# Chapter 9. Environmental Effects: Ozone Effects on Vegetation and Ecosys t ems

9.1. In tro d u c tio n

1 This chapter synthesizes and evaluates the most policy

- relevant science to help form the



### 2 scientific foundation for the re view of a vegetation

- and ecologically

- based secondary NAAQS for



### 3 O 3 . The secondary NAAQS are based on welfare effects. The Clean Air Act (CAA) definition of



### 4 welfare effects includes, but is not limited to, effects on soils, water, wildlife, vegetation, vis ibility,



### 5 weather, and climate, as well as effects on materials, economic values, and personal comfort and



### 6 well

- being. The effects of O 3 as a greenhouse gas and its direct effects on climate are discussed in



### 7 Chapter 10 of this document.



### 8 The intent of the IS A, according to the CAA, is to “accurately reflect the latest scientific



### 9 knowledge expected from the presence of [a] pollutant in ambient air” (42U.S.C.7408 (1990,



### 10 080701 ) 42U.S.C.7409 (1990, 037658 ) ). This chapter of the ISA includes scienti fic research from



### 11 biogeochemistry, soil science, plant physiology, and ecology conducted at multiple scales (e.g.,



### 12 organ, individual, population, community, ecosystem). Key information and judgments formerly



### 13 found in the AQCDs regarding O 3 effects on veget ation and ecosystems are found in this chapter.



### 14 This chapter of the O 3 ISA serves to update and revise Chapter 9 and AX9 of the 2006 O 3 AQCD



### 15 (U.S. EPA, 2006, 088089 ).



### 16 Numerous studies of the effects of O 3 on vegetation and ecosystems were reviewed in the



### 17 2006 O 3 AQCD. That document concluded that the effects of ambient O 3 on ve getation and



### 18 ecosystems appear to be widespread across the U.S., and experimental studies demonstrated



### 19 plausible mechanisms for these effects. Ozone effect studies published from 2005 to September 2010



### 20 are reviewed in this document in the context of the previous O 3 AQCDs (U.S. EPA, 2006,



### 21 088089 ) (U.S. EPA, 1996, 080827 ) (U.S. EPA, 1984, 029711 ) (U.S. EPA, 1978, 040586 ) . From 2005



### 22 to 2010, some areas have had ver y little new research published and the reader is referred back to



### 23 sections of the 2006 O 3 AQCD for a more comprehensive treatment of those subjects. This chapter is



### 24 focused on studies of vegetation and ecosystems that occur in the U.S. and that report endpoints or



### 25 processes most relevant to the review of the secondary standard. Many studies have been published



### 26 about vegetation and ecosystems outside of the U.S. and North America, largely in Europe and Asia.



### 27 This document includes discussion of studies of vegetation and ecosystems outside of North America



### 28 only if those studies contribute to the general understanding of O 3 effects across species and



### 29 ecosystems. For example, studies outside North America are discussed that consider physiological

Note: Hyperlinks to the reference citations throughout this document will take you to the NCEA HERO database (Health and

Environmental Research Online) at http://epa.gov/hero . HERO is a database of scientific literature used by U.S. EPA in the process of developing science assessments such as the Integrated Science Assessments (ISAs) and the Integrated Risk Information System ( IRIS).

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### 1 and biochemical processes that contribute to the understanding of effects of O 3 across species. Also,



### 2 ecosystem studies outside of North America that contribute to the understanding of O 3 effects on



### 3 general ecosystem processes are discussed in the chapter.



### 4 This chapter is organized in sections that discuss exposure methods, followed by effects on



### 5 vegetation and ecosystems at various scales and ends with policy

- relevant discussions of exposure



### 6 indices and exposure

- response. First, Section 9.2 presents summary information and conclusions



### 7 based on the literature assessed in this chapter and the assessments and conclusions of the previous



### 8 O 3 AQCDs. A brief overview of various methodologies that have been, and continue to be, central to



### 9 quantifying O 3 effects on vegetation is p rovided in Section 9.3 ( AX9.1 of the 2006 O 3 AQCD for



### 10 more detailed discussion ) (U.S. EPA, 2006, 088089 ) . Sections 9.4 through 9.6 begin with a



### 11 discussion of effects at the cellular and subcellu l ar level followed by consideration of the whole



### 12 plant and finally, O 3 impacts on ecosystem

- level processes (Figure 9 -1 ). In Section 9.4, research is



### 13 reviewed from the molecular to the biochemical and physiological levels in impacted plants, offering



### 14 insight into the mode of action of O 3 . Section 9.5 provides a review of the effects of O 3 exposure on



### 15 major endpoints at the whole plant s cale including growth, reproduction, visible foliar injury and leaf



### 16 gas exchange in woody and herbaceous plants in the U.S., as well as a brief discussion of O 3 effects



### 17 on agricultural crop yield and quality. The response of plants to O 3 as influenced by n umerous



### 18 environmental biotic and abiotic factors is also discussed Section 9.5. In Section 9.6, available



### 19 research for assessing the effect of O 3 on ecosystems is reviewed, along with data potentially



### 20 available for estimating the loss of various ecosystem services. The development of indices of O 3



### 21 exposure and dose modeling is discussed in Section 9.7. Finally, exposure

- response relationships for



### 22 a number of tree species, native vegetation, and crop species and cultivars are reviewed, tabulated,



### 23 and compare d in Section 9.8 to form the basis for an assessment of the potential risk to vegetation



### 24 from current ambient levels of O 3

.



# 9.2. S u mm a ry a n d In te g ra tio n

9.2.1. Introd uc tion

25 The subsequent sections of this chapter will present the most policy

- relevant information



### 26 rela ted to this review of the NAAQS for the effects of O 3 on vegetation and ecosystems. This section



### 27 integrates the key findings from the disciplines evaluated in this current assessment of the O 3



### 28 scientific literature, which includes plant physiology, biochem istry, whole plant biology, ecosystems



### 29 and exposure

- response.



### 30 Ozone effects at small scales, such as the leaf of an individual plant, can result in effects at a



### 31 continuum of larger scales. Figure 9 -1 is a simplified diagram of the major pathway through whi ch



### 32 O 3 enters plants and the major endpoints O 3 may affect from small to large scales. The subsequent



### 33 sections in this chapter are organized around this paradigm of effects at the cellular and subcellu l ar



### 34 level followed by consideration of the whole plant a nd finally, O 3 impacts on ecosystem

- level

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7 processes. Ozone enters leaves through stomata, and can alter stomatal conductance and disrupt CO 2 fixation (Section 9.4). These effects can change rates of leaf gas exchange, growth and reproduction

at the individ ual plant level (Section 9.5). Those O 3 - induced effects can translate from the individual

plant level to the ecosystem level, and cause changes in ecosystem services, such as C storage, water

production, nutrient cycling, and community composition (Section 9.6). The EPA framework for

causal determinations described in Chapter 1 has been applied to the body of scientific evidence to

collectively examine effects attributed to O 3 exposure (See Table 9

- 1).

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### Figure 9

- 1. The effects of ozone at leaf, plant and ecosystem scales

.

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### Table 9

- 1 Summary of ozone causal determinations for vegetation and ecosystem effects

Vegetation and Ecosystem Effects Causality Determination

Reduced Vegetation Growth Causal

Alteration of Vegetation Reproduction Causal

Visible Folia r Injury Effects on Vegetation Causal

Alteration of Leaf Gas Exchange in Vegetation Causal

Reduced Yield and Quality of Agricultural Crops Causal

Reduced Productivity in Terrestrial Ecosystems Causal

Reduced Carbon (C) Sequestration in Terrestrial Eco systems Likely Causal

Alteration of Terrestrial Ecosystem Water Cycling Likely Causal

Alteration of Below

- ground Biogeochemical Cycles Causal

Alteration of Terrestrial Community Composition Likely Causal



## 9.2.2. Me ch a nis ms Go ve rnin g Res p on s e

1 Section 9.4 focu ses on the effects of O 3 stress on plants and their responses to that stress on the



### 2 molecular, biochemical and physiological levels. Many of the studies focus on the molecular



### 3 mechanisms that underlie the observed biochemical and physiological changes obse rved in many



### 4 plant species in response to O 3 exposure. The results support and strengthen those reported in the



### 5 2006 O 3 AQCD. The most significant change in this section from the 2006 O 3 AQCD is the emphasis



### 6 on molecular mechanisms as new techniques, such as those used in evaluating transcriptomes (total



### 7 set of RNA transcripts in a particular cell at a particular time) and proteomes (total set of proteins



### 8 expressed in a particular cell at a particular time), have been utilized to perform very comprehensive



### 9 analyses of changes in gene transcription and protein expression in plants exposed to O 3 . These



### 10 newer molecular studies not only provide very important and wide

- ranging information regarding



### 11 the many mechanisms of plant responses to O 3 , they also allow for the analysis of interactions



### 12 between various biochemical pathways which are induced in response to O 3 . However, many of



### 13 these studies are conducted in artificial conditions with model plants which are typically exposed to



### 14 very high, short doses of O 3 . The refore, additional work remains to elucidate whether these plant



### 15 responses are transferable to other plant species exposed to more realistic ambient conditions.



### 16 Ozone is taken up into leaves through open stomata. Once inside the substomatal cavity, O 3 is



### 17 thought to rapidly react with the aqueous layer surrounding the cell (apoplast) to form breakdown

) and peroxy · ), hydroxyl radicals (HO ־ 

### 18 products such as hydrogen peroxide (H 2 O 2 ), superoxide (O 2



### 19 radicals (HO 2 · ). These radicals may play a role in signaling processes and may also interact with



### 20 sensitive molecules both outside and inside the cell to cause damage. This process was very

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39 comprehensively described i n the 2006 O 3 AQCD and is only summarized in this document in

Section 9.4.2.

As plants have been shown to respond to O 3 exposure very rapidly, this response must result

from a plant's ability to sense the presence of O 3 and/or its breakdown products and th en

communicate that information to the nucleus to initiate changes in gene expression. While it remains

unclear what the exact mechanism is by which the plant senses the presence of O 3 , whether there are

multiple simultaneous mechanisms by which O 3 can be sensed, and how much variation exists in O 3 sensing between species and exposure conditions, some progress has been made in the understanding

of this process since the 2006 O 3 AQCD. Experimental evidence described in Section 9.4.3.1

suggests that O 3 and/or its breakdown products may be directly sensed by apoplastic receptor

proteins (although they have not yet been identified). Additionally, a change in cellular redox state

due to plant exposure to O 3 could be the manner in which plants sense the presence o f the pollutant.

Once the plant has sensed the presence of the pollutant, there is much evidence to suggest that

mitogen

- activated protein kinases (MAPK) play an important role in communicating signals to the

nucleus that result in gene expression changes in response to O 3 . Calcium has also been implicated to

play a role in the signal transduction processes. To summarize, the evidence to date suggests there

may be several mechanisms by which plants sense the presence of O 3 and then communicate this

signal t o the nucleus to induce changes in gene expression.

New technologies have allowed for the evaluation of changes in the entire transcriptome and

proteome, rather than analyzing the modification of the expression of individual genes and proteins;

the result s of these studies are presented in Section 9.4.3.2. While transcriptome and proteome

analyses per se were not previously addressed, the 2006 O 3 AQCD did provide much information

regarding changes in gene expression and protein quantity of individual genes and proteins in

O 3 - treated plants. In the transcriptome and proteome studies described here, O 3 exposure conditions

(concentration, duration of exposure), plant species and sampling times vary significantly; however,

functional classification of the genes and proteins that are either up

- or down

- regulated by plant

exposure to O 3 exhibit common trends. In summary, genes involved in plant defense, signaling, and

those associated with the synthesis of plant hormones and secondary metabolism are generally up

r egulated in plants exposed to O 3 , while those related to photosynthesis and general metabolism are

typically down

- regulated. Proteome studies support these results by demonstrating concomitant

increases or decreases in the proteins encoded by these genes. The transcriptome and proteome

results support and enhance the findings of the 2006 O 3 AQCD.

The 2006 O 3 AQCD included a discussion on the role of phytohormones, including salicylic

acid, ethylene and jasmonic acid, in plant response to O 3 . Many additional studies using microarray

technology (used to determine changes in the transcriptome) and a variety of Arabidopsis mutants

are described in Section 9.4.3.3 and support the conclusions from the 2006 O 3 AQCD. Transcriptome

analysis has also illuminated the complex interactions that exist between these hormones to better

define plant response to O 3 . To summarize, the results indicate that while ethylene and salicylic acid

are needed to develop O 3 - induced leaf lesions, jasmonic acid acts antagonistically to et hylene and

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39 salicylic acid to limit the spread of the lesions. Abscisic acid, in addition to its role in regulating

stomatal aperture, may also act antagonistically to the jasmonic acid signaling pathway. Nitric oxide

has also been proposed to play a role i n regulating O 3 - induced changes in gene expression; however,

its role is not yet well defined. Changes in phytohormones and the interactions between them reveal

some of the complexity of plant responses to an oxidative stressor such as O 3

.
Antioxidant met abolites, such as ascorbate and glutathione, and the enzymes that regenerate

them are a critical part of plant defense responses to oxidative stress. The role of ascorbate, which is

located in several cellular compartments and also in the apoplast, was com prehensively evaluated in

the 2006 O 3 AQCD as a first line of defense (due to its location in the apoplast) against oxidative

stress. Ascorbate has also been the focus of studies investigating differences in O 3 tolerance between

plant species or cultivars/ genotypes within one species. While the studies evaluated for the current

document support the important role of ascorbate, several studies suggest that ascorbate quantity,

especially in the apoplast, is not the primary factor in determining plant toleranc e to O 3 . In summary,

antioxidant metabolites and enzymes increase in quantity in plants exposed to O 3 . In most cases,

there is a correlation between the degree to which these defensive systems are induced and the

ability of the plant to tolerate exposure to O 3 . This up

- regulation of antioxidant defenses and the

need to keep antioxidant metabolites in a reduced state requires a significant shift in C metabolism

away from growth and reproduction to sustain the energy needs of the plant for defense.

While decl ines in C fixation as a result of plant exposure to O 3 were extensively described in

the 2006 O 3 AQCD, some recent studies (described in Section 9.4.5.1) of O 3 - induced declines in

photosynthesis have focused also on O 3 effects on the light reactions. Decli nes in the Fv/Fm ratio (a

measure of the maximum efficiency of the light reactions of photosynthesis) were observed in

several studies using a variety of plant species and exposure conditions. Additionally, O 3 increased

the coefficient of non

- photochemical quenching in several species, an indication that defense and

repair mechanisms of a non

- photochemical nature are activated in these plants while less absorbed

light is being used to drive photosynthesis. This indicates a shift away from photosynthesis to

defense, resulting in negative impacts on growth and reproduction.

Section 9.4.5.2 evaluates the effects of O 3 on respiration. While C assimilation declines in O 3 exposed plants, respiration is generally up

- regulated. These increases in respiration are th ought to

result from a plant's greater energy needs for defense (maintaining its antioxidant metabolites in a

reduced state) and repair. The increased energy needs will negatively impact plant growth and

reproduction.

Secondary metabolism is most often up

- regulated in a variety of species exposed to either

acute or chronic O 3 exposures as a part of a generalized plant defense mechanism. Changes in gene

expression, quantity and activity of enzymes associated with secondary metabolism and alterations

in secon dary metabolite quantity have been documented in plants exposed to O 3 . Some secondary

metabolites, such as flavonoids and polyamines, are of particular interest as they are known to have

antioxidant properties. Investigations on the importance of isoprenes in plant response to O 3 have

revealed conflicting results; however, there is some evidence to suggest that they may play a

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### 1 protective role. In summary, secondary metabolites increase in quantity in O 3 - treated plants as part of



### 2 a generalized plant defense response. Some secondary metabolites are of particular importance in



### 3 O 3 - treated plants as they may have antioxidant functions. Increased synthesis of secondary



### 4 metabolites represents a large energy investment of the plant into defense responses and away fr om



### 5 growth and reproduction.



### 6 Section 9.4.6 focuses on O 3 - induced changes in stomatal function. Stomata play a critical role



### 7 in limiting O 3 uptake into the plant by reducing stomatal aperture. Declines in stomatal conductance



### 8 in response to O 3 have been documented for many plant species, and much evidence suggests that



### 9 this results from increases in intercellular CO 2 concentration due to reductions in C fixation.



### 10 Additionally, sensitivity of some plants to O 3 has been related to a sluggish stomatal response, in



### 11 which plants are unable to close their stomata rapidly in response to O 3 . To summarize, stomatal



### 12 response to O 3 can help to determine plant sensitivity to the pollutant, and the decreases in stomatal



### 13 conductance are thought to be related to declines in C fixation rates. Reduced stomatal conductance



### 14 will decrease rates of C assimilation and lead to diminished growth and reproduction in plants.



## 9.2.3. Na ture o f Effec ts on Veg eta tio n

15 Ambient O 3 concentrations have long been known to cause visible foliar injury, d ecreases in



### 16 photosynthetic rate, decreases in growth, and decreases in the quality and yield of some plant species



### 17 (U.S. EPA, 2006, 088089 ) (U.S. EPA, 1996, 080827 ) (U.S. EPA, 1984, 029711 ) (U.S. EPA, 1978,



### 18 040586 ) . Numerous studies have related O 3 exposure to plant responses, with most research effort



### 19 focused on the growth of tree seedlings and the yield of crops as endpoints. The response of a plant



### 20 species to O 3 exposure depends upon many factors, including genetic characteristics, biochemical



### 21 and physiological status, and previous and current exposure to other stressors. The associated



### 22 sections in Section 9.5 focus mainly on studies published since the release of the 2006 O 3 AQCD



### 23 (U.S. EPA, 2006, 088089 ) . However, because much O 3 research was conducted prior to the 2006 O 3



### 24 AQCD, the conclusions presented below are collectively based on this ISA as well as the 1978,



### 25 1986, 1996, and 2006 AQCDs (U.S. E PA, 2006, 088089 ) (U.S. EPA, 1996, 080827 ) (U.S. EPA, 1984,



### 26 029711 ) (U.S. EPA, 1978, 040586 ).



## 9.2.3.1. Effe c ts o n Wo ody a nd He rba c e ous Ve ge ta tio n

Growth and Biomass Allocation

27 The previous O 3 AQCDs concluded that there is strong and consistent evidence that ambient



### 28 concentrations of O 3 decrease growth in numerous plant species across the U.S. Studies published



### 29 since the last review continue to support that conclusion (Section 9.5.2.1).



### 30 In a recently published meta

- analysis of 263 studies, Wittig et al. (2009, 191631 ) reported that



### 31 current ambient O 3 concentrations (~40 ppb) significantly decreased annual total biomass growth of



### 32 forest species by an average of 7%, with potentially greater decreases (11

- 17%) in areas that have

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1 higher O 3 concentrations and as background O 3 increases in the future. This meta -analysis



### 2 demonstrates the coherence of O 3 effects across numerous studies and species using a variety of



### 3 experimental techniques. In a study conducted on mature forest trees, McLaughlin et al. (2007,



### 4 090348 ) reported that the cumulative effects of ambient levels of O 3 decreased seasonal stem growth

by 30

- 50% for most of the species in a high O 3 year in comparison to a low O 3 year.



