 AQCD, highlighting key issues in the validity of these assessments (U.S. Environmental
8 Protection Agency, 1996). First, the evidence should reflect how crop yields will respond under
9 actual field conditions to O3 exposures. Second, the air quality data used to frame current or
10 hypothetical effects of O3 on crops should represent actual exposures sustained by crops at
11 individual sites or production areas. Finally, the assessment methodology into which such data
12 are entered should (1) capture the economic behavior of producers and consumers as they
13 adjust to changes in crop yields and prices that may accompany changes in O3 air quality;
14 (2) accurately reflect institutional considerations, such as regulatory programs and income
15 support policies (e.g., provisions of federal Farm Bill legislation), that may result in market
16 distortions; and (3) use measures of well-being that are consistent with economic principles.
17 Assessments of O3 damage to agricultural crops reported in the 1996 AQCD employed
18 procedures for calculating economic losses that met the conditions described above. More
19 specifically, the assessments provided 23 quantitative estimates of the economic consequences
20 of exposures to O3 and other air pollutants to agriculture that reflect producer-consumer
21 decision-making processes, associated market adjustments, and some measure of distributional
22 consequences between affected parties. Many of the economic assessments reviewed in
23 previous O3 documents also focused on O3 effects in specific regions, primarily California and
24 the Corn Belt (e.g., Garcia et al., 1986). This regional emphasis in the earlier literature may be
25 attributed to the relative abundance of data on crop response and air quality for selected U.S.
26 regions, as well as the importance of some agricultural regions (such as California) for the U.S.
27 agricultural economy.
28 Two U.S. national studies described in previous O3 criteria documents that are worthy of
29 additional comment are Kopp et al. (1985) and Adams et al. (1986). These were judged to be
30 "adequate" in terms of the three critical areas of data inputs in the 1996 AQCD. Together, it was
31 argued, they provide a reasonably comprehensive estimate of the economic consequences of
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1 changes in ambient air O3 levels on agriculture. Because of their central role in the 1996 criteria
2 document, these two studies are reviewed briefly below.
3 The Kopp et al. (1985) and Adams et al. (1986) studies indicated that ambient levels of O3
4 were imposing substantial economic costs of ~$3.4 billion (in 2000 U.S. dollars) on agriculture.
5 Both were judged to suffer from several sources of uncertainty, but the document concluded that
6 these possible improvements in future assessments were not likely to greatly alter the range of
7 agricultural benefit estimates arising from O3 reductions for several reasons. First, the studies
8 covered about 75 to 80 % of U.S. agricultural crops (by value). For inclusion of the other 20%
9 to significantly change the estimates would require that their sensitivities to O3 be much greater
10 than for the crops that have been included to date. Second, model-sensitivity analyses reported
11 in past studies indicate that changes in plant exposure response relationships must be substantial
12 to translate into major changes in economic benefits estimates. For example, it was assumed
13 unlikely that use of different exposure measures, or inclusion of interaction effects, would
14 greatly alter the magnitude of the economic estimates. Third, it was believed that countervailing
15 effects would mitigate against large swings in the estimates, e.g., longer O3-exposure periods
16 may predict greater yield losses, but O3-water stress interactions tend to reduce the
17 yield estimates.
18 Other national assessments reported in the 1996 AQCD provided general corroboration of
19 the results of Kopp et al. (1985) and Adams et al. (1986). An evaluation of these studies in
20 terms of the adequacy of information from plant exposure studies, and aerometric and economic
21 data was presented in the 1996 AQCD, along with estimates of benefits or damages associated
22 with changes in O3. Most of the studies added onto either Kopp et al. (1985) or Adams et al.
23 (1986). A relevant question was whether subsequent studies provided any "surprises" in terms
24 of magnitude of economic effects.
25 Common themes or findings from these and earlier O3 and other air pollution studies were
26 summarized in two synthesis papers, Adams and Crocker (1989) and Segerson (1991). The
27 major conclusion is that the agricultural effects of tropospheric O3 at ambient levels impose
28 economic costs to society or, conversely, that reductions in ambient O3 should result in societal
29 benefits.
30 Several studies contained in the 1996 AQCD still are of interest. For example, one finding
31 pertains to the relationship between federal farm programs and air pollution regulations
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1 (McGartland, 1987). In each case, the inclusion of farm programs in the economic models
2 resulted in modest reductions in the economic benefits of O3 control due to increased farm
3 program costs. As Segerson (1987) noted, however, it is not clear that these increased costs
4 should be charged against the potential benefits of an O3-regulatory standard, but rather,
5 considered as an additional cost associated with the inefficiencies of the farm program. It should
6 also be noted that the nature of federal farm programs was changed dramatically by Congress in
7 1996 in an attempt to reduce the federal government's role in agriculture. Although more recent
8 federal legislation, such as the 2002 Farm Bill (U.S. Congress, 2002), appears to be restoring the
9 federal government's role in the farming sector, this issue currently is not as important as
10 suggested by earlier studies, due to the declining reliance on deficiency payments to farmers,
11 which tend to distort resource allocation.
12 Another national study (Adams et al., 1988) analyzed economic benefits under a regulatory
13 alternative involving a seasonal (crop growing season) O3-exposure index measured as a 12-h
14 mean, instead of hourly levels or percent changes from ambient as reported in earlier studies.
15 Specifically, a seasonal average of 50 ppb O3 (measured as a 12-h seasonal mean) with a
16 95% compliance level, is reported in Adams et al. (1988). The result (a $2.9 billion benefit in
17 2000 dollars) is similar to the assumed 25% reduction across all regions reported by Adams et al.
18 (1986). At least one study also combined environmental stressors (e.g., O3, UV-B radiation) in
19 performing economic assessments. Adams and Rowe (1990), using the same model as Adams
20 et al. (1986), reported that a 15% depletion of stratospheric O3 (resulting in a 13% increase in
21 tropospheric O3) would cause an economic loss of ~$1.4 billion in 2000 dollars attributed to the
22 tropospheric O3 increase. Reducing VOCs/NOx motor vehicle emissions by 10% would result in
23 a benefit of ~$0.3 billion, while a complete elimination of motor vehicle emissions would yield a
24 benefit of ~$3.4 billion (1990 dollars). The range of these numbers is consistent with values
25 reported in Adams et al. (1986), Kopp et al. (1985), and other national-level analyses, i.e.,
26 estimates of from $1.0 to 2.0 billion for reductions in ambient O3 of 25 to 40%.
27
28 AX9.7.3.2 Forests (Tree Species) and Natural Ecosystems
29 The long-term nature of air pollution effects on perennial species such as trees creates
30 challenges to plant scientists in attempts to sort out the effects of specific individual stressors
31 such as O3 from among the many other potential causal factors (Skelly, 1988). It also creates
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1 problems in terms of measuring the impacts on direct economic value of goods, such as
2 reductions in board-feet of lumber produced per unit of time.
3 Most of the literature in the 1996 AQCD dealing with forest species reported the effects
4 of O3 exposures in terms of foliar injury (Davis and Skelly, 1992; Freer-Smith and Taylor, 1992;
5 Simini et al., 1992; Taylor and Hanson, 1992). This emphasis on foliar effects in the forest
6 effects literature (rather than marketable yield) is similar to the state of science for agricultural
7 crops prior to 1980. More recent studies address the effects of air pollutants on forest tree
8 species diversity (Bringmark and Bringmark, 1995; Vacek et al., 1999; Weiner et al., 1997).
9 However, such information is of limited use in economic assessments. The exception is in
10 measuring the economic value of aesthetic changes in a forest stock, where changes in species
11 composition may affect recreational values (Crocker, 1985).
12 The data concerning changes in marketed output such as board-feet of lumber or changes
13 in growth rates in managed forests, the affects on the growth of almond, peach, apricot, pear and
14 plum trees in orchards cited in the 1996 document (U.S. Environmental Protection Agency,
15 1996) have not been quantified. In addition, the economic impact of reductions in growth of
16 seedlings of evergreen trees, e.g., slash pine, presented in the same document have not been
17 valued. The few studies which attempted to measure economic losses arising from exposures
18 to O3 or other pollutants circumvented the lack of plant science data by assuming (often
19 arbitrary) reductions in forest species growth or harvest rates (Adams, 1986; Callaway et al.,
20 1986; Crocker and Forster, 1986; Haynes and Adams, 1992). Although the economic estimates
21 reported in the 1996 AQCD are comparable to those reported for agriculture (e.g., $2.6 billion
22 for eastern Canada forests, $2.9 billion for eastern U.S. forests in 2000 dollars), the lack of yield
23 and/or growth effects data makes these studies only suggestive at best, of the economic
24 consequences of forest effects directly attributable to O3 exposures questionable. Recent
25 developments in forestry economic modeling capabilities, in support of climate change research,
26 have enhanced the ability to measure the effects of environmental stressors on this sector
27 (Adams et al., 1996; McCarl et al., 1998). However, these models need data on changes in either
28 timber production or growth rates, both of which are lacking for forest species under alternative