### 6 Since the 2006 O 3 AQCD

, several studies were published based on the Aspen free

- air carbon



### 7 dioxide/ozone enrichment (FACE) experiment using “free air”, O 3 , and CO 2 exposures in a forest in



### 8 Wisconsin. It was found that O 3 caused reductions in total biomass relative to the control in aspen,



### 9 paper birch, and sugar maple communities during the first seven years of stand development.

Overall, the studies at the Aspen FACE experiment were consistent with many of the open -top



### 11 chamber (OTC) studies th at were the foundation of previous O 3 NAAQS reviews. These results



### 12 strengthen our understanding of O 3 effects on forests and demonstrate the relevance of the



### 13 knowledge gained from trees grown in open

- top chamber studies.



### 14 In recent studies, O 3 was shown to have either negative, non

- significant, or positive effects on

root biomass and root:shoot ratio. While the findings of individual studies were mixed, recent meta


### 16 analyses have generally indicated that O 3 reduced C allocated to roots (Grantz et al., 2006,



### 17 191545 ) (Wittig et al., 2009, 191631 ).



### 18 For some annual species, particularly crops, the endpoint for an assessment of the risk of O 3



### 19 exposure can be defined as yield or growth, e.g., production of grain. For plants grown in mixtures

such as hayfields, and natural or semi

- natural grasslands (including native nonagricultural species),



### 21 endpoints other than production of biomass may be important. Such endpoints i nclude biodiversity



### 22 or species composition, and measures of plant quality. Effects may also result from competitive



### 23 interactions among plants in mixed

- species communities. Most of the available data on non -crop



### 24 herbaceous species are for grasslands with many of the recent studies conducted in Europe.

Evidence is sufficient to conclude that there is a causal relationship between O 3 exposure



### 26 and reduced growth of woody and herbaceous vegetation.



### Reproduction

27 Studies during recent decades have demonstrated O 3 effects on different stages of plant



### 28 reproduction (Section 9.5.2.2). Several recent studies published since the 2006 O 3 AQCD further



### 29 demonstrate the effects of O 3 on reproductive processes in herbaceous and woody plant species.

The impacts of O 3 on reprodu ctive development can occur by influencing (1) age at time of



### 31 initial flowering, particularly in long

- lived trees that often have long juvenile periods of early growth



### 32 without flower and seed production; (2) flower bud initiation and development; (3) polle n



### 33 germination and pollen tube growth; and (4) seed, fruit, or cone yields and seed quality.



### 34 Evidence is sufficient to conclude that there is a causal relationship between O 3 exposure

and changes in reproduction of vegetation.

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### Visible Foliar Injury

1 Visible foliar injury resulting from exposure to O 3 has been well characterized and



### 2 documented over several decades of research on many tree, shrub, herbaceous, and crop species



### 3 ( U.S. EPA, 2006, 088089 ) (U.S. EPA, 1996, 080827 ) (U.S. EPA, 1984, 029711 ) (U.S. EPA, 1 978,



### 4 040586 ) (Section 9.5.2.3). Ozone

- induced visible foliar injury symptoms on certain bioindicator plant

species are considered diagnostic as they have been verified experimentally in exposure

- response



### 6 studies, using exposure methodologies such as continuous stirred tank reactors (CSTRs), OTCs, and



### 7 free

- air fumigation. Experimental evidence has clearly established a consistent association of visible



### 8 injury wi th O 3 exposure, with greater exposure often resulting in greater and more prevalent injury.



### 9 Since the 2006 O 3 AQCD, several multiple

- year field surveys of O 3 - induced visible foliar injury

have been conducted at National Wildlife Refuges in Maine, Michigan, New Jersey, and South



### 11 Carolina. New sensitive species showing visible foliar injury continue to be identified from field



### 12 surveys and verified in controlled exposure studies.



### 13 The use of biological indicators in field surveys to detect phytotoxic levels of O 3 is a



### 14 longstanding and effective methodology. The USDA Forest Service through the Forest Health

Monitoring (FHM) Program (1990

- 2001) and currently the Forest Inventory and Analysis (FIA)



### 16 Program has been collecting data regarding the incidence and severi ty of visible foliar injury on a



### 17 variety of O 3 sensitive plant species throughout the U.S. The network has provided evidence that O 3



### 18 concentrations were high enough to induce visible symptoms on sensitive vegetation. From repeated



### 19 observations and measurem ents made over a number of years, specific patterns of areas experiencing

visible O 3 injury symptoms can be identified.



### 21 In addition, a study by Kohut (2007, 093289 ) assessed the risk of O 3 - induced visible foliar



### 22 injury on bioindicator plants (NPS, 2006, 677536 ) in 244 national parks in support of the National



### 23 Park Service's Vital Signs Monitoring Network (NPS, 2007, 677537 ) . Kohut (2007, 093289 )



### 24 concluded that the risk of visible foliar injury was high in 65 parks (27%), moderate in 46 parks

(19%), and low in 131 parks (54%). Some of the well

- known parks with a high risk of O 3 -induced



### 26 visible foliar injury include Gettysburg, Valley Forge, Delaware Water Gap, Cape Cod, Fire Island,



### 27 Antietam, Harpers Ferry, Manassas, Wolf Tr ap Farm Park, Mammoth Cave, Shiloh, Sleeping Bear



### 28 Dunes, Great Smoky Mountains, Joshua Tree, Sequoia and Kings Canyon, and Yosemite.



### 29 Evidence is sufficient to conclude that there is a causal relationship between ambient O 3
exposure and the occurrence of O 3 - induced visible foliar injury on sensitive vegetation across



### 31 the U.S.



### Gas Exchange

32 There is strong experimental evidence over several decades of research that exposure to O 3



### 33 reduces photosynthesis and alters stomatal conductance in a wide variety of plant species. The mode



### 34 of action, as characterized in Section 9.4 and in previous reviews, provides biological plausibility for

O 3 effects on leaf gas exchange.

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### 1 In compiling more than 55 studies, Wittig et al. (2007, 191695 ) reported that current O 3



### 2 concentrations in the northern hemisphere are decreasing photosynthesis (11%) and stomatal



### 3 conductance (13%) across tree species. It was also found that younger trees (<4 year ) were affected



### 4 less by O 3 than older trees. Further, the authors also found that decreases in photosynthesis are



### 5 consistent with the cumulative uptake of O 3 into the leaf. In contrast, several studies reported that O 3



### 6 exposure may result in loss of stomatal control, incomplete stomatal closure at night and a



### 7 decoupling of photosynthesis and stomatal conductance, which may have implications for whole


### 8 plant water use (Section 9.6.3).



### 9 Evidence is sufficient to conclude that there is a causal relationship between O 3 exposure



### 10 and the alteration of leaf gas exchange in vegetation.



## 9.2.3.2. Agric ultura l Cro ps

Yield and Crop Quality

11 The detrimental effect of O 3 on crop production h as been recognized since the 1960’s and a



### 12 large body of research has subsequently stemmed from those initial findings. Previous O 3 AQCDs



### 13 have extensively reviewed this body of literature (U.S. EPA, 2006, 088089 ) . Recent experimental



### 14 studies of O 3 effects on crops are discussed in Section 9.5.3 and summarized in Table 9

- 3 and 9 -16



### 15 Current O 3 concentrations across the U.S. are high enough to cause yield loss for a variety of



### 16 agricultural crops including, but not limited to, soybean, wheat, cotton, potato, watermelon, beans,



### 17 turnip, onion, lettuce, and tomato. Continued increases in O 3 concentration may further decrease



### 18 yield in these sensitive crops while also initiating yield losses in less sensitive crops. Despite the



### 19 well

- documented yield losses due to increasing O 3 concentration, there is still a knowledge gap



### 20 pertaining to the exact mechanism of O 3 - induced yield loss. Research has linked increasing O 3



### 21 concentration to decreased photosynthetic rates and accelerated senescence, which are related to



### 22 yield.



### 23 Recent modeling research has correlated satellite air

- column observations w ith direct air



### 24 sampling O 3 data and modeled the yield

- loss due to O 3 over the continuous tri

- state area of Illinois,



### 25 Iowa and Wisconsin. This modeling data correlates well with the previous results from FACE

- type



### 26 experiments and OTC experiments.



### 27 New resear ch is beginning to consider the mechanism of damage caused by long, lower O 3



### 28 concentration (so

- called chronic exposure) compared to short, very high O 3 concentration (so

- called



### 29 acute exposure). Both types of O 3 exposure cause damage to agricultural crops, but through very



### 30 different mechanisms. Until recently, most research on the mechanism of O 3 damage has used acute



### 31 exposure studies. It has become clear that the same cellular and biochemical processes involved in



### 32 the response to acute O 3 exposure are not i nvolved in response to chronic O 3 exposure, yet both



### 33 cause yield

- loss in agriculturally important crops.



### 34 In addition, new research has highlighted the effects of O 3 on crop quality. Increasing O 3



### 35 concentration decreases nutritive quality of grasses, decrea ses macro

- and micro -nutrient

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1 concentrations in fruits and vegetable crops, and decreases cotton fiber quality. These areas of



### 2 research require further investigation to determine the mechanism and dose

- responses.



### 3 Evidence is sufficient to conclude that the re is a causal relationship between O 3 exposure



### 4 and reduced yield and quality of agricultural crops.



## 9.2.3.3. Fa c tors Tha t Mo dify Fu nc tio na l a nd Gro wth Re s pons e

Many biotic and abiotic factors, including insects, pathogens, root microbes and fungi,



### 6 temperature, wat er and nutrient availability, and other air pollutants, as well as elevated CO 2 , 

### 7 influence or alter plant response to O 3 . These modifying factors were comprehensively reviewed in



### 8 the 2006 O 3 AQCD. A limited number of studies published since 2006 provide fu rther support for



### 9 our understanding of the role of these interactions in modifying O 3 - induced plant responses and are

discussed in Section 9.5.4.



## 9.2.4. Ecos ys tems and Services

11 Ozone has been found to alter plant physiological processes such as growth, biomass



### 12 al location, reproduction and gas exchange (Section 9.5). Those O 3 - induced effects at the individual



### 13 plant scale have the potential to translate to effects at the ecosystem level, and cause changes in



### 14 biogeochemical cycling and community composition. Informat ion presented in the associated section

(Section 9.6) was collected at multiple scales, ranging from responses at the population level to the



### 16 ecosystem level. The effects of O 3 on ecosystem productivity, C sequestration, water cycling,



### 17 nutrient cycling, an d community composition are reviewed.



## 9.2.4.1. Productivity and Carbon Se ques tration

18 During the previous NAAQS reviews, there were very few studies that investigated the effect



### 19 of O 3 exposure on ecosystem productivity and C sequestration. Recent studies from long

- term FACE

experiments provided evidence of the association of O 3 exposure and reduced productivity at the



### 21 ecosystem level. Elevated O 3 reduced stand

- level biomass by 13

- 23% at Aspen FACE after 7 years



### 22 of O 3 exposure (King et al., 2005, 191701 ) , and annual volume growth by 9.5 m 3 /ha at the Kranzberg



### 23 Forest (Germany) FACE (Pretzsch et al., 2010, 580435 ) . Studies at the leaf and plant scales showed



### 24 that O 3 reduced photosynthesis and plant growth, which provided coherence and biological

plausibility for the decrease in ecosystem productivity. Results across different ecosystem models



### 26 were consistent with the FACE experimental evidence, which showed that O 3 reduced ecosystem



### 27 productivity.



### 28 Although O 3 generally causes negative effects on plant growth, the magnitude of the response



### 29 varies among plant communities. For example, O 3 had little i mpact on white fir, but greatly reduced

growth of ponderosa pine in southern California (Weinstein et al., 2005, 179965 ) . Ozone decreased



### 31 net primary production (NPP) of most forest types by 7

- 8% in Mid

- Atlantic region, but had small



### 32 impacts on spruce

- fir forest, which was decreased by only 1% (Pan et al., 2009, 596032 ) . Among

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1 crop species, the estimated yield loss for whea t (7

- 12%) and soybean (6

- 16%) were higher than rice



### 2 (3

- 4%) and maize (3

- 5%) (Van Dingenen et al., 2009, 199765 )

. 

### 3 In addition to plant growth, other indicators that are typically estimated by model studies



### 4 include net ecosystem CO 2 exchange (NEE), C sequestration, and crop yield. Model simulations

consistently found th at O 3 exposure caused negative impacts on those indicators (Section 9.6.2,



### 6 Table 9 -5 ), but the severity of these impacts was influenced by multiple interactions of biological



### 7 and environmental factors. For example, the largest O 3 - induced crop yield losses occurred in high


### 8 production areas exposed to high O 3 concentrations, such the Midwest and the Mississippi Valley



### 9 regions of the U.S. (Van Dingenen et al., 2009, 199765 )

. The suppression of ecosystem C sinks results in more CO 2 accumulation in the atmosphere.



### 11 Globally, the indirect radiative forcing, reported in Watts/ square meter (W/m 2 ), caused by O 3



### 12 exposure through lowering ecosystem C sink (0.62

- 1.09 W/m 2 ) could have an even greater impact



### 13 on global warming than the direct radiative forcing of O 3 (0.89 W/m 2 ) (Sitch et al., 2007, 093294 )

. 

### 14 Ozone could also affect regional C budgets through interacting with multiple factors, such as N

deposition, elevated CO 2 and land use history. Model simulations suggested that O 3 partially offset



### 16 the growth stimulation caused by elevated CO 2 and N deposition in both Northeast

- and Mid



### 17 Atlantic

- region forest ecosystems of the U.S. (Ollinger et al., 2002, 180189 ) (Pan et al., 2009,



### 18 596032 ).



### 19 The evidence is sufficient to infer that there is a causal relationship between O 3 exposure

and reduced productivity, and a likely causal relationship between O 3 exposure and reduced



### 21 ca rbon sequestration in terrestrial ecosystems.



## 9.2.4.2. Wa te r Cyc ling

22 Although the evidence was from a limited number of field and modeling studies, these



### 23 findings showed an association of O 3 exposure and the alteration of water cycle at the ecosystem



### 24 level. Field s tudies conducted by McLaughlin et al. (2007, 090348 ) (2007, 090347 ) suggested that

peak hourly O 3 exposure increased the rate of water loss from several tree species, and led to a



### 26 reduction in the late

- season modeled stream flow in three forested wate rsheds in eastern Tennessee.



### 27 Evidence of sluggish stomatal responses during O 3 exposure was found in their study and several



### 28 other studies (Section 9.6.3), which provided biological plausibility for the observed higher water



### 29 loss at the ecosystem level. Ho wever, many experiments, mostly based on short

- term O 3 exposure,

found that O 3 generally reduced stomatal conductance. The O 3 - induced reduction in stomatal



### 31 aperture is the biological assumption for most process

- based models. Therefore, results of those



### 32 mod els normally found that O 3 reduced water loss. For example, Felzer (2009, 191460 ) foun d that



### 33 O 3 damage and N limitation together reduced evapotranspiration and increase runoff.



### 34 Although the direction of the response differed among studies, the evidence is sufficient to

conclude that there is likely to be a causal relationship between O 3 exp osure and the alteration



### 36 of ecosystem water production.

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## 9.2.4.3. Below

- Ground Processes

1 Since the 2006 O 3 AQCD, more evidence has shown that although the responses are often



### 2 species specific, O 3 altered the quality and quantity of C input to soil, microbial communi ty



### 3 composition, and C and nutrient cycling. Biogeochemical cycling of below

- ground processes is



### 4 driven by C input from plants. Studies at the leaf and plant level have provided biologically plausible

mechanisms, such as reduced photosynthetic rates, increased metabolic cost, and reduced root C



### 6 allocation (Section 9.6.4) for the association of O 3 exposure and the alteration of below -ground



### 7 processes.



### 8 Results from Aspen FACE and other experimental studies consistently found that O 3 reduced



### 9 litter production and altered C chemistry, such as soluble sugars, soluble phenolics, condensed

tannins, lignin, and macro/micro nutrient concentration in litter (Liu et al., 2005, 187005 ) (Parsons et



### 11 al., 2008, 191853 ) (Kasurinen et al., 2006, 191269 ) . The changes in substrate quality and quantity



### 12 could alter microbial metabolism unde r elevated O 3 , and therefore soil C and nutrient cycling.



### 13 Several studies indicated that O 3 generally suppressed soil enzyme activities (Chung et al., 2006,



### 14 191729 ) (Pritsch et al., 2009, 626808 ) . However, the impact of O 3 on litter decomposition was

inconsistent and varied among species, sites and exposure length. Ozone had small impact on



### 16 dynamics of micro and macro nutrients, except for N. Ozone was found to reduce N release from



### 17 leaf litter and decrease gross N mineralization, which could potentially decrease N availability to



### 18 plants (Holmes et al., 2006, 191372 ) (Liu et al., 2007, 093286 ).



### 19 Studies from the Aspen FACE experiment suggested that the response of below -ground

C cycle to O 3 exposure, such as litter decomposition, soil respiration and soil C content, changed



### 21 over time. For example, in the early part of the experiment (1998

- 2003), O 3 had no impact on soil



### 22 respiration but reduced the formation rates of total soil C under elevated CO 2 . However, after 10

­



### 23 11 yr of exposure, O 3 was found to increase soil respiration but have no significant impact on soil



### 24 C formation under elevated CO 2 (Section 9.6.4. 3 ).

The evidence is sufficient to in fer that there is a causal relationship between O 3 exposure



### 26 and the alteration of below

- ground biogeochemical cycles.



## 9.2.4.4. Community Compos ition

27 In the 2006 O 3 AQCD, the impact of O 3 exposure on species competition and community



### 28 composition was assessed. Ozone was found to cause a significant decline in ponderosa and Jeffrey



### 29 pine in the San Bernardino Mountains in southern California. O zone exposure also tended to shift

the grass

- legume mixtures in favor of grass species (U.S. EPA, 2006, 088089 ) . Since the 2006 O 3



### 31 AQCD, more evidence has shown that O 3 exposure changed the competitive interactions and led to



### 32 loss of O 3 sensitive species or genotypes. Studies at plant level found that the severity of O 3 damage



### 33 on growth, reproduction and foliar injury varied among species (Section 9.6.5), which provided the



### 34 biological plausibility for t he alteration of community composition. Additionally, research since the

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1 last review has shown that O 3 can alter community composition and diversity of soil microbial



### 2 communities.



### 3 The decline of conifer forests under O 3 exposure was continually observed in several regions.



### 4 Ozone damage was believed to be an important causal factor in the dramatic decline of sacred fir in

the valley of Mexico (de Lourdes de Bauer and Hernandez

- Tejeda, 2007, 196891 ) , as well as



### 6 c embran pine in southern France and Carpathian Mountains (Wieser et al., 2006, 191391 ) . Results



### 7 from the Aspen FACE site indicated that O 3 could alter community composition of broadleaf forests



### 8 as well. At the Aspen FACE site, O 3 reduced growth and increased mortality of a sensitive aspen



### 9 clone, while the O 3 tolera nt clone emerged as the dominant clone in the pure aspen community. In

the mixed aspen

- birch and aspen

- maple communities, O 3 reduced the competitive capacity of aspen



### 11 compared to birch and maple (Kubiske et al., 2007, 191336 ).



### 12 The tendency for O 3 - exposure to shift the biomass of grass

- legume mixtures in favor of grass



### 13 species, was reported in the 2006 O 3 AQCD (U.S. EPA, 2006, 088089 ) and has been generally



### 14 confirmed b y recent studies. However, in a high elevation mature/species

- rich grass

- legume pasture,

O 3 fumigation showed no significant impact on community composition (Bassin et al. , 2007,



### 16 191534 ).



### 17 Ozone exposure not only altered community composition of plant species, but also



### 18 microorganisms. The shift in community composition of bacteria and fungi has been observed in



### 19 both natural and agricultural ecosystems, although no general patterns could be identified (Kanerva

et al., 2008, 191264 ) (Morsky et al., 2008, 191507 ) (Kasurinen et al., 2005, 191245 )

. 

### 21 The evidence is sufficient to conclude that there is likely a causal relationship between O 3



### 22 exposure and the alteration of community composition.



## 9.2.5. Air Qu ality Indic es

23 Expo sure indices are metrics that quantify exposure as it relates to measured plant damage



### 24 (i.e., reduced growth). They are summary measures of monitored ambient O 3 concentrations over

time intended to provide a consistent metric for reviewing and comparing ex posure- response effects



### 26 obtained from various studies. No new information is available since 2006 that alters the basic



### 27 conclusions put forth in the 2006 and 1996 O 3 AQCDs (U.S. EPA, 1996, 080827 ) (U.S. EPA, 2006,



### 28 088089 ) . These AQCDs focused on the research used to develop various exposure indices to help



### 29 quantify effects on growth and yield in crops, perennials, and trees (primarily seedlings). The

performance of indices was compared through regression analyses of earlier studies designed to



### 31 support the estimation of predictive O 3 exposure

- response models for growth and/or yield of crops



### 32 and tree (seedling) species.



### 33 The main conclusions from the 1996 and 2006 O 3 AQCDs (U.S. EPA, 1996,



### 34 080827 ) (U.S. EPA, 2006, 088089 ) regarding an index based on ambient exposure are still valid.

These key conclusions can be restated as follows:



### 36  O 3 effects in plants are cumulative;

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1  higher O 3 conc entrations appear to be more important than lower concentrations in



### 2 eliciting a response;



### 3  plant sensitivity to O 3 varies with time of day and plant development stage; and



### 4  exposure indices that cumulate hourly O 3 concentrations and preferentially weight the

higher concentrations have better statistical fits to growth/yield response data than do the



### 6 mean and peak indices.



### 7 Various weighting functions have been used, including threshold

- weighted (e.g., SUM06) and



### 8 continuous sigmoid

- weighted (e.g., W126) functions. Based on statistical goodness -of- fit tests, these



### 9 cumulative, concentration

- weighted indices could not be differentiated from one another using data

from previous exposure studies. Additional statistical forms for O 3 exposure indices have been



### 11 discusse d in Lee et al. (1988, 042136 ) and in Section 9.7 of this ISA. The majority of studies



### 12 published since the 2006 O 3 AQCD (200 6, 088089 ) do not change earlier conclusions, including the



### 13 importance of peak concentrations, and the duration and occurrence of O 3 exposures in altering pl ant



### 14 growth and yield.

Given the current state of knowledge and the best available data, exposure indices that



### 16 cumulate and differentially weight the higher hourly average concentrations and also include the



### 17 mid

- level values continue to offer the most defen sible approach for use in developing response



### 18 functions and comparing studies, as well as for defining future indices for vegetation protection.



## 9.2.5.1. Mo de le d Ozone De p os ition or "Flux"

19 Another approach for improving risk assessment of vegetation response to amb ient O 3 is based

on determining the O 3 concentration from the atmosphere that enters the leaf (i.e., flux or



### 21 deposition). Interest has been increasing in recent years, particularly in Europe, in using



### 22 mathematically tractable flux models for O 3 assessments at the regional, national, and European



### 23 scale (Matyssek et al., 2008, 191262 ) (Paoletti and Manning, 2007, 180174 ) (Emberson et al., 2000,



### 24 040350 ) (Embe rson et al., 2000, 042537 ) (ICP M&M, 2004, 677471 ) . While some efforts have been

made in the U.S. to calculate O 3 flux int o leaves and canopies (Grantz et al., 1997, 026664 ) (Gran tz



### 26 et al., 1995, 026659 ) (Grulke et al., 2004, 042646 ) (Turnipseed et al., 2009, 588752 ) (Uddling et al.,



### 27 2009, 596219 ) (Bergweiler et al., 2008, 191656 ) (Hogg et al., 2007, 199349 ) , little information has



### 28 been published relating these fluxes to effects on vegetation. There is also concern that not all O 3



### 29 stomatal uptake results in a yield reduction, which depends t o some degree on the amount of internal

detoxification occurring with each particular species. Those species having high amounts of



### 31 detoxification potential may, in fact, show little relationship between O 3 stomatal uptake and plant



### 32 response (Musselman and Massman, 1999, 040706 ) . The lack of data in the U.S. and the lack of



### 33 understanding of detoxification processes have made this technique less viable for vulnerability and



### 34 r isk assessments in the U.S.

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## 9.2.5.2. Night

- Time Exp os ures

1 A 12

- h (8 :00 a.m. to 8 :00 p.m. ) daylight period for a W126 cumulating exposure was proposed



### 2 in 2007 and 2009 following the release of the 2006 O 3 AQCD and was based primarily on evidence



### 3 that the conditions for uptake of O 3 into the plant occur mainly during the daytime hours. Plants have



### 4 the highest stomatal conductance during the daytime and atmospheric turbulent mixing is greatest

then as well (U.S. EPA, 2006, 088089 ) (Uddling et al., 2010, 387073 ) . Recent reviews of the



### 6 literature reported that a large number of species had varying degrees of nocturnal stomatal



### 7 conductance (Caird et al., 2007, 199337 ) (Dawson et al., 2007, 670381 ) (Musselman and Minnick,



### 8 2000, 011612 ) . In general, stomatal conductance at night is at a much lower rate compared to



### 9 daytime conductance (Cai rd et al., 2007, 199337 ) . For significant nocturnal stomatal flux and O 3 effects to occur, specific conditions must exist. A susceptible plant with nocturna l stomatal



### 11 conductance and low defense must be growing in an area with relatively high night

- time O 3 and



### 12 appreciable nocturnal turbulence. It is unclear how many areas there are in the U.S. where these



### 13 conditions occur. More information is needed in these locations in order to assess the local O 3



### 14 patterns, micrometeorology and responses of potentially vulnerable plant species.



## 9.2.6. Expos ure

- Response

None of the information on effects of O 3 on vegetation published since the 2006 O 3 AQCD has



### 16 modified the assessme nt of quantitative exposure

- response relationships that was presented in that



### 17 document (U.S. EPA, 2006, 088089 ) . This assessment updates the 2006 exposure

- response models



### 18 by computing them using the W126 metric, cumulated over 90 days. Almost all of the experimental



### 19 research on the effects of O 3 on growth or yield of plants pub lished since 2006 used only two levels

of exposure. In addition, hourly O 3 concentration data that would allow calculations of exposure



### 21 using the W126 scale are generally unavailable. However, two long

- term experiments, one with a



### 22 crop species (soybean), o ne with a tree species (aspen), have produced data that can be used to



### 23 validate the exposure

- response models presented in the 2006 O 3 AQCD, and methodology used to



### 24 derive them.

Quantitative characterization of exposure

- response in the 2006 O 3 AQCD was bas ed on



### 26 experimental data generated for that purpose by the National Crop Loss Assessment Network



### 27 (NCLAN) and EPA National Health and Environmental Effects Research Laboratory, Western



### 28 Ecology Division (NHEERL

- WED) projects, using OTCs to expose crops and tr ees seedling to O 3

. 

### 29 In recent years, yield and growth results for two of the species that had provided extensive exposure

response information in those projects have become available from studies that used FACE



### 31 technology, which is intended to provide cond itions much closer to natural environments (Dickson et



### 32 al., 2000, 628220 ; Morgan et al., 2004, 072764 ; Morgan et al., 2006, 079186 ; Pregitzer et al., 2008,



### 33 191677 ) . The robust methods that were used previously with e xposure measured as SUM06 were



### 34 applied to the NCLAN and NHEERL

- WED data with exposure measured as W126, in order to

derive single

- species median models for soybean and aspen from studies involving different

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### 1 genotypes, years, and locations. The resulting mo dels were used to predict the change in yield of



### 2 soybean and biomass of aspen between the two levels of exposure reported in current FACE



### 3 experiments. Results from these new experiments were exceptionally close to predictions from the



### 4 models. The accuracy of model predictions for two widely different plant species provides support



### 5 for the validity of the corresponding multiple

- species models for crops and trees in the NCLAN and



### 6 NHEERL

- WED projects. However, variability among species in those projects indica tes that the



### 7 range of sensitivity is likely quite wide. This was confirmed by a recent experiment with cottonwood



### 8 in a naturally occurring gradient of exposure (Gregg et al., 2006, 186961 ) , which established the



### 9 occurrence of species with responses substantially more severe under currently existing conditions



### 10 than are predicted by the median model for multiple species.



### 11 Re sults from several meta

- analyses have provided approximate values for responses of yield



### 12 of soybean, wheat, rice and other crops under broad categories of exposure, relative to charcoal



### 13 filtered air (Ainsworth, 2008, 191646 ; Feng et al., 2008, 191453 ; Morgan et al., 2003, 055527 )

. 

### 14 Likewise, Feng and Kobayashi (2009, 199223 ) have summarized yield data for six crop species



### 15 under various broad comparative exposure categories, while Wittig et al. (2009, 191631 ) reviewed



### 16 263 studies that reported effects on tree biomass. However, these analyses have proved difficult to



### 17 compare with e xposure

- response models, especially given that exposure was not expressed on the



### 18 same W126 scale.



# 9.3. Exp e rime n ta l Expo s u re Me th o do lo gie s

9.3.1. Introd uc tion

19 A variety of methods for studying plant response to O 3 exposures have been developed over



### 20 the last several d ecades. Methodological advancements since 2006 have not fundamentally altered



### 21 our understanding of O 3 effects on plants or ecosystems. The majority of methodologies currently



### 22 used have been discussed in detail in the 1996 O 3 AQCD (Section 5.2; U.S. EPA, 1996, 080828 ) and



### 23 2006 O 3 AQCD (Section AX9.1; U.S. EPA, 2006, 088089 ) . This section will serve as a short



### 24 overview of the methodologies and the reader is re ferred to the previous O 3 AQCDs for more in



### 25 depth discussion.



## 9.3.2. “Ind o or,” Con trolled En viro n men t, an d Gre en ho u s e Ch a mb ers

26 The earliest experimental investigations of the effects of O 3 on plants utilized simple glass or



### 27 plastic

- covered chambers, often located within greenhouses, into which a flow of O 3 - enriched air or



### 28 oxygen could be passed to provide the exposure. The types, shapes, styles, materials of construction,



### 29 and locations of these chambers have been numerous. Hogsett et al. (1987, 043465 ) have



### 30 summarized the construction and performance of more elaborate and better instrumented chambers



### 31 since the 1960s, inclu ding those installed in greenhouses (with or without some control of



### 32 temperature and light intensity).

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1 One greenhouse chamber approach that continues to yield useful information on the



### 2 relationships of O 3 uptake to both physiological and growth effects employs continuous stirred tank



### 3 reactors (CSTRs) first described by Heck et al. (1978, 037673 ) . A lthough originally developed to



### 4 permit mass

- balance studies of O 3 flux to plants, their use has more recently widened to include

short- term physiological and growth studies of O 3 × CO 2 interactions (Heagle et al., 1994,



### 6 026699 ) (Loats and Rebbeck, 1999, 029709 ) (Rao et al., 1995, 030221 ) (Reinert and Ho, 1995,



### 7 030247 ) (Reinert et al., 1997, 030252 ) , and validation of visible foliar injury on a variety of plant



### 8 species (Kline et al., 2009, 196918 ) (Orendovici et al., 2003, 049080 ) . In many cases, supplementary



### 9 lighting a nd temperature control of the surrounding structure have been used to control or modify the

environmental conditions (Heagle et al., 1994, 026699 ).



### 11 Many investigations have utilized commercially available controlled environment chambers



### 12 and walk

- in rooms adapted to permit the introduction of a flow of O 3 into the controlled air

- volume.