29 O3 levels.
30
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1 AX9.7.4 Studies Since 1996 of Ozone Exposure Effects on the Economic
2 Value of Agriculture, Forests, and Ecosystems
3 Of the few current (post-1996) economic studies addressing agricultural effects, none offer
4 new insights of value in determining the economic cost of O3 exposures. These post-1996
5 studies used variants of the economic methods from earlier assessments and measure yield
6 changes from response functions arising from the NCLAN or similar data. For example, Kim
7 et al. (1998) used a mathematical programming model of the San Joaquin Valley agricultural
8 sector in California, combined with crop yield response functions, to assess the economic effects
9 of O3 on California crops. Their results showed net benefits from reductions in ambient O3
10 levels, a finding consistent with all previous economic assessments. In another study,
11 Westenbarger and Frisvold (1994) measured agricultural exposures to O3 (and acid precipitation)
12 in the United States. Though not an economic analyses of the costs of ambient exposures, they
13 identified areas of the United States of greatest potential economic damage based on the
14 interface between regional pollution levels and the value of crop production in each region.
15 A study by Murphy et al. (1999) of the economic effects of tropospheric O3 on U.S.
16 agriculture is of note here, because it confirms the general magnitude of economic effects
17 reported by the two key studies performed a decade earlier (Adams, 1986; Adams et al., 1985).
18 Specifically, Murphy et al. (1999) evaluated benefits to eight major crops associated with several
19 scenarios concerning the reduction or elimination of O3 precursor emissions from motor vehicles
20 in the United States. Their analysis reported a $2.8 to 5.8 billion (1990 dollars) benefit from
21 complete elimination of O3 exposures from all sources, i.e., ambient O3 reduced to a background
22 level assumed to be 0.025 to 0.027 ppm. While the analytical framework is similar to Adams
23 et al. (1986) in the use of NCLAN-based yield response functions and a mathematical
24 programming-based economic optimization model, the study is novel in its focus on the role of
25 motor vehicle emissions of VOCs/NOx in total anthropogenic O3 levels. The study is also
26 notable in its careful attention to federal farm program effects, particularly the deficiency
27 payment component.
28 In addition to these studies in peer-reviewed journals, a number of site-specific effects
29 studies have been performed, primarily by consulting companies for state public utility
30 commissions. Although perhaps of use to public utility commissioners concerned with effects
31 from single power plants or other localized sources, these regional studies generally contribute
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1 little to the assessment of air pollution effects at the national level. Also, such reports are not
2 peer reviewed. Therefore, they are not discussed here.
3 There have been a number of recent studies of air pollutant effects on tree species in the
4 literature. Some have reported changes in total biomass and focused on European species
5 (Kurczynska et al., 1997). Other studies have assessed changes in composition of forest species
6 (biodiversity) or forest health due to exposure to air pollutants (Bringmark and Bringmark, 1995;
7 McLaughlin and Percy, 1999; Vacek et al., 1999). As noted previously, changes in forest
8 biomass and composition are more difficult to value than marketable products. However,
9 measures of forest composition or health have implications for an area of increasing policy
10 concern, that being the effect of air pollutants and other environmental stressors on unmanaged
11 (natural) ecosystems and the services they provide (Goulder and Kennedy, 1997; Pimentel et al.,
12 1997). Considerable discussion has occurred among ecologists and economists as to the
13 appropriate means for valuing these services (Anderson, 1990; Carpenter and Dixon, 1985;
14 Common and Perrings, 1992). A number of conceptual articles have been published on this
15 issue in both economic and ecological journals (Bergstrom, 1990; Castle, 1993; Pearce, 1993;
16 Suter, 1990).
17 A continuing empirical challenge concerns the lack of information on how changes in
18 biodiversity affect ecosystem performance resulting from O3 stresses, and the problem of
19 establishing economic values for such changes (Cairnes and Lackey, 1992; Norton, 1987; Pimm,
20 1997; Polasky, 2001; Randall, 1991). As noted in the 1996 AQCD, and more recently by Daily
21 (1999, 2000) and Polasky (2001), there continues to be a lack of empirical studies that actually
22 assess the economic value of changes in biodiversity or in service flows due to any
23 environmental stressors. While some studies report monetary estimates, the estimates are
24 generally for expository purposes and those would not be as defensible as the agricultural studies
25 described earlier. For example, Costanza et al. (1997) assigned a value to the world's
26 ecosystems, but the procedures used render this an exploratory study at best. As assessed by
27 Polasky (2001), "In general, the field of valuation of ecosystem services is in its infancy."
28 He attributed the lack of empirical studies due to both a lack of the understanding of ecology of
29 ecosystem services and to the absence of reliable methods to estimate the value of these services.