### 13 Such chambers continue to find use in genetic screening and in physiological and bioc hemical



### 14 studies aimed primarily at improving our understanding of modes of action. For example, some of

the studies of the O 3 responses of common plantain ( Plantago major ) populations have been



### 16 conducted in controlled environment chambers (Rei ling and Davison, 1994, 035373 ) (Whitfield et



### 17 al., 1996, 055544 ).



### 18 More recently, some researchers have been interested in direct O 3 effects on reproductive



### 19 processes, separate from the effects on vegetative processes (Black et al., 2010, 625575 ) . For this

purpose, controlled exposure systems have been employed to expose the reproductive structures of



### 21 annual plants to gaseous pollutants independently of the vegetative component (Black et al., 2010,



### 22 625575 ) (Stewart et al., 1996, 036384 ).



## 9.3.3. Field Chambers

23 In general, field chamber studies are dominated by the use of various versions of the open top



### 24 chamber (OTC) design, first described by Heagle et al. (1973, 038348 ) and Mandl et al. (1973,

039967 ) . The OTC method continues to be a widely used technique in the U.S. and Europe for



### 26 exposing plan ts to varying levels of O 3 . Most of the new information confirms earlier conclusions



### 27 and provides additional support for OTC use in assessing plant species and in developing exposure



### 28 response relationships. Chambers are generally ~3 m in diameter with 2.5 -m- high walls. Hogsett et



### 29 al. (1987, 043780 ) described in detail many of the various modifications to the original OTC designs

that appeared subsequently, e.g., the use of larger chambers for exposing small trees (Kats et al.,



### 31 1985, 055511 ) or grapevines (Mandl et al., 1989, 043987 ) , the addition of a conical baffle at the top



### 32 to improve ventilation (Kats et al., 1976, 039799 ) , a frustum at the top to reduce ambient air



### 33 incursions, and a p lastic rain

- cap to exclude precipitation (Hogsett et al., 1985, 039383 ) . All versions



### 34 of OTCs included the discharge of air via ports in annular ducting or interiorly perforated double

layered walls at the base of the chambers to provide turbulent mixing and the upward mass flow of



### 36 air.

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38 Chambered systems, including OTCs, have several advantages. For instance, they can provide

a range of treatment levels including charcoal

- filtered (CF), clean

- air control, and several above

ambient concentrations for O 3 experiments. Depending on experimental intent, a replicated, clean

- air

control treatment is an essential component in many experimental designs. Th e OTC can provide a

consistent, definable exposure because of the constant wind speed and delivery systems. Statistically

robust concentration

- response (C

- R) functions can be developed using such systems for evaluating

the implications of various alternati ve air quality scenarios on vegetation response. Nonetheless,

there are several characteristics of the OTC design and operation that can lead to exposures that

might differ from those experienced by plants in the field. First, the OTC plants are subjected to

constant air flow turbulence, which, by lowering the boundary layer resistance to diffusion, may

result in increased uptake. This may lead to an overestimation of effects relative to areas with less

turbulence (Krupa et al., 199 5, 038895 ) (Legge et al., 1995, 040689 ) . Conversely, however, other

research has found that OTC's may slightly change vapor pressure deficit (VPD) in a way that may

decrease the uptake of O 3 into leaves (Piikki et al., 2008, 199265 ) . As with all methods that expose

vegetation to modified O 3 concentrations in chambers, OTCs create internal environments that differ

from ambient air. This so

- called “chamber effect” refers to the modification of microclimatic

variables, including reduced and uneven light intensity, uneven rainfall, constant wind speed,

reduced dew formation, and increased air temperatur es (Fuhrer, 1994, 055549 ) (Manning and Krupa,

1992, 044155 ) . However, in at least one case where canopy resistance was quantified in OTCs and in

the field, it was determined that gaseous pollutant exposure to crops in OTCs was similar to that

which would have occurred at the same concentration i n the field (Unsworth et al., 1984,

041239 ) (Unsworth et al., 1984, 040024 ) . Because of the standardized methodology and protocols

used in National Crop Loss Assessment Network (NCLAN) and other programs, the database can be

assumed to be internally consistent.

While it is clear that OTCs can alter some aspects of the microenvir onment and plant growth,

it is important to establish whether or not these differences affect the relative response of a plant to

O 3 . As noted in the 1996 O 3 AQCD (U.S. EPA, 1996, 080828 ) , evidence from a number of

comparative studies of OTCs and other exposure systems suggested that responses were essentially

the same regardless of exposure system used and chamber effects did not significantly affect

response. For example, a study of chamber effects examined the responses of tolerant and sensitive

white clover clones ( Trifolium repens ) to ambient O 3 in greenhouse, open top, and ambient plots

(Heagle et al., 1996, 042660 ) . The response found in OTCs was the same as in ambient plots.

Another type of field chamber called a “ terracosm ” has been developed and used in recent

studies (Lee et al., 2009, 595904 ) . Concern over the need to establish realistic plant

- li tter

- soil

relationships as a prerequisite to studies of the effects of O 3 and CO 2 enrichment on ponderosa pine

( Pinus ponderosa ) seedlings led Tingey et al. (1996, 055536 ) to develop closed, partially

environmentally controlled, sun

- lit chambers (“terracosms”) incorporating 1 -m- deep lysimeters

containing forest soil in which the appropriate horizon structure was retained.

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1 Other researchers have recently published studies using another type of out

- door chamber



### 2 called recirculating Outdoor Plant Environment Chambers (OPECs) (Flowers et al., 2007, 191852 )

. 

### 3 These closed chambers are approximately 2.44 m×1.52 m with a growth volume of approximately



### 4 3.7 m 3 in e ach chamber. These chambers admit 90% of full sunlight and control temperature,

humidity and vapor pressure (Fiscus et al., 1999, 672964 ).



## 9.3.4. Plu me an d FACE

- Type Sys te ms

6 Plume systems are chamberless exposure facilities in which the atmosphere surrounding plants



### 7 in the field is modified by the injection of pollutant gas into the air above or around them from



### 8 multiple orifices s paced to permit diffusion and turbulence, so as to establish relatively homogeneous



### 9 conditions as the individual plumes disperse and mix with the ambient air. They can only be used to

increase the O 3 levels in the ambient air.



### 11 The most common plume system used in the U.S. is a modification of the free

- air carbon



### 12 dioxide/ozone enrichment (FACE) system (Hendrey and Kimball, 1994, 040397 ) (Hendrey et al.,



### 13 1999, 042641 ) . Although originally designed to provide chamberless field facilities for studying the



### 14 CO 2 effects of climate change, FACE systems have been adapted to include the dispensing of O 3 (Karnosky et al., 1999, 035307 ) . This method has been employed in Illinois (SoyFACE) to study



### 16 soybeans (Morgan et al., 2004, 072764 ) (Rogers et al., 2004, 079201 ) and in Wisconsin (Aspen



### 17 FACE) to study trembling aspen ( Populus tremuloides ), birch ( Betula papyrifera ) and maple ( Acer



### 18 saccharum ) (Karnosky et al., 1999, 035307 ) . Volk et al. (2003, 055568 ) also described a similar



### 19 system for exposing grasslands that uses 7

- m diameter plots. FACE systems discharge the pollutant

gas (O 3 and/or CO 2 ) through orifices spaced along an a nnular ring (or torus) or at different heights on



### 21 a ring of vertical pipes. Computer

- controlled feedback from the monitoring of gas concentration



### 22 regulates the feed rate of enriched air to the dispersion pipes. Feedback of wind speed and direction



### 23 informat ion ensures that the discharges only occur upwind of the treatment plots, and that discharge



### 24 is restricted or closed down during periods of low wind speed or calm conditions. The diameter of

the arrays and their height (25

- 30 m) in some FACE systems requir es large throughputs of enriched



### 26 air per plot, particularly in forest tree systems. The cost of the throughputs tends to limit the number



### 27 of enrichment treatments, although Hendrey et al. (1999, 042641 ) argued that the cost on an enriched



### 28 volume basis is comparable to that of chamber systems.



### 29 Although plume systems make virtually none of the modifications to the physical environment

that are inevitable with c hambers, their successful use depends on selecting the appropriate numbers,



### 31 sizes, and orientations of the discharge orifices to avoid “hot-spots” resulting from the direct



### 32 impingement of jets of pollutant

- enriched air on plant foliage (Werner and Fabian, 2002, 053040 )

. 

### 33 Because mixing is unassisted and completely dependent on wind turbulence and diffusion, local



### 34 gradients are inevitable especially in l arge

- scale systems. FACE systems have provisions for shutting

down under low wind speed or calm conditions and for an experimental area that is usually defined



### 36 within a generous border in order to strive for homogeneity of the exposure concentrations withi n the



### 37 treatment area. They are also dependent upon continuous computer

- controlled feedback of the O 3

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### 2 systems also are unable to reduce O 3 levels below ambient in a reas where O 3 concentrations are



### 3 phytotoxic.



## 9.3.5. Ambient Gradients

4 Ambient O 3 gradients that occur in the U.S. hold potential for the examination of plant

responses over multiple levels of exposure that are occurring. However, few such gradients can be



### 6 found that meet the rigorous statistical requirements for comparable site characteristics such as soil



### 7 type, temperature, rainfall, radiation, and aspect (Manning and Krupa, 1992, 044155 ) ; although with



### 8 small plants, soil variability can be avoided by the use of plants in large pots. The use of soil



### 9 monoliths transported to various locations along natural O 3 gradi ents is another possible approach to

overcome differences in soils; however, this approach is also limited to small plants.



### 11 Studies in the 1970s used the natural gradients occurring in southern California to assess yield



### 12 losses of alfalfa and tomato) (Oshima et al., 1976, 038475 ) (Oshima et al., 1977, 038938 ) . A transect



### 13 study of the impact of O 3 on the growth of white clover and barley in the U.K. was confounded by



### 14 differences in the concurrent gradients of SO 2 and NO 2 pollution (Ashmore et al., 1988, 037038 )

. Studies of forest tree species in national parks in the eastern U.S. (Winner et al., 1989, 043403 )



### 16 revealed increasing gradients of O 3 and visible foliar injury with increased elevation.



### 17 Several studies have used the San Bernardino Mountains Gradient Study in southern



### 18 California to study the effects of O 3 and N deposition on forests dominate d by ponderosa and Jeffrey



### 19 pine (Arbaugh et al., 2003, 052925 ) (M iller and Elderman, 1977, 038488 ) (Grulke, 1999,

052983 ) (Jones and Paine, 2006, 191301 ) . However, it is difficult to separate the effects of N and O 3



### 21 in some instances in these studies (Arbaugh et al., 2003, 052925 ) . An O 3 gradient in Wisconsin has



### 22 been used to study foliar injury in a series of trembling aspen clones ( Populus tremuloides ) differing



### 23 in O 3 sensitivity (Karnosky et al., 1999, 035307 ) (Maňkovská et al., 2005, 672965 )

. 

### 24 More recently, some studies have been published that have used natural gradients to study a

variety of endpoints and spec ies. For example, Gregg et al. (2003, 046996 ) studied cottonwood



### 26 saplings grown in an urban to rural gradient of O 3 in the New Yor k City area. The secondary nature



### 27 of the reactions of O 3 formation and NO X titration reactions within the city center resulted in



### 28 significantly higher cumulative O 3 exposures in the rural sites. The results of this gradient study were



### 29 similar to those of a parallel OTC study. Also, the U.S. forest service Forest Inventory and Analysis

(FIA) program uses large

- scale O 3 exposure patterns across the continental U.S

. to study occurrences



### 31 of foliar injury due to O 3 exposure (Smith et al., 2003, 044183 ) ( Section 9.5.2.3). Finally,



### 32 McLaughlin et al. (2007, 090348 ) (2007, 090347 ) used spatial and temporal O 3 gradients to study



### 33 forest growth and water use in the southern Appalachians. These studies found varying O 3 exposures



### 34 between years and between sites.

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## 9.3.6. Comparative Studies

All experimental approaches used to expose plants to O 3 have shortcomings. The use of

laboratory, greenhouse, or field chambers raises concerns for the roles of chamber effects on

micrometeorology. In contrast, plume, FACE and gradient systems suffer from limited exposure

levels, few replicates and an inability to reduce O 3 levels below ambient in areas where O 3 concentrations are phytotoxic.

While it is clear that chambers can alter some aspects of plant growth, it is important to

establish whether or not these differences affect plant response to O 3 . As noted in the 1996 O 3 AQCD

(U.S. EPA, 1996, 080828 ) , evidence from the comparative studies of OTCs and from closed

chamber and O 3 - exclusion exposure systems on the growth of alfalfa ( Medicago sativa ) by Olszyk et

al. (1986, 055530 ) suggested that, since significant differences were found for fewer than 10% of the

growth parameters measured, the responses were, in general, essentially the same regardless of

exposure system used ; and chamber effects did not significantly affect response. In 1988, Heagle et

al. (1988, 043559 ) concluded: “Although chamber effects on yield are common, there are no results

showing that this will result in a changed yield response to O 3 .” A study of chamber effects examined

t he responses of tolerant and sensitive white clover clones ( Trifolium repens ) to ambient O 3 in

greenhouse, open

- top, and ambient plots (Heagle et al., 1996, 042660 ) . For individual harvests,

greenhouse O 3 exposure reduced the forage weight of the sensitive clone 7 to 23% more than in

OTCs. However, the response in OTCs was the same as in ambient plots. Several studies have

shown very similar response of yield to O 3 for plants grown in pots or in the groun d, suggesting that

even such a significant change in environment does not alter the proportional response to O 3 , at least

as long as the plants are well watered (Heagle, 1979, 039329 ) (Heagle et al., 1983, 039372 ).

A few recent studies have compared results of O 3 experiments between OTCs, FACE

experiments

, and gradient stu dies. For example, a series of studies undertaken at Aspen FACE

(Isebrands et al., 2000, 044174 ) (Isebrands et al., 2001, 036345 ) showed that O 3 - symptom expression

was generally similar in OTCs, FACE, and ambient -O 3 gradient sites, and supported the previously

observed variation among trembling aspen clones using OTCs (Karnosky et al., 1999,

035307 ) (Maňkovská et al., 2005, 672965 ) . In the SoyFACE experiment in Illinois, soybean (Pionee r

93B15 cultivar) yield loss data from a two

- year study was published (Morgan et al., 2006, 079186 )

. This cultivar is a recent selection and, like most modern cultivars, has been selected under an already

high current O 3 exposure. It was found to have average sensitivity to O 3 compared to 22 other

cultivars tested at SoyFACE. In th is experiment, ambient hourly O 3 concentrations were increased by

approximately 20% and measured yields were decreased by 15% in 2002 as a result of the increased

O 3 exposure ( Morgan et al., 2006, 079186 ) . To compare these results to chamber studies, Morgan et

al. (2006, 079186 ) calculated the expected yield loss from a linear relationship constructed from

chamber data using 7

- h seasonal averages (Ashmore, 2002, 672967 ) . They calculated an 8%

expected yield loss from the 2002 O 3 exposure using that linear relationship. In another study, Gregg

et al. (2003, 046996 ) (2006, 186961 ) found similar O 3 effects on cottonwood sapling biomass growth

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### 1 and physiology along an ambient O 3 g radient in the New Yor k City area and a parallel OTC study.



### 2 Additionally , Section 9.8.3 of this document presents comparisons of exposure

- response in from



### 3 OTC studies in trees and crops with results from more recent FACE experiments.



# 9.4. Me c h a n is ms Go ve rn in g Veg e ta tio n Re s p o n s e to

Ozone

9.4.1. Introd uc tion

4 This s ection focuses on the effects of O 3 stress on plants and their responses to that stress on



### 5 the molecular, biochemical and physiological levels. First, the pathway of O 3 uptake into the leaf and



### 6 the initial chemical reactions occurring in the substomatal cavity and apoplast will be described



### 7 (Section 9.4.2). Once O 3 has entered the substomatal cavity and apoplast, it is thought that the cell



### 8 must be able to sense the presence of O 3 or its breakdown products in o rder to initiate the rapid



### 9 changes in gene expression that have been measured in O 3 - treated plants. While an “O 3 sensor” still



### 10 remains elusive, much progress has been made in examining several different mechanisms that may



### 11 contribute both to sensing the pr esence of O 3 and its breakdown products, and also transducing a



### 12 signal to the nucleus to initiate changes in gene transcription, which will be described in Section



### 13 9.4.3.1. The next section focus es on changes in gene expression in response to O 3 exposure, with



### 14 particular emphasis on results from transcriptome and proteome analyses (Section 9.4.3.2).



### 15 Subsequently, the role of phytohormones such as salicylic acid (SA), ethylene (ET), jasmonic acid



### 16 (JA), and abscisic acid (ABA) and their interactions in both s ignal transduction and determining



### 17 plant response to O 3 is discussed in Section 9.4.3.3. After O 3 uptake and sensing, plants can respond



### 18 to the oxidative stress to minimize damage. These mechanisms of detoxification, with particular



### 19 emphasis on antioxidant enzymes and metabolites, are reviewed in Section 9.4.4. The next section



### 20 focus es on the effects of O 3 on primary and secondary metabolism in plants, looking at



### 21 photosynthesis, respiration and several secondary metabolites, some of which may also act as



### 22 an tioxidants and protect the plant from oxidative stress (Section 9.4.5). The last section focus es on



### 23 the mechanisms underlying changes in stomatal function (Section 9.4.6). For many of these topics,



### 24 information from the 2006 O 3 AQCD (U.S. EPA, 2006, 088089 ) has been summarized, as this



### 25 information is still valid and supported by more recent findings. For other topics, such as genomics



### 26 and proteomics, which have arisen due to the availability of new technologies, the information is



### 27 based solely on new publications with no reference to the 2006 O 3 AQCD.



### 28 As Section 9.4 focuses on me chanisms underlying vegetation response to O 3 , the conditions



### 29 that are used to study these mechanisms are often artificial and do not necessarily reflect conditions



### 30 that a plant may be exposed to in an agricultural setting or natural ecosystem. The goal of many of



### 31 these studies is to elicit a plant response to O 3 in a relatively short period of time and not always to



### 32 simulate ambient O 3 exposures. Therefore, plants are often exposed to unrealistically high O 3



### 33 concentrations for several hours or days (acute exposure), and only in a few cases to ambient or

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35 slightly elevated O 3 concentrations for longer time periods (chronic exposure). Additionally, the

plant species utilized in these studies are often not agriculturally important or commonly found as

part of natural ecosystems. Model organisms such as Arabidopsis thaliana are frequently used as

they are easy to work with, and mutants or transgenic plants are easy to develop or have already been

developed. Furthermore, the Arabidopsis genome has been sequenced, and much is known about the

molecular basis of many biochemical and cellular processes.