30 In summary, the studies of crop and forest responses in the economic literature indicate
31 that O3 reduces crop yields and imposes economic costs. The economic literature also indicates
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1 that O3 adversely influences the physiological performance of tree species and demonstrates, as
2 expected, that changes in growth have economic consequences. However, the economic data
3 and literature available on ecosystem effects of O3 exposures are not sufficient to determine the
4 economic costs.
5
6 AX9.7.5 Limitations of Scientific Studies and Economic Information
7 The 1996 O3 AQCD discussed the need for additional research on both ecological
8 functions and economic methodology in order to better understand the economic implications of
9 air pollutants on ecosystem services. As noted by Daily (1999, 2000), Polasky (2001), and
10 others, this research agenda continues to be important. Despite the large number of discussion
11 and survey articles published since 1996, there still are not sufficient data by which to estimate
12 confidently the magnitude of economic effects of O3 to forests and natural ecosystems. Nor is it
13 apparent that ongoing research is adequate to answer this question in the next criteria document
14 cycle. Specifically, there do not appear to be any comprehensive, ecological studies underway
15 that attempt to measure changes in ecosystem outputs under alternative O3 or other air pollutant
16 levels. Thus, in the near term, ecosystem services can only be discussed in qualitative terms.
17 However plausible the likelihood of economic damages to ecosystems, the available scientific
18 and economic information does not provide specific guidance on the magnitudes of these effects.
19 Beside the need to improve our understanding of the effects of O3 exposures on natural
20 ecosystems, a number of areas of research could help assess the full economic consequences of
21 such pollutants on managed ecosystems. The first of these is the relationship between O3
22 exposure levels and the variability of crop yields or changes in forest biodiversity. Most
23 assessments are based on the average or expected yield response of a crop to air pollution
24 exposure. However, the variability in yields (the spread or dispersion around the mean) appears
25 to be affected by the nature of plant exposure to pollutants (Hogsett et al., 1985; Musselman
26 et al., 1983). Plants exposed to the same mean dose but with different second moments of the
27 distribution of exposure may have different mean yields (Altshuller and Lefohn, 1996; Lefohn
28 and Benedict, 1982). In addition, the variability of the yield of the plant may also be increased
29 by greater variability in exposures (e.g., a higher frequency of extreme events). The economic
30 significance of higher yield varieties is such that variability may impose additional economic
31 costs, because most farmers have been reported to be averse to risk and prefer less variability for
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1 a given yield (or profit). To date, no economic assessments of O3 damages to agriculture or
2 vegetation in general include risk-averse behavior (studies cited here assumed risk neutrality).
3 To assess the economic consequences of a relationship between farmers' risk preferences
4 and O3-induced changes in yield variability will require more information on the potential
5 effects of changes in O3 on crop yield variability. While no economic studies were found on the
6 effects of O3-induced changes in yield variability, a number of recent studies of climate change
7 effects on crop yields have indicated increased economic costs in the presence of increased
8 climatic variability (e.g., Mearns et al., 1997; Dixon and Segerson, 1999). Analogous economic
9 costs would be expected for changes in air pollution distributions; and these effects need to be
10 examined and quantified.
11 Another research area concerns the need improvement in our understanding of temporal
12 (dynamic) and spatial characteristics of O3 exposures and their implications for crop yields,
13 production and producer profits. Most economic studies are static, in that they compare two
14 states of the world (e.g., economic activity at one O3 level versus at an alternative level).
15 In addition, most national-level studies (the type needed to evaluate Secondary National
16 Ambient Air Quality Standards [SNAAQS]) display coarse regional-level resolution in terms of
17 crop response, O3 exposure, and economic behavior. The responses of producers to changes in
18 yields due to changes in O3 levels are generally assumed to be similar over geographical areas up
19 to several states in size. However, the changes between air quality scenarios are more likely to
20 be characterized by transient changes in exposure levels, which means the producer responses
21 are also likely to be gradual, rather than abrupt. Similarly, the lack of finer scale (regional-level)
22 data and modeling capabilities suggests that important micro-level physical and economic effects
23 are ignored. To what extent these abstractions influence net economic effects is an empirical
24 question. Research on these types of abstractions and assumptions within other economic
25 settings, such as climatic change, have shown that they have implications for economic
26 measurements (Adams, 2002).
27 Another issue is the natural or background level of O3 (or other pollutants of interest)
28 assumed in economic studies. While many economic studies focus on changes in pollution
29 levels from current conditions, some studies have measured the economic damages between an
30 assumed, or pristine, level and current levels in agricultural areas. Such an analysis, it is
31 reasoned, will provide a measure of the net damages due to anthropogenic sources. The
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1 challenge here is to have a correct measure of the background level of the pollutant. Recent
2 research by Lefohn et al. (2001) has suggested that background levels may be considerably
3 higher than assumed in some of the previous economic assessments reported in the 1996 AQCD
4 (25 to 30 ppb in most studies). For example, Lefohn et al. (Lefohn et al., 2001) detected hourly
5 readings of from 40 to 80 ppb during winter and spring months in remote areas of the United
6 States. If background O3 levels are in the range, then the economic damages estimated in studies
7 with lower background levels will be overstated. The issue of the background O3 level is
8 important to all assessments of vegetative damages due to O3.
9 In terms of expanding economic methods for future assessment, analysts should consider
10 using more "reduced form" estimation methods, particularly in situations where the availability
11 of dose-response functions is limited. This estimation approach is exemplified by Garcia et al.
12 (1986). Specifically, their econometric study of the impact of O3 on producer profits used
13 such a reduced-form approach. In this approach, farmer actions are modeled as a function of
14 ambient O3 levels (along with other explanatory variables) without the direct use of dose
15 response functions. The advantage of this procedure is that one source of modeling uncertainty,
16 the need for dose-response functions, including time-consuming crop experiments to generate
17 data, is reduced. Actual responses of farmers' profits across air pollution gradients of ambient
18 pollution are observed instead of hypothesized. Although this procedure has not been widely
19 used in air pollution economic assessments, it has been used in a number of relatively recent
20 climate change studies (e.g., Mendelsohn et al., 1994). The reduced-form method suffers from
21 the fact that if proposed O3 levels are lower than those observed in the estimation sample, then
22 the prediction accuracy of the method deteriorates. Also, some dose-response information is
23 needed, if only to establish the plausibility of the economic estimates.
24 Another area that may improve economic assessments is incorporating consideration of
25 livestock issues. To date, most agricultural economic assessments of O3 impacts have ignored
26 the livestock sector. Presumably this is because ambient O3 levels do not noticeably affect meat
27 yields. However, if feed prices and pasture conditions are affected by ambient O3 levels, then a
28 more accurate estimate of economic impacts would be forthcoming by including this link to
29 livestock in the assessment. These types of feed production and feed price effects are included
30 in the mathematical programming model used in Adams et al. (1986), but not in most other O3
31 effects assessments. The significance of livestock-feed linkages are demonstrated in a recent
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1 study in the Netherlands by Kuik et al (2000). Using a mathematical programming model
2 similar to that in Adams et al. (1986), Kuik et al. (2000) found that livestock effects were
3 prominent, due mainly to improved pasture yields under reduced ambient O3 levels.
4
5 AX9.7.6 Conclusions
6 Substantial progress has been made over the past two decades in our understanding of the
7 effects of O3 and other oxidants on vegetation, particularly for agriculturally important plant
8 species. The physical and economic effects on agriculture are well documented and provide
9 useful information for the setting of SNAAQS. Effects on forests and natural ecosystems remain
10 problematic, due to limitations in natural science data and economic methods. The problem is
11 most acute for valuing natural ecosystem service flows.
12 The current limitations surrounding forests and natural ecosystems present a rich research
13 agenda. However, not all research needs are likely to lead to better policies. Thus, areas of
14 greatest potential value in terms of regional policymaking need to be prioritized. Such priority
15 setting can be assisted by sensitivity analyses with existing economic models. By measuring the
16 changes in economic effects arising from changes in key parameters, research data gaps most
17 likely to affect economic values can be identified.
18
19
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21 28:2895-2907.
22
August 2005 AX9-410 DRAFT-DO NOT QUOTE OR CITE
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Please make all necessary changes in the below label, PRESORTED STANDARD
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EPA/600/R-05/004CB
August 2005
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