Many of the studies described in this section focus on changes in the expression of genes in

O 3 - treated plants. However, changes in gene expression (i.e., either up - o r down

- regulation of gene

expression) do not always translate into changes in protein quantity and/or activity, as there are many

levels of post

- transcriptional and post

- translational modifications which impact protein quantity and

activity. Frequently, th ese studies do not evaluate whether the observed changes in gene expression

lead to changes at the protein level and, therefore, it is not always clear how relevant the changes in

gene expression are in determining plant response to O 3

. The advent of new technologies, such as those employed in genomics and proteomics, has

allowed for a more comprehensive analysis of the many molecular and biochemical mechanisms of

plant response to O 3 and how all these responses interact with or affect each other to determine the

ultimate response of plants to a stressor such as O 3 . While the studies of transcriptome changes are

very valuable, further work needs to be done to evaluate whether the transcriptome changes result in

concomitant changes in the proteome. A few rec ent studies have evaluated proteome changes in

response to O 3 , and those results are also discussed in Section 9.4.3.2.

The most significant change in this section in relation to the 2006 O 3 AQCD (U.S. EPA, 2006,

088089 ) is the increased emphasis on the understanding of molecular mechanisms underlying plant

responses to O 3 , as a significant number of the newer studies that were evaluated for this section

focus on changes in gene expression in plants exposed to elevated O 3 . Conclusions from the 2006 O 3 AQCD have been supported by these new studies, and the advent of new technolo gies has allowed

for a more comprehensive understanding of the mechanisms governing plant response to O 3

. In summary, the goal of many of these new studies reported on in this section was to increase

knowledge of the mechanisms of plant response to O 3 by using artificial exposure conditions and

model organisms. Th is information adds to the understanding of the basic biology of plant response

to oxidative stress in the absence of any other potential stressors. The results of these studies are

important and valid, even though they may not always directly translate into effects observed in

other plants under more realistic exposure conditions. They represent one step in the process of

comprehensively understanding plant responses to oxidative stress, which the n need to be followed

up with additional experiments using other plant species exposed to O 3 under more natural

conditions.

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## 9.4.2. Ozone Uptake into the Leaf

AX 9.2.3 of the 2006 O 3 AQCD clearly describe d the process by which O 3 enters plant leaves

through open s tomata (U.S. EPA, 2006, 088089 ) . This information continues to be valid a nd is only

summarized here.

Ozone moves into the leaf interior by diffusing through open stomata, and environmental

conditions which promote high rates of gas exchange will favor the uptake of the pollutant by the

leaf. Factors that may limit uptake inclu de boundary layer resistance and the size of the stomatal

aperture (Figure 9 -2 ) (U.S. EPA, 2006, 088089 ) . Once inside the substomatal cavity, O 3 is thought to

rapidly react with the aqueous apoplast to form breakdown products known as reactive oxygen

species (ROS), such as hydrogen peroxide (H 2 O 2 ), superoxide (O 2 ), hydroxyl radicals (HO · ) and

peroxy radicals (HO 2 · ) (Figure 9 -3 ). Hydrogen peroxide is not only a toxic breakdown product of O 3 , but has been shown to function as a signaling molecule, which is activated in response to both biotic

and abiotic stressors. The role of H 2 O 2 in signaling was described in detail in the 2006 O 3 AQCD

(U.S. EPA, 2006, 088089 ) . Additional organic molecules present in the apoplast or cell wall, such as

those containing double bonds or sulfhydryls that are sensitive to oxidation, could also be converted

to oxygenated molecules after interacting with O 3 ( Figure 9 -4 ). These reactions are not only pH

dependent but are also influenced by the presence of other molecules in the apoplast (U.S. EPA,

2006, 088089 ) . The 2006 O 3 AQCD provide d a comprehensive summary of what is known about the

possible interactions of O 3 with other biomolecules (U.S. EPA, 2006, 088089 ) . It is in the apoplast

that initial detoxification reactions by antioxidant metabolites and enzymes take place, and these

initial reactions are critical to reduce concentrations of the oxidative breakdown products of O 3 ;

these reactions are described in more detail in Section 9.4.4 of this document.

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### While details among species vary, the general ﷒ The microarchitecture of a dic ot leaf. Figure 9 -2

. overview remains the same. Light that drives photosynthesis generally falls upon the upper (adaxial) leaf surface. Carbon dioxide and ozone enter through the stomata on the lower (abaxial) leaf surfa ce, while water vapor exits through the stomata (transpiration).

Figure 9 -3. Possible reactions of ozone within water. ﷒ (a) Ozone reacts at the double bonds to form carbonyl groups. (b) Under certain circumstances, peroxides are generated.

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### (a) The typical Crigee ﷒ The Crigee mechanism of ozone attack of a double bond. Figure 9 -4

. mechanism is shown in which several reactions paths from the initial product is shown. (b) Typical reaction of ascorbic acid with ozone

.

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## 9.4.3. Cellular to Sys temic Res pons es

9.4.3.1. Ozone Sens ing a nd Signal Trans duc tio n

New technologies allowing for large

- scale analysis of oxidative stress

- induced changes in

gene express ion have facilitated the study of signal transduction processes associated with the

perception and integration of responses to the stress. Many of these studies have been conducted

using Arabidopsis or tobacco plants, for which a variety of mutants are available and/or which can be

easily genetically modified to generate either loss -of- function or over

- expressing genotypes. Several

comprehensive review articles provide an overview of what is known of O 3 - induced signal

transduction processes and how they may help to explain differential sensitivity of plants to the

pollutant (Kangasjarvi et al., 2005, 180341 ) (Ludwikow and Sadowski, 2008, 191426 ) (Baier et al.,

2005, 186866 ) . Additionally, analysis of several studies of transcriptome changes has also allowed

for the compilation of these data to determine an initial time

- course for O 3 - induced activation of

various signaling compounds (Kangasjarvi et al., 2005, 180341 ).

A number of different mechanisms for plant sensing of O 3 have been proposed; however, there

is still much that is not known about this process. Ozone and/or the ROS breakdown products could

be sensed by an apoplastic receptor protein, which would either be directly modified by O 3 or ROS

or which might sense O 3 or ROS modifications in other apoplastic components (Kangasjarvi et al.,

2005, 180341 ) (Baier et al., 2005, 186866 ) . Some of the earliest events that occur in plant response to

O 3 have been described in the guard cells of stomata. Reactive oxygen species were observed in the

chloroplasts of guard cells in the O 3 tolerant Col

- 0 Arabidopsis thaliana ecotype plants within

5 min utes of plant exposure to 350 ppb O 3 (Joo et al., 2005, 191307 ) . Reactive oxygen species from

the breakdown of O 3 in the apoplast are believed to activate GTPases (G

- proteins), which, in turn,

activate several intracellular sources of ROS, including ROS derived from the chloroplasts.

G- proteins are also believed to play a role in activating membrane

- bound NADPH oxidases to

produce ROS and, as a result, propagate the oxidative burst to neighboring cells (Joo et al., 2005,

191307 ) . Therefore, G

- proteins are recognized as important molecules involved in plant responses to

O 3 and may play a role in perceiving ROS from the breakdown of O 3 in the apoplast (Booker et al.,

2004, 020581 ) (Kangasjarvi et al., 2005, 180341 )

. A change in the redox state of the plant and the oxidation of sensitive molecules in itself may

represent a means of perception and signaling of oxidative stress in plants. Disulfide

- thiol

conversions i n proteins and the redox state of the glutathione pool are likely to be important

components of redox sensing and signal transduction (Foyer and Noctor, 2005, 191555 ) (Foyer and

Noctor, 2005, 631198 )

. Calcium (Ca 2+ ) has also been implicated in the transduction of signals to the nucleus in

response to oxidative stress. The influx of Ca 2+ from the apoplast into the cell occurs early in plant

response to O 3 , and it is thought to play a role in regulating the activity of protein kinases, which are

discussed below (Hamel et al., 2005, 191214 ) (Ba ier et al., 2005, 186866 ) . Calcium channel blockers

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39 inhibited O 3 - induced activation of protein kinases in tobacco suspension cells exposed to 500 ppb O 3

for 10 minutes, indicating that the opening of Ca 2+ channels is an important upstream signaling event

or that the as yet unknown upstream process has a requirement for Ca 2+ (Samuel et al., 2000,

625706 )

. Once ROS are generated by NADPH oxidase, signals are further transmitted to the nucleus to

initiate changes in gene expression associated with plant defense responses. Integral to these signal

cascades are m itogen

- activated protein kinases (MAPK), which phosphorylate proteins and activate

various cellular responses (Hamel et al., 2005, 191214 ) . Mitogen

- activated protein kinases are

induced in several different plant species in response to O 3 exposure, including tobacco (Samuel et

al., 2005, 199316 )

, Arabidopsis (Ludwikow et al., 2004, 595939 ) , the shrub Phillyrea latifolia

(Paolacci et al., 2007, 191422 ) and poplar (Hamel et al., 2005, 191214 ) . In tobacco, the MAPK that

is induced by plant exposure to 500 ppb O 3 is a salicylic acid (SA) induced protein kinase (SIPK),

which was found to positively regulate O 3 - induced ethylene (ET) product ion and negatively regulate

SA accumulation (Samuel et al., 2005, 199316 ) . In poplar suspension c ells exposed to 500 ppb O 3 ,

O 3 - induced activation of two distinct MAP kinases was dependent on ROS formation and the

activity of Ca 2+ channels (Hamel et al., 2005, 191214 )

. Arabidopsis mutants with suppressed activity

of M A PK3 and M A PK6 (an ortholog of SIPK in tobacco) were more susceptible to exposure t o

500 ppb O 3 , as evidenced by tissue death resembling the hypersensitive response (Miles et al., 2005,

191648 ) . Similar results of increased susceptibility to 500 ppb O 3 were found in tobacco lines either

over

- expressing SIPK or with suppressed SIPK function. The authors concluded that these tobacco

lines were unable to cope with increased oxidative stress due to the alteration of the normal

O 3 - induced MAPK signal transduction process (Samuel and Ellis, 2002, 625703 ) . The Arabidopsis

MA PK3 and M A PK6 are also differentially induced in Col

- 0 and the O 3 - sensitive Arabidopsis

mutant radical induced cell death (rcd1) exposed to 250

- 300 ppb O 3 for 6 hour s , indicating a role for

these kinases in plant response to oxidative stress (Overmyer et al., 2005, 191596 ) . Similarly,

components of the MAPK cascades were down

- regulated in the sensitive Wassilewskija (Ws)

Arabidopsis ecotype after exposure to 300 ppb O 3 for 6 h, suggesting that the sensitive ecotypes may

not activate these defense responses (Mahalingam et al., 2006, 191221 ) . Expression of MAPK5 was

also reduced in the Ws ecotype in response to chronic O 3 exposures; Ws Arabidopsis plants were

exposed to O 3 concentrations 20

- 25% above ambient for 8

- 12 days in the SoyFACE site (Li et al.,

2006, 191332 )

. The cysteine

- rich RLKs (CRKs), which are p art of the receptor

- like/Pelle kinase (RLKs)

group, are thought to be involved in the regulation of defense responses and cell death in Arabidopsis

(Wrzaczek et al., 2010, 644190 ) . CRKs were up

- regulated by treatments that resulted in apoplastic

ROS production, such as O 3 (250 ppb O 3 for 6 hour s ) and pathogen treatments, but either remained

unchanged or were down

- regulated by treatments resulting in ROS production in other cellular

compartments, such as the mitochondria or chloroplasts. Although their function remains unclear, it

has been p ostulated that a conserved cysteine motif in the CRKs could serve as a sensor for redox

modifications in the cell resulting from ROS production (Wrzaczek et al., 2010, 644190 )

.

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1 In conclusion, experimental evidence suggests that there may be several different mechanisms



### 2 involved in sensing the presence of O 3 or its breakdown products. These mechanisms may vary by



### 3 species or developmental stage of the plant or may co

- exist and be activated by different exposure



### 4 conditions. Calcium and protein kinases are likely involved in the transduction o f the initial signal to

the nucleus and other cellular compartments to initiate the changes in gene transcription discussed in



### 6 Section 9.4.3.2.



## 9.4.3.2. Gene Expres s ion Changes in Res pons e to O zone

7 The advent of DNA microarray technology has allowed for the study of gene expression in



### 8 cells on a large scale. Rather than assessing changes in gene expression of individual genes, DNA



### 9 microarrays facilitate the evaluation of entire transcriptomes, providing a comprehensive picture of

alterations in gene expression. In addition, these studies have provided more insight into the complex



### 11 interactions between molecules and signal pathways, which result in the regulation of plant



### 12 responses to stresses such as O 3 (Ludwikow and Sadowski, 2008, 191426 ) . Transcriptome analysis



### 13 of O 3 - treated plants has been performed in several species, including Arabidopsis thaliana (To sti et



### 14 al., 2006, 191425 ) (Heidenreich et al., 2005, 191260 ) (Li et al., 2006, 191332 ) (Mahalingam et al.,

2005, 191693 ) (Tamaoki et al., 2003, 080053 ) , pepper ( Capsicum annuum ) (Lee and Yun, 2006,



### 16 191592 ) , clover ( Medicago truncatula ) (Puckette et al., 2008, 191698 )

, Phillyrea latifolia (Paolacci



### 17 et al., 2007, 191422 ) , and European beech ( Fagus sylvatica ) (Olbrich et al., 2005, 191697 ) (Olbrich



### 18 et al., 2009, 596020 ) (Olbrich et al., 2010, 625424 ) . In some cases, researchers compared



### 19 trans criptomes of two or more cultivars, ecotypes or mutants that differed in their sensitivity to O 3
(Lee and Yun, 2006, 191592 ) (Tamaoki et al., 2003, 080053 ) (Li et al., 2006, 191332 ) (Puckette et al.,



### 21 2008, 191698 ) (Rizzo et al., 2007, 19144 7 ) . Species, O 3 exposure conditions (concentration, duration



### 22 of exposure) and sampling times varied significantly in these studies. However, functional



### 23 classification of the genes that were either up

- or down

- regulated by plant exposure to O 3 exhibit ed



### 24 c ommon trends. Genes involved in plant defense, signaling and those associated with the synthesis

of plant hormones and secondary metabolism were generally up

- regulated, while those related to



### 26 photosynthesis and general metabolism were generally down -regula ted in O 3 - treated plants (Tosti et



### 27 al., 2006, 191425 ) (Olbrich et al., 2005, 191697 ) (Tamaoki et al., 2003, 080053 ) (Lee and Yun, 2006,



### 28 191592 ) (Puc kette et al., 2008, 191698 ) (Li et al., 200 6, 191332 )

. 

### 29 Analysis of the transcriptome has been used to evaluate differences in gene expression

between O 3 sensitive and tolerant plants. In pepper, 67% of the 180 genes studied that were affected



### 31 by O 3 were differentially regulated in the sensitive and tolerant cultivars. At both 0 hour s and



### 32 48 hour s after a 3

- day exposure at 150 ppb, O 3 responsive genes were either up

- or down -regulated



### 33 more markedly in the sensitive than in the tolerant cultivar (Lee and Yun, 2006, 191592 )

. 

### 34 Transcriptome analysis also revealed differences in timing and magnitude of changes in gene

expression between sensitive and tolerant clovers. Acute exposure (300 ppb O 3 for 6 hour s ) led to the



### 36 production of an oxidative burst in both clovers (Puckette et al., 2008, 191698 ) . However, the



### 37 sensitive Jemalong cultivar exhibited a sustained ROS burst and a concomitant down

- regulation of

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39 defense response genes at 12 hour s after the onset of exposure, while the tolerant JE 154 accession

showed much more rapid and large

- sc ale transcriptome changes than the Jemalong cultivar (Puckette

et al., 2008, 191698 )

. Arabidopsis ecotypes WS and Col

- 0 were exposed to 1.2 × ambient O 3 concentrations for

8- 12 days at the SoyFACE site (Li et al., 2006, 191332 ) . The sensitive WS ecotype showed a far

greater number of changes in gene expression in response to this low

- level O 3 exposure than the

tolerant Col

- 0 ecotype. Exposure of the WS ecotype to 300 ppb O 3 for 6 hour s showed a rapid

induction of genes leading to cell death, such as proteases, and down

- regulation or inactivation of

cell signaling genes, demonstrating an ineffective defense response in this O 3 sensitive ecotype

(Mahalingam et al., 2006, 191221 )

. The temporal response of plants to O 3 exposure was evaluated in the Arab idopsis Col- 0

ecotype during a 6

- h exposure at 350 ppb O 3 and for 6 hour s after the exposure was completed.

Results of this study, shown in Figure 9 -5 , indicate that genes associated with signal transduction and

regulation of transcription were in the clas s of early up

- regulated genes, while genes associated with

redox homeostasis and defense/stress response were in the class of late up

- regulated genes

(Mahalingam et al., 2005, 191693 )

. A few studies have been conducted to evaluate transcriptome changes in response to longer

term chronic O 3 exposures in woody plant species. Longer term exposures resulted in the up

regulation of genes associated with secondary metabolites, including isoprenoids, polyamines and

phenylpropanoids in 2- year

- old seedlings of the Mediterranean shrub Phillyrea latifolia exposed to

110 ppb O 3 for 90 days (Paolacci et al., 2007, 191422 ) . In 3- year

- old European beech saplings

exposed to O 3 for 20 mo nths , with monthly average twice ambient O 3 concentrations ranging from

11 to 80 ppb, O 3 - induced changes in gene transcription were similar to those observed for

herbaceous species, including genes associated with plant stress response, primary metabolism,

hor mone synthesis, cell structure and premature senescence (Olbrich et al., 2009, 596020 )

. In

another study, the magnitude of these transcriptional changes was far greater in beech saplings than

in adult trees exposed to the same O 3 concentrations for the same time period, indicating that adult

trees may be less respo nsive to this stressor than younger plants (Olbrich et al., 2010, 625424 ).

These results have been substantiated by results from proteome analysis in rice, poplar, wheat,

and soybean. Exposure of soybean to 120 ppb O 3 for 12 h/day for 3 days in growth chambers

resulte d in decreases in the quantity of proteins associated with photosynthesis, while proteins

involved with antioxidant defense and C metabolism increased (Ahsan et al., 2010, 644189 ) . Young

poplar plants exposed to 120 ppb O 3 in a growth chamber for 35 days also showed significant

c hanges in proteins involved in C metabolism (Bohler et al., 2007, 199408 ) . Declines in enzymes

associated with C fixation, the Calvin cycle and photosystem II were measured, while ascorbate

peroxidase and enzymes associated with glucose catabolism increased in abundance. Two

- week -old

rice seedlin gs exposed to varying levels of O 3 (4, 40, 80, 120 ppb) in a growth chamber for 9 days

showed reductions in expression of proteins associated with photosynthesis and energy metabolism,

and increases in some antioxidant and defense related proteins (Feng et al., 2008, 191626 ) . A

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26 subsequent study of O 3 - treated rice seedlings (exposed to 200 ppb O 3 for 24 h) focusing on the

integration of transcriptomics and proteomics, supported and further enhanced these results (Cho et

al., 2008, 603254 ) . The authors found that of the 22,000 genes analyzed from the rice genome, 1, 535

were differentially regulated by O 3 . Those genes were functionally categorized as transcription

factors, MAPK cascades, those encoding for enzymes involved in the synthesis of JA

, ET , shikimate,

tryptophan and lignin, and those involved in glycolysis , citric acid cycle, oxidative respiration and

photosynthesis. The authors determined that the proteome and metabolome analysis supported the

results of the transcriptome changes described above (Cho et al., 2008, 603254 ) . This type of study,

which ties together results from changes in gene expression, protein quantity and activity, and

metabolite levels, provides the most complete picture of the molecular and biochemical changes

occurring in plants exposed to a stressor such as O 3 . Sarkar et al. (2010, 657214 ) compared two

cultivars of wheat grown in OTCs at several O 3 concentrations, including filtered air, ambient O 3
(mean concentration 47 ppb), ambient + 10 ppb and ambient + 20 ppb for 5 h/day for 50 days.

Declines in the rate of photosynthesis and stomatal conductance were related to decreases in proteins

involved in C fixation and electron transport, and evidence of increased proteolysis of photosynthetic

proteins such as the large subunit of ribulose

- 1,6

- bisphosphat e carboxylase/oxygenase ( Rubisco )

. Enzymes that take part in energy metabolism, such as ATP synthesis, were also down -regulated,

while defense/stress related proteins were induced with O 3 treatment. In comparing the two wheat

cultivars, Sarkar et al. (2010, 657214 ) found that while the qualitative changes in protein expression

between the two cultivars was similar, the magnitude of these changes differed between the sensitive

and tolerant wheat cultivars.

All of these studies describe common trends for changes in g ene and protein expression which

occur in a variety of plant species in response to O 3 . While genes associated with C assimilation and

general metabolism are down

- regulated, genes associated with signaling, catabolism, and defense are

up

- regulated. The mag nitude of these changes in gene and protein expression appears to be related to

plant sensitivity or tolerance to O 3

.

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### Figure 9 -5 Composite diagram of major themes in the temporal evolution of the genetic (A) Temporal profile of the oxidative stress response to ﷒ response to ozone stress.

ozone . The biphasic ozone

- induced oxidative burst is represented in black, with the ROS control measurements shown as a broken line. Average transcript profiles are shown for early up

- regulated genes (yellow, peaks at 0.5 -1 hour s ), and the 3 hour s (blue), 4.5 hour s (red) and 9

- 12 h our s (green) late up

- regulated genes and for the

down

- regulated genes coding for photosynthesis proteins (brown). (B) Diagrammatic representation of redox regulation of the oxidative stress response.



## 9.4.3.3. Role of P hytoh ormo ne s in Pla n t Re s p ons e to O zone

1 Many s tudies of O 3 effects on plants have analyzed the importance of plant hormones such as



### 2 SA, ET and JA in determining plant response to O 3 ; some of the roles of these hormones were



### 3 described in the 2006 O 3 AQCD (U.S. EPA, 2006, 088089 ) . Transcriptome analysis and the use of a



### 4 variety of mutants have allowed for further elucidation of the complex interactions between SA, ET,



### 5 JA and the role of abscisic acid (ABA) in mediating plant response to O 3 (Ludwikow and Sadowski,



### 6 2008, 191426 ) . In addition to their roles in signaling pathways, phytohormones also appear to

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38 regulate, and be regulated by, the MAPK signaling cascades described previously. Most evidence

suggests that while ET and SA are needed to develop O 3 - induced leaf lesions, J A acts

antagonistically to SA and ET to limit the lesions (Figure 9 -6) (Kangasjarvi et al., 2005, 180341 )

. The rapid production of ET in response to O 3 has been described in many plant species and

has been further characterized through the use of a variety of mutants that either over

- produce or are

insensitive to ET. Production of stress ET in O 3 - treated plants, which is thought to be a wounding

response, was found to be correlated to the degree of injury development in leaves (U.S. EPA, 2006,

088089 ) . More recent studies ha ve supported these conclusions and have also focused on the

interactions occurring between several oxidative

- stress induced phytohormones. Yoshida et al.

(2009, 191385 ) determined that ET likely amplifies the oxidative signal generated by ROS, thereby

promoting lesion formation. By analyzin g the O 3 - induced transcriptome of several Arabidopsis

mutants of the Col

- 0 ecotype, Tamaoki et al. (2003, 080053 ) determined that at 12 hour s after

initiating the O 3 exposure (200 ppb for 12 hour s ), the ET and JA signaling pathways were the main

pathways used to activate plant defense responses, w ith a lesser role for SA. The authors also

demonstrated that low levels of ET production could stimulate the expression of defense genes,

rather than promoting cell death when ET production is high. Tosti et al. (2006, 191425 ) supported

these findings by showing that O 3 not only activates the biosynthetic pathways of ET, JA and SA, but

also increases t he expression of genes related to the signal transduction pathways of these

phytohormones in O 3 - treated Arabidopsis plants (300 ppb O 3 for 6 hour s ). Conversely, in the O 3 sensitive Ws ecotype, its sensitivity may, in part, be due to intrinsically high ET levels leading to SA

accumulation, and the high ET and SA may act to repress JA

- associated genes, which would serve to

inhibit the spread of lesions (Mahalingam et al., 2006, 191221 ) . Ogawa et al. (2005, 191653 ) found

that an O 3 - induced increase in SA leads to the formation of leaf lesions in tobacco plants exposed to

200 ppb O 3 for 6 hour s . Furthermore, several genes encoding for enzymes in the biosyntheti c

pathway of SA were suppressed in transgenic tobacco plants with reduced levels of O 3 - induced ET

production, suggesting that SA levels are controlled by ET in the presence of O 3

. Exposure of the Arabidopsis mutant rcd1 to acute doses of O 3 (250 ppb O 3 for 8 h/day for

3 days) resulted in programmed cell death (PCD) and the formation of leaf lesions. Salicylic acid

was required for the initiation of PCD in these mutants, and increased ET synthesis resulted in

amplification of cell death, while JA was thought to contain the spreading of lesions (Overmyer et

al., 2000, 036361 ) . In addition, the O 3 - treated rcd1 mutants showed higher SA and JA accumulation

as compared to the Col

- 0 ecotype. High SA levels could be involved in amplifying cell death in these

mutants. Jasmonic acid, which is thought to accumulate as a direct result of cell death, may represent

an autocatalytic mechanism for limiting cell death, by which the increased cell death observed in the

rcd1 mutant would determine the high amount of JA needed to contain lesion formation (Overmyer

et al., 2005, 191596 ) . In cotton plants expo sed to a range of O 3 concentrations (0

- 120 ppb) and

methyl jasmonate (MeJA), Grantz et al. (2010, 625419 ) determined that endogenous applications of

MeJA did not protect plants from chronic O 3 exposure.

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30 There are distinct patterns of gene expression when evaluating early and later O 3 responsive

genes (Mahalingam et al., 200 5, 191693 ) . While ET and JA pathways were stimulated rapidly after

Arabidopsis Col- 0 exposure to O 3 , at 48 hour s postexposure , JA and ET synthesis in O 3 - tre ated

Arabidopsis were reduced, while the synthesis of SA was stimulated (D'Haese et al., 2006, 191448 )

.
Abs cisic acid has been investigated for its role in regulating stomatal aperture and also for its

contribution to signaling pathways in the plant. The role of ABA and the interaction between ABA

and H 2 O 2 in O 3 - induced stomatal closure was described in the 2006 O 3 AQCD (U.S. EPA, 2006,

088089 ) . More recently, it was determined that synthesis of ABA was induced in O 3 - treated

Arabidopsis plants (250

- 350 ppb O 3 for 6 hour s ), with a more pronounced induction in several O 3 sensitive rcd mutants as compared to the wildtype Col

- 0 (Overmyer et al., 2008, 191283 ) . Ludwikow

et al. (2009, 199440 ) used Arabidopsis ABI1td mutants, in which a key negati ve regulator of ABA

action (abscisic acid insensitive1 protein phosphatase 2C) has been knocked out, to examine O 3 responsive genes in this mutant compared to the Arabidopsis Col- 0. Results of this study indicate a

role for ABI1 in negatively regulating the synthesis of both ABA and ET in O 3 - treated plants

(350 ppb O 3 for 9 hour s ). Additionally, ABI1 may stimulate JA

- related gene expression, providing

evidence for an antagonistic interaction between ABA and JA signaling pathways (Ludwikow et al.,

2009, 199440 ).

Nitric oxide (NO) has also been shown to play a role in regulating O 3 - induced gene expression

in plants. However, little is known to date about NO and its role in the complex interactions of

molecules in response to O 3 . Exposure of tobacco to O 3 (150 ppb for 5 hour s ) stimulated NO and

NO

- depe ndent ET production, while NO production itself did not depend on the presence of ET

(Ederli et al., 2006, 191479 ) . Analysis of O 3 - treated Arabidopsis indicated the possibility of a dual

role for NO in the initiation of cell death and later lesion containment (Ahlfors et al., 2009, 191533 )

. While much work remains to be done to better elucidate how plants sense O 3 and how signals

are communicated to the nucleus to generate plant responses to oxidative stress, it is clear that the

mechanism for O 3 sensing and signal transduction is very complex. Many of the phytohormones and

other signaling molecules thought to be involved in these processes are interactive and depend upon

a variety of other factors, which could be either internal or external to the plant. This results in a

highly dynamic and complex system, capable of generating a variety of plant responses to oxidative

stre ss.

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### Ozone

- derived radicals induce endogenous ROS ﷒ The oxidative cell death cycle

. Figure 9 -6. production (1) which results in salicylic acid (SA) accumulation and programmed

cell death ; (2) Cell death triggers ethylene (ET) production, which is required for the continuing ROS production responsible for the propagation of cell death ; (3)

Jasmonates counteract the progression of the cycle by antagonizing the cell death promoting function of SA and ET ; (4) Abscisic acid (ABA) antagonizes ET function in many situations and might also have this role in ozone

- induced cell death ; (5) Mutually antagonistic interactions between ET, SA and jasmonic acid (JA) are

indicated with red bars.



## 9.4.4. De toxific a tio n

9.4.4.1. Overview o f Ozone

- In duc e d De fe ns e Me c ha nis ms

1 Plants are exposed to an oxidizing environment on a continual basis, and many reactions that



### 2 are part of the basic metabolic processes, such as photosynthesis and respiration, generate ROS. As a



### 3 result, there is an extensive and complex mechanism in place to detoxify these oxidizing radicals,



### 4 including both enzymes and metabolites, which are located in several locations in the c ell and also in



### 5 the apoplast of the cell. As O 3 enters the leaf through open stomata, the first point of contact of O 3



### 6 with the plant is thought to be in the apoplast, where it breaks down to form oxidizing radicals such



### 7 as H 2 O 2 , O 2 - , HO· and HO 2 . Another source of oxidizing radicals is an oxidative burst, generated by



### 8 a membrane

- bound NADPH oxidase enzyme, which is thought to be part of the plant’s defense



### 9 system against pathogens (Schraudner et al., 1998, 056358 ) . Antioxidant metabolites and enzymes



### 10 located in the apoplast are thought to form a first line of defense by detoxifying O 3 and/or the ROS



### 11 that are formed as breakdown products o f O 3 (Section 9.4.2.). However, even with the presence of



### 12 several antioxidants, including ascorbate, the redox buffering capacity of the apoplast is far less than

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1 that of the cytoplasm, as it lacks the regeneration systems necessary to retain a reduced pool of



### 2 antioxidants (Foyer and Noctor, 2005, 631198 )

. 

### 3 Redox homeostasis is regulated by the presence of a pool of anti oxidants, which are typically



### 4 found in a reduced state and detoxify ROS produced by oxidases or electron transport components.

As ROS increase due to environmental stress such as O 3 , the antioxidant pool will no longer be able



### 6 to maintain its reduced state (Foyer and Noctor, 2005, 631198 ) . As such, not only the quantity and



### 7 types of antioxidant enzymes and metabolites pr esent, but also the cellular ability to regenerate those



### 8 antioxidants are important considerations in mechanisms of plant tolerance to oxidative stress



### 9 (Dizengremel et al., 2008, 191587 ) . Molecules such as glutathione (GSH), thioredoxins and NADPH

play very important roles in this regeneration process; additionally, alterations in C metabolism will



### 11 be necessary to supply the needed reducing power for antioxidant regeneration (Dizengremel et al.,



### 12 2008, 191587 ) . Increases in the activity of catabolic pathways allow the cell to generate more



### 13 NADPH (Section 9.4.5).



## 9.4.4.2. Role of An tioxida n ts in Pla nt De fe ns e Re s po n s e s

14 Ascorbate has been the focus of many different studies as an antioxidant metabolite that

protects plants from exposure to O 3 . It is found in several cellular locations, including the



### 16 chloroplast, the cytosol and the apoplast (Noctor and Foyer, 1998, 657213 ) . Ascorbate is synthesized



### 17 in the cell and transported to the apoplast. Apoplastic ascorbate can be oxidized to dehydroascorbate



### 18 (DHA) with exposure to O 3 and is then transported back to the cytoplasm. Here, DHA is reduced to



### 19 ascorbate by the enzyme dehydroascorbate reductase (DHAR) and r educed GSH , which is part of the

ascorbate

- glutathione cycle (Noctor and Foyer, 1998, 657213 ) . Many studies have focused on



### 21 evaluating whether ascorbate is the determining factor in differential sensitivity of plants to O 3

. 

### 22 Cheng et al. (2007, 191499 ) exposed two soybean cultivars to elevated O 3 (77 ppb) a nd filtered air



### 23 for 7 h/day for 6 days. The differences in sensitivity between the two cultivars could not be



### 24 explained by differential O 3 uptake or by the fraction of reduced ascorbate present in the apoplast.

However, total antioxidant capacity of the ap oplast was two fold higher in the tolerant Essex cultivar



### 26 as compared to the sensitive Forrest cultivar, indicating that there may be other compounds in the



### 27 leaf apoplast that scavenge ROS. D'Haese et al. (2005, 191551 ) exposed the NC

- S (sensitive) and



### 28 NC

- R (resistant) clones of white clover ( Trifolium repens ) to 60 ppb O 3 for 7 h/day for 5 days in



### 29 environmental chambers. Surprisingly, the NC

- S clone had a higher constitutive concentration of

apoplastic ascorbate with a higher redox status than the NC

- R clone. However, the redox status of



### 31 symplastic GSH was higher in NC

- R, even t hough the concentration of GSH was not higher than in



### 32 NC

- S. In addition, total symplastic antioxidative capacity was not a determining factor in differential



### 33 sensitivity between these two clones. Severino et al. (2007, 199293 ) also examined the role of



### 34 antioxidants in the differential sensitivity of the two white clover clones by growing them in the field

for a growing season and then e xposing them to elevated O 3 (100 ppb for 8 h/day for 10 days) in



### 36 OTC at the end of the field season. The NC

- R clone had greater quantities of total ascorbate and total



### 37 antioxidants than the NC

- S clone at the end of the experiment. While the second study in dicates a

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39 possible relationship between O 3 tolerance and ascorbate levels, the first study indicates that there

are other factors besides ascorbate that determine plant tolerance to O 3 . In snap bean, plants of the O 3 tolerant Provider cultivar had greater total ascorbate and more ascorbate in the apoplast than the

sensitive S156 cultivar after exposure to 71 ppb O 3 for 10 days in OTC (Burkey et al., 2003,

630251 ) . While most of the apoplastic ascorbate was in the oxidized form, the ratio of reduced

ascorbate to total ascorbate was higher in Provider than S156, indicating that Provider is better able

to maintain this ratio to maximize plant protection from oxidative stress.

While the quantities of antioxidant metabolites such as ascorbate are an important indicator of

plant tolerance to O 3 , the ability of the plant to recycle oxidized ascorbate efficiently plays a large

role in determining t he plant’s ability to deal with a sustained exposure to oxidative stress. Tobacco

plants over

- expressing DHAR were better protected from exposure to either chronic (100 ppb O 3

4 h/day for 30 days) or acute (200 ppb O 3 for 2 hour s ) conditions than control p lants and those with

reduced expression of DHAR. The DHAR over

- expressing plants exhibited an increase in guard cell

ascorbic acid, leading to a decrease in stomatal responsiveness to O 3 and an increase in stomatal

conductance and O 3 uptake. Despite this, the presence of higher levels of ascorbic acid led to a lower

oxidative load and a higher level of photosynthetic activity in the DHAR over

- expressing plants

(Chen and Gallie, 2005, 191465 ) . A subsequent study with tobacco plants over

- expressing DHAR

confirmed some of these results. Levels of ascorbic acid were higher in the transgenic tobacco

plants, and they exhibited greater tolerance to O 3 exposure (200 ppb O 3 ) as demonstrated by higher

photosynthetic rates in the transgenic plants as compared to the control plants (Eltayeb et al., 2006,

191377 ) . Over

- expression of monodehydroascorbate reductase (MDAR) in tobacco plants also

showed enhanced stress tolerance in response to O 3 exposure (200 ppb O 3 ), with higher rates of

photosynthesis and higher levels of reduced ascorbic acid as compared to controls (Eltayeb et al.,

2007, 191553 ) . Results of these studies show the importanc e of ascorbic acid as a detoxification

mechanism, but more importantly emphasize that the recycling of oxidized ascorbate and

maintenance of a reduced pool of ascorbate is critical in determining plant tolerance to oxidative

stress.

The roles of other antioxidant metabolites and enzymes, including GSH , catalase (CAT), and

superoxide dismutase (SOD), were comprehensively reviewed in the 2006 O 3 AQCD (U.S. EPA,

2006, 088089 ) . Additional studies have supported the findings reported in that document. Superoxide

dismutase (SOD) and peroxidase (POD) activities were measured in both th e tolerant Bel B and

sensitive Bel W3 tobacco cultivars exposed to ambient O 3 concentrations for 2 weeks 3 times

throughout a growing season (Borowiak et al., 2009, 191247 )

. Tulip poplar ( Liriodendron tulipifera )

trees exposed to increasing O 3 concentrations (from 100 to 300 ppb O 3 during a 2- week period)

showed increases in activities of SOD, ascorbate peroxidase (APX), glutathione reductase (G R),

MDAR, DHAR, CAT and POD in the 2- week period, although individual enzyme activities

increased at different times during the 2- week period (Ryang et al., 2009, 191267 ).

Longer, chronic O 3 exposures in trees revealed some distinctive patterns of increases in SOD

and APX activity that were measured in Quercus mongolica after 45 days of plant exposure to

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### 1 80 ppb O 3 , which were followed by declines in the activities and quantities of these enzymes after



### 2 75 days of exposure (Yan et al., 2010, 628514 ) . Similarly, activities of SOD, APX, DHAR, MDAR,



### 3 and GR increased in Gingko biloba trees during the first 50 days of exposure to 80 ppb O 3 , followed



### 4 by decreases in activity below control values after 50 days of exposure (He et al., 2006, 199430 )

. 

### 5 Soybean plants exposed to 70 or 100 ppb O 3 for 4 h/day over the course of a growing season showed



### 6 elevated POD activity and a decrease in CAT activity at 40 and 60 days after germination (Singh et



### 7 al., 2010, 386275 ).



### 8 Antioxidant enzymes appear to increase in quantity in O 3 - treated plants as a defense



### 9 mechanism against oxidative stress. Ho wever, it appears that plants cannot maintain these elevated



### 10 levels of antioxidant enzymes for an extended period of time, likely due to the high metabolic costs



### 11 involved. Therefore, plants exposed to chronic O 3 conditions may show more symptoms of exposur e



### 12 as defensive mechanisms are down

- regulated over time.



## 9.4.5. Effe cts on P rimary a n d S ec on d ary Meta bolis m

9.4.5.1. Lig ht a nd Da rk Re a c tio ns o f Ph o tos yn the s is

13 Declines in the rate of photosynthesis and stomatal conductance in O 3 - treated plants have been



### 14 documented for ma ny different plant species (U.S. EPA, 2006, 088089 ) (Booker et al., 2009, 191569 )



### 15 (Wittig et al., 2007, 191695 ) . Much of the literature regarding O 3 effects on photosynthesis has



### 16 focused on C assimilation; however, more recently, attention has also been focused on deleterious



### 17 effects on the light reactions. Chlorophyll fluorescence provides a useful measure of changes to the



### 18 photosynthetic process from exposure to oxidative stress. Decreases in the Fv/Fm ratio (a measure of



### 19 the maximum efficiency of Photosystem II) in dark adapted leaves indicate a decline in the



### 20 efficiency of the PSII photosystems and a conco mitant increase in non

- photochemical quenching



### 21 (Guidi and Degl'lnnocenti, 2008, 191571 ) (Scebba et al., 2006, 191219 ) . Changes in these parameters



### 22 have been correlated to differential sensitivity of plants to the pollutant. In a study to evaluate the



### 23 response of 4 maple species to O 3 (exposed to an 8 -h avg of 51 ppb for ambient and 79 ppb for



### 24 elevated treatment in OTC), the 2 species which were most sensitive based on visible injury and



### 25 declines in CO 2 assimilation also showed the greatest decreases in Fv/Fm in symptomatic leaves. In



### 26 asymptomatic leaves, CO 2 assimilation decreased significantly but there was no significant decline



### 27 in Fv/Fm (Calatayud et al., 2007, 191411 ) . Degl 'Innocenti et al. (2007, 191350 ) measured



### 28 significant decreases in Fv/Fm in young and symptomatic leaves of a resistant tomato genotype (line



### 29 93.1033/1) in response to O 3 exposure (150 ppb O 3 for 3 hour s in a growth chamber), but only minor



### 30 decreases in a symptomatic leaves with no associated changes in net photosynthetic rate. In the O 3



### 31 sensitive tomato cultivar Cuor Di Bue, the Fv/Fm ratio did not change, while the photosynthetic rate



### 32 declined significantly in asymptomatic leaves (Degl'Innocenti et al., 2007, 191350 ) . In two soybean



### 33 cultivars, Fv/Fm also declined significantly with plant exposure to O 3 (Singh et al., 2009, 199427 ) . It

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1 appears that in asymptomatic leaves, photoinhibition, as indicated by a decrease in Fv/Fm, is not the



### 2 main reason for a decline in photosynthesis.



### 3 An evaluation of photosynthetic parameters of two white clover ( Trifolium repens cv. Regal)



### 4 clones that differ in their O 3 sensitivity revealed that O 3 (40

- 110 ppb O 3 for 7 h/day for 5 days)

increased the coefficient of non

- photochemical quenching (q NP ) in both the resistant (NC

- R) and



### 6 sensitive (NC

- S) clones, however q NP was significantly lower for the sensitive clone (Crous et al.,



### 7 2006, 199321 ) . Sensitive Acer clon es had a lower coefficient of non

- photochemical quenching,



### 8 while exposure to O 3 increased q NP in both sensitive and tolerant clones (Calatayud et al., 2007,



### 9 191411 ) . While exposure to O 3 also increased q NP in tomato, there were no differences in the

coefficient of photochemical quenching between cultivars thought to be differentially sensitive to O 3

. 

### 11 (Degl'Innocenti et al., 2007, 191350 ) . Higher q NP as a result of exposure to O 3 indicates a reduction



### 12 in the proportio n of absorbed light energy being used to drive photochemistry. A lower coefficient of



### 13 non

- photochemical quenching in O 3 sensitive plants could indicate increased vulnerability to ROS



### 14 generated during exposure to oxidative stress (Crous et al., 2006, 199321 )

. Several measures of the light reactions of photosynthesis are sensitive to exposure to O 3 , 

### 16 however, photosynthetic C assimilation is generally considered to be more affected by pollutant



### 17 exposure, resulting in an overall decline in photosynthesis (Heath, 2008, 195632 ) (Fiscus et al., 2005,



### 18 079155 ) (Guidi and Degl'lnnocenti, 2008, 191571 ) . Loss of C assimilation capacity has been shown



### 19 to result primarily from declines in the quantity of Rubisco (Calatayud et al., 2007, 191411 ) (Singh et

al., 2009, 199427 ) . Experimental evidence suggests that both decreases in Rubisco synthesis and



### 21 enhanced degradation of the protein contribute to the measured reduction in its quantity (U.S. EPA,



### 22 2006, 088089 ) . Reduced C assimilation has been linked to reductions in biomass and yield (Keutgen



### 23 et al., 2005, 191295 ) (He et al., 2007, 199789 ) (Novak et al., 2007, 194630 ) (Gregg et al., 2006,



### 24 186961 )

, (Wang et al., 2009, 199303 )

.
Most of the research on O 3 effects on photos ynthesis has focused on C3 (Calvin cycle) plants



### 26 because C4 (Hatch

- Slack) plants have lower stomatal conductance and therefore assumed to be less



### 27 sensitive to O 3 stress. However, a few studies have been conducted to evaluate the effects of O 3 on



### 28 C4 photosy nthesis. In older maize leaves, Leitao et al. (2007, 191456 ) (2007, 191263 ) found that the



### 29 activity, quantity and transcript levels of both Rubisco and phosphoenolp yruvate carboxylase (PEPc)

decreased as a function of rising O 3 concentration. In younger maize leaves, the quantity, activity,



### 31 and transcript levels of the carboxylases were either increased or unaffected in plants exposed to



### 32 40 ppb O 3 for 7 h/day for 28

- 33 days, but decreased at 80 ppb (Leitao et al., 2007, 191263 ) (Leitao et



### 33 al., 2007, 199379 )

.



## 9.4.5.2. Re s pira tio n a n d Da rk Re s pira tion

34 While much research emphasi s regarding O 3 effects on plants has focused on the negative

impacts on C assimilation, other studies have measured impacts on catabolic pathways such as



### 36 respiration and photorespiration. Generally, respiration has been found to increase in plants exposed



### 37 to O 3 . Bean plants exposed to ambient (average 12

- h mean 43 ppb) and twice ambient (average 12 -h

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1 mean 80 ppb) O 3 showed increases in respiration. When mathematically partitioned, the maintenance



### 2 coefficient of respiration was significantly increased by O 3 exposure, while the growth coefficient of



### 3 respiration was not affected (Amthor, 1988, 041870 ) . Loblolly pines were exposed to ambient (12 -h



### 4 daily mean was 45 ppb) and twice ambi ent (12 hour s daily mean was 86 ppb) O 3 for 12 h/day for

approximately seven mon ths per year for 3 and 4 y ea rs . While photosynthetic activity declined with



### 6 the age of the needles and increasing O 3 concentration, enzymes associated with respiration showed



### 7 h igher levels of activity with increasing O 3 concentration (Dizengremel et al., 1994, 187217 ) . In



### 8 their review on the role of metabolic changes in plant redox st atus after O 3 exposure, Dizengremel et



### 9 al. (2009, 199424 ) summarize d multip le studies in which several different tree species were exposed

to O 3 concentrations ranging from ambient to 200 ppb O 3 for at least several weeks. In all cases, the



### 11 activity of enzymes, including phosphofructokinase, pyruvate kinase and fumarase, which ar e part of



### 12 several catabolic pathways, were increased in response to O 3 exposure.



### 13 Photorespiration is a light

- stimulated process which consumes O 2 and releases CO 2 . While it



### 14 has been regarded as a wasteful process, more recent evidence suggests that it may play a role in

photoprotection during photosynthesis (Bagard et al., 2008, 191593 ) . The few studies that have been



### 16 conducted on O 3 effects on photorespiration suggest that rates of photorespiration decline



### 17 concomitantly with rates of photosynthesis. Soybean pl ants were exposed to ambient (daily averages



### 18 43

- 58 ppb) and 1.5 ambient O 3 (daily averages 63

- 83 ppb) O 3 in OTCs for 12 h/day for 4 months.



### 19 Rates of photosynthesis and photorespiration and photorespiratory enzyme activity declined only at

the end of the gr owing season and did not appear to be very sensitive to O 3 exposure (Booker et al.,



### 21 1997, 026425 ) . Young h ybrid poplars exposed to 120 ppb O 3 for 13 h/day for 35 days in phytotron



### 22 chambers showed that effects on photorespiration and photosynthesis were dependent upon the



### 23 developmental stage of the leaf. While young leaves were not impacted, reductions in photo synthesis



### 24 and photorespiration were measured in fully expanded leaves (Bagard et al., 2008, 191593 )

.



## 9.4.5.3. Se c on da ry Me ta bolis m

Transcriptome analysis of Arabidopsis plants has revealed modulation of several genes



### 26 involved in plant secondary metabolism (Ludwikow and Sadowski, 2008, 191426 ) . Phenylalanine



### 27 ammonia lyase (PAL) has been the focus of many studies involving plant responses to O 3 due to its



### 28 importance in linking the phenylpropanoid pathway of plant secondary metabolism to primary



### 29 metabolism in the form of the shikimate pathway. Genes encoding several enzymes of the

phenylpropanoid pathway and lignin biosynthesis were up

- regulated in transcriptome analysis of



### 31 Arabidopsis plants (Col

- 0) exposed to 350 ppb O 3 for 6 hour s , while 2 genes involved in flavonoid



### 32 biosynthesis were down -regulated (Ludwikow et al., 2004, 5 95939 ) . Exposure of Arabidopsis



### 33 (Col

- 0) to lower O 3 concentrations (150 ppb for 8 h/day for 2 days) resulted in the induction of 11



### 34 transcripts involved in flavonoid synthesis. In their exposure of 2

- year

- old Mediterranean shrub

Phillyrea latifolia to 11 0 ppb O 3 for 90 days, Paolacci et al. (2007, 191422 ) identified four clones



### 36 that were up

- regulated and corresponded to genes involved in the synthesis of secondary



### 37 metabolites, such as isoprenoids, polyamines and phenylpropanoids. Up

- regulation of genes

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38 involved in isoprene synthesis was als o observed in Medicago trunculata exposed to 300 ppb O 3 for

6 hour s, while genes encoding enzymes of the flavonoid synthesis pathway were either up

- or down

regulated (Puckette et al., 2008, 191698 ) . Exposure of red clover to 1.5 × ambient O 3 (average

concentrations of 32.4 ppb) for up to 9 weeks in an open field exposure system resulted in increases

in leaf total phenolic content. However, the types of phenolics that were increased in response to O 3 exposure differed depending upon the developmental stage of the plant. While almost all of the 31

different phenolic compounds measured increased in quantity initially during t he exposure, after

3 weeks the quantity of isoflavones decreased while other phenolics increased (Saviranta et al., 2010,

102177 ) . Exposure of beech saplings to ambient and 2 × ambient O 3 concentrations over 2 growing

seasons resulted in the induction of several enzymes which contribute to lignin formation, while

enzymes involved in flavonoid biosynthesis were down

- regulated (Olbrich et al., 2009, 596020 )

. Exposure of tobacco Bel W3 to 160 ppb O 3 for 5 hours showed up

- regulation of almost all genes

encoding for enzymes which are part of the prechorismate pathway (Janzik et al., 2005, 191581 )

. Isoprenoids can serve as antioxidant compounds in plants exposed to oxidative stress (Paolacci et al.,

2007, 191422 )

. The prechorismate pathway is the pathway leading to the formation of chorismate, a precursor

to the formation of the aromatic amino acids tryptophan, tyrosine and phenylalanine. These amino

acids are precursors for the formation of many secondary aromatic compounds, and, therefore, the

prechorismate pathway represents a branch

- point in the regulation of metabolites into either primary

or secondary metabolism (Janzik et al., 2005, 191581 ) . Exposure of the O 3 sensitive Bel W3 tobacco

cultivar at 160 ppb for 5 hour s showed an increase in transcript levels of most of the genes encoding

enzymes of the prechorismate pathway. However, shikimate kinase (SK) did not show any change in

tra nscript levels and only one of three isoforms of DAHPS (3

- deoxy -D- arabino

- heptulosonat -7­

phosphate synthase), the first enzyme in this pathway, was induced by O 3 exposure (Janzik et al.,

2005, 191581 ) . Differential induction of DAHPS isoforms was also observed in European beech

after 40 days of exposure to 150

- 190 ppb O 3 . At this time point in the beech experiment , transcript

levels of shikimate pathway enzymes, including SK, were generally strongly induced after an only

weak initial induction after the first 40 days of exposure. Both soluble and cell

- wall bound phenolic

metabolites showed only minimal increases in response to O 3 for the duration of the exposure period

(Alonso et al., 2007, 199289 ) . Total leaf phenolics decreased with leaf age in Populus nigra exposed

to 80 ppb O 3 for 12 h/day for 14 days . Ozone increased the concentration of total leaf phenolics in

newly expanded leaves, with the most significant increases occu rring in compounds such as

quercitin glycoside, which has a high antioxidant capacity (Fares et al., 2010, 628522 ) . While O 3 exposure induced the activity of several phenylpropanoid pathway enzymes, the degree of induction

differed in the two poplar clones exposed to 60 ppb for 5 h/day for 15 days. In the tolerant I

- 214

clone, PAL activity increased ninefold in O 3 - treated plants as compared to controls, while there was

no significant difference in PAL activity in the sensitive Eridano clone (Di Baccio et al., 2008,

199850 )

.

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39 Polyamines such as putrescine, spermidine and spermine play a variety of roles in plants and

have been implicated in plant defense responses to both abiotic and biotic stresses. They exist in both

a free form and conjugated to hydroxycinnamic acids. Investigations on the role of polyamines have

found that levels of putrescine increase in response to oxidative stress. This increase stems largely

from the increase in the activity of arginine decarboxylase (ADC), a key enzyme in the synthesis of

putrescine (Groppa and Benavides, 2008, 191616 ) . Langebartels et al. (1991, 043506 ) described

differences in putrescine accumulation in O 3 - treated tobacco plants exposed to several O 3 concentrations, ranging from 0

- 400 ppb for 5 -7 hour s . A large and rapid increase in putrescine

occurred in the tolerant Bel B cultivar and only a small increase in the Bel W3 cultivar, which

occurred only after the formation of necrotic leaf lesions. Van Buuren et al. (2002, 631199 ) further

examined the role of polyamines in these two tobacco cultivars during an acute (130 ppb O 3 for 7 h

in a growth chamber) exposure. They found that while free putrescine accumulated in undamaged

tissue of both cultivars, conjugated putrescine predominantly accumulated in tissues undergoing cell

death after plant exposure to O 3 (van Buuren et al., 20 02, 631199 ) . The authors suggest that while

free putrescine may not play a role in conferring tolerance in the Bel B cultivar, conjugated

putrescine may pla y a role in O 3 - induced programmed cell death in Bel W3 plants.

Isoprene is emitted by some plant species and represents the predominant biogenic source of

hydrocarbon emissions in the atmosphere (Guenther et al., 2006, 607080 ) . In the atmosphere, the

oxidation of isoprene by hydroxyl radicals can enhance O 3 formation i n the presence of NO X , thereby impacting the O 3 concentration that plants are exposed to. While isoprene emission varies

widely between species, and it has been proposed to stabilize membranes and provide those plant

species that produce it with a mechanis m of thermotolerance (Sharkey et al., 2008, 191492 ) . It has

also been suggested that isoprene may act as an antioxidant compound to scavenge O 3 (Loreto and

Velikova, 2001, 657212 ) . Recent studies using a variety of plant species have shown confli cting

results in trying to understand the effects of O 3 on isoprene emission. Acute doses of O 3 (300 ppb for

3 h) stimulated isoprene emissions in detached leaves of Phragmites australis (Velikova et al., 2005,

199410 ) . Similarly, isoprene emissions were stimulated in Populus nigra after exposure to 100 ppb

O 3 for 5 days continuously (Fares et al., 2008, 191683 ) . Isoprene emission in attached leaves of

Populus alba , which were exposed to 150 ppb O 3 for 11 h/day fo r 30 days inside cuvettes, was

inhibited, while isoprene emission and transcript levels of isoprene synthase mRNA were increased

in the leaves exposed to ambient O 3 (40 ppb), which were located above the leaves enclosed in the

exposure cuvettes (Fares et al., 2006, 191455 ) . Exposure of 2 genotypes of hybrid poplar to 120 ppb

O 3 for 6 h/day for 8 days resulted in a significant reduction in isoprene emission in the O 3 - sensitive

but not the tolerant genotype (Ryan et al., 2009, 191299 ) . Similarly, O 3 treatment (80 ppb 12 h/day

for 14 days) of Populus nigra sh owed that isoprene emission was reduced in the treated plants

relative to the control plants (Fares et al., 2010, 628522 ) . Based on results of this and other studies,

Fares et al

. (2010, 628522 ) concluded that the isoprenoid pathway may be induced in plants exposed

to acute O 3 doses, while at lower doses isoprene emission may be inhibited. Vickers et al. (2009,

191497 ) developed transgenic tobac co plants with the isoprene synthase gene from Populus alba and

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1 exposed them to 120 ppb O 3 for 6 h/day for 2 days. They determined that the wildtype plants showed



### 2 significantly more O 3 damage, including the development of leaf lesions and a decline in



### 3 phot osynthetic rates, than the transgenic, isoprene

- emitting plants. Transgenic plants also



### 4 accumulated less H 2 O 2 and had lower levels of lipid peroxidation following exposure to O 3 than the

wildtype plants (Vickers et al., 2009, 191497 ) . These results indicate that isoprene may have a



### 6 protective role f or plants exposed to oxidative stress.



## 9.4.6. Changes in Stomatal Function

7 There has been some debate as to whether O 3 - induced reductions in photosynthesis result from



### 8 changes in stomatal conductance or direct effects on C assimilation. A review of the literatur e



### 9 suggests there may be species

- specific differences in how O 3 affects photosynthesis through either

direct or indirect mechanisms. Recent studies utilizing a new simultaneous O 3 exposure/gas



### 11 exchange device have demonstrated that exposure of Arabidopsis p lants to 150 ppb O 3 resulted in a



### 12 60

- 70% decline in stomatal conductance within 9

- 12 min utes of beginning the exposure. Twenty to



### 13 thirty minutes later, stomatal conductance had returned to its initial value, even with continuing



### 14 exposure to O 3 , indicating a rapid direct effect of O 3 on stomatal function (Kollist et al., 2007,

191539 ) . The contributions of stomatal versus non

- stomatal factors in reducing net photosynthesis



### 16 have been investigated in a number of tree species. Ginkgo biloba trees exposed to ambient O 3



### 17 (40 ppb) a nd elevated O 3 (80 ppb) in OTCs for 90 days were investigated over the course of seven



### 18 months. Initially, stomatal limitation restricted C assimilation and, along with higher rates dark



### 19 respiration, served to protect against oxidative stress. However, as the season progressed, declines in

C assimilation resulted from direct oxidative damage to the photosynthetic apparatus (He et al.,



### 21 2007, 199789 ) . In other studies focusing on O 3 effects on adult trees, including Quercus mongolica



### 22 (exposed to 80 ppb O 3 for 9 h/day for 4 mo nths in OTCs) and Fagus sylvatica (exposed to the



### 23 2 × ambient concentrations, with a mean of 57 ppb O 3 for 6 mo nths in a free air fumigation system)



### 24 O 3 - induced reductions in stomatal conductance were accompanied by a decrease in intercellular CO 2 concentration, indicating that stomatal closure did not result from high in ternal CO 2 but rather from a



### 26 direct effect of O 3 on the guard cells (Wang et al., 2009, 199303 ) (Kitao et al., 2009, 191331 ) . In a



### 27 related study, Populus deltoides grown in rural areas showed a greater O 3 - induced decline in biomass



### 28 allocation than those grown in urban areas (with means in O 3 concentrations ranging from 20

- 36 ppb



### 29 in urban areas and 36

- 47 ppb in rural areas). This decline in biomass was determined to result from

significantly higher rates of stomatal conductance in the rural trees, indicating a loss of stomatal



### 31 control (Gregg et al., 2006, 186961 ) . An investigation of the differences between O 3 - sensitive and



### 32 insensitive cutleaf coneflower ( Rudbeckia laciniata var. digitata ) revealed differences in stomatal



### 33 beh avior (Grulke et al., 2007, 186963 ) . Cutleaf conefl ower plants growing in Great Smoky Mountain



### 34 National Park (exposed to mean O 3 concentration of 42 ppb from mid

- May through mid

- September)

showed variable stomatal responses to changes in environmental conditions, such as light and vapor



### 36 pressure deficit. I t is likely that the O 3 - sensitive coneflower plants have a set of traits, such as a

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### 1 sluggish stomatal response to changes in light intensity, which predispose them to being more



### 2 sensitive to O 3 exposure (Grulke et al., 2007, 186963 ) (Paoletti and Grulke, 2010, 628561 )

. 

### 3 Feng et al. (2007, 191641 ) determined that reductions in net photosynthesis in O 3 -exposed



### 4 winter wheat ( Triticum aestivum ), exposed to a mean concentration of either 52 or 105 ppb O 3 for



### 5 6 weeks in OTCs, likely occurred as a result of both stomatal and non

- st omatal factors. Other studies



### 6 suggest that O 3 impacts net photosynthesis only through non

- stomatal factors. A direct effect of O 3



### 7 on C fixation as described above would lead to a buildup of internal CO 2 , resulting in a reductions in



### 8 stomatal conductance th rough stomatal closure. In Acer opalus , a significant reduction in light



### 9 saturated photosynthesis was accompanied by significant increases in internal CO 2 concentration and



### 10 decreases in water use efficiency without appreciable changes in stomatal conductan ce (Calatayud et



### 11 al., 2007, 191411 ) . A similar decrease in photosynthesis, accompanied by an increase in internal CO 2



### 12 concentrati on, was measured in soybean plants exposed to O 3 . Singh et al. (2009, 199427 ) attributed



### 13 the declines in photosynthesis to direct damage to the photosynthetic apparatus rather than to



### 14 stomatal limitations.



# 9.5. Na tu re o f Effe c ts o n Ve g e ta tio n

9.5.1. Introd uc tion

15 Ambient O 3 concentrations have long been known to cause visible symptoms, decreases in



### 16 photosynthetic rates, decreases in growth, and decreases in the yield of plants (U.S. EPA, 1978,



### 17 040586 ) (U.S. EPA, 1986, 017607 ) (U.S. EPA, 1996, 080828 ) (U.S. EPA, 2006, 088089 ) . Numerous



### 18 studies have r elated O 3 exposure to plant responses, with most effort focused on the yield of crops



### 19 and the growth of tree seedlings. Most experiments exposed individual plants grown in pots or soil



### 20 under controlled conditions to known concentrations of O 3 for a segment of daylight hours for some



### 21 portion of the plant’s life span (Section 9.3). This section focuses on the responses of plants to



### 22 seasonal or multi

- year exposures to known amounts of O 3 . Quantitative responses include changes in



### 23 growth and biomass allocation, changes in reproduction, onset of visible foliar injury, and changes in



### 24 leaf gas exchange. The response of a plant species or variety to O 3 exposure depends upon many



### 25 factors, including biochemical and physiological status (Section 9.4), genetic character istics, and



### 26 previous and current exposure to other stressors (Section 9.5.4.). Because of the available



### 27 information, most of this section focuses on the response of individual plants, especially tree



### 28 seedlings and crops, with limited discussion of mixtures of herbaceous species. Responses at the



### 29 ecosystem scale are discussed in Section 9.6.



### 30 This section will focus mainly on studies published since the release of the 2006 O 3 AQCD



### 31 (U.S. EPA, 2006, 088089 ) . However, because much O 3 research was conducted prior to the 2006 O 3



### 32 AQCD, the present discussion of vegetation response to O 3 exposure is largely based on the



### 33 conclusions of the 1978, 1986, 1996, and 2006 O 3 AQCDs (U.S. EPA, 1978, 040586 ) (U.S. EPA,



### 34 1986, 017607 ) (U.S. EPA, 1996, 080828 ) (U.S. EPA, 2006, 088089 ).

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## 9.5.2. Effe cts on Wo od y an d Herb ac e ou s Ve ge ta tio n

9.5.2.1. Growth and Bi o ma s s Alloc a tion

The previous O 3 AQCDs concluded that there is strong evidence that exposure to O 3 decreases

growth in numerous plant species. Studies published since the last review support those conclusions

and are summarized below.

In a recently publis hed meta

- analysis, Wittig et al. (2009, 191631 ) quantitatively compiled

peer reviewed studies from the past 40 y ea rs on the effect of current and future O 3 exposures on the

physiology and growth of forest species. Wittig et al. (2009, 191631 ) reported that current ambient

O 3 concentrations (~40 ppb) significantly decreased annual total biomass growth (7%) across 263

studies. However, this effect could be greater (11 to 17%) in areas that have higher O 3 concentrations

and as background O 3 increases in the future (Wittig et al., 2009, 191631 ) . This meta -analysis

demonstrates the coherence of O 3 effects across numerous studies and species using a variety of

experimental techniques.

In two companion papers, McLaughlin et al. (2007, 090348 ) (2007, 090347 ) investigated the

effects of ambient O 3 on tree growth and hydrology at forest sites in the southern Appalachian

Mountains. The authors reported the cumulative effects of ambient levels of O 3 decreased seasonal

stem growth by 30

- 50% for most trees species in a high O 3 year in comparison to a low O 3 year

(McLaughlin et a l., 2007, 090348 ) . The authors also report that high ambient O 3 concentrations can

disrupt whole

- tree water use and in turn reduce late

- season streamflow (McLaughlin et al., 2007,

090347 ) ; see Section 9.6.3 for more on water cycling.

Since the 2006 O 3 AQCD, several ne w studies based on the Aspen FACE “free air” O 3 and

CO 2 exposure experiment in a forest in Wisconsin were published (Darbah et al., 2007,

093288 ) (Darbah et al., 2008, 196890 ) (King et al., 2005, 191701 ) (Kubiske et al., 2006,

093284 ) (Kubiske et al., 2007, 191336 ) (Riikonen et al., 2008, 191258 ) . King et al. (2005, 191701 )

found that O 3 fumigation over the first seven years of stand development reduced total biomass

relative to the control by 23, 13, and 14% in the aspen, aspen

- birch, and asp en

- maple communities,

respectively. Over the same time period, Kubiske et al. (2006, 093284 ) observed that elevated O 3 decreased tree heights, diameters, and main stem volumes in the aspen community by 11, 16, and

20%, respectively. In addition, Kubiske et al. (2007, 191336 ) reported that elevated O 3 may change

the intra

- and inter

- species competition. For example, O 3 treatments increas ed the rate of conversion

from a mixed aspen

- birch community to a birch dominated community. In another study at this site,

Percy et al. (2007, 093287 ) suggested that negative growth effects were seen below the previous 8 -h

O 3 standard level of 0.084 ppm, but the informativeness of the study was diminished by severe

methodological problem s.

Several studies at the Aspen FACE site also considered other growth

- related effects of elevated

O 3 . Darbah et al. (2007, 093288 ) (2008, 196890 ) reported that O 3 t reatments decreased paper birch

seed weight and seed germination and that this would likely lead to a negative impact of

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1 regeneration for that species. Riikonen et al. (2008, 191258 ) found that elevated O 3 decreased the



### 2 amount of starch in birch buds by 16%, and reduced aspen bud size, which may have been related to



### 3 the observed delay in spring leaf development. The results suggest that elevated O 3 concentrations



### 4 have the potential to alter C metabolism of overwintering buds, which may have carry

- over effects in

the subsequent growing season (Riikonen et al., 2008, 191258 ).



### 6 Effects on growth of understory vegetation were also investigated at Aspen FACE. Bandeff et



### 7 al. (2006, 191733 ) found that the effects of elevated CO 2 and O 3 on understory species composition,



### 8 total and individual species biomass, N cont ent, and 15 N recovery were a result of overstory



### 9 community responses to those treatments; however, there were no apparent direct treatment e ff ects

due to high variability of the data. Total understory biomass increased with increasing light and was



### 11 greatest under the open canopy of the aspen/maple community, as well as the more open canopy of



### 12 the elevated O 3 treatments (Bandeff et al., 2006, 191733 ) . Similarly, data from a study by Awmack et



### 13 al. (2007, 191415 ) suggest that elevated CO 2 and O 3 may have indirect growth effects on red



### 14 ( Tr ifolium pratense ) and white ( Trifolium repens ) clover in the understory via overstory community

effects; however, no direct effects of elevated O 3 were observed.



### 16 Overall, the studies at the Aspen FACE experiment are consistent with many of the OTC



### 17 studies that were the foundation of previous O 3 NAAQS reviews. These results strengthen our



### 18 understanding of O 3 effects on forests and demonstrate the relevance of the knowledge gained from



### 19 trees grown in open

- top chamber studies.

For some annual species, particu larly crops, the endpoint for an assessment of the risk of O 3



### 21 exposure can be defined as yield or growth, e.g., production of grain. For plants grown in mixtures



### 22 such as hayfields, and natural or semi

- natural grasslands (including native nonagricultural sp ecies),



### 23 endpoints other than production of biomass may be important. Such endpoints include biodiversity



### 24 or species composition, and effects may result from competitive interactions among plants in mixed

species communities. Most of the available data on n on

- crop herbaceous species are for grasslands



### 26 with many of the recent studies conducted in Europe. See Section 9.6.5 for a review of the recent



### 27 literature on O 3 effects on competition and biodiversity in grasslands.



### Root Growth

28 Although O 3 does not penetra te soil, it could alter root development by decreasing



### 29 C assimilation via photosynthesis (Andersen, 2003, 041673 ) . The response of root development to

O 3 exposure depends on available photosynthate and could vary over time. Many biotic and abiotic



### 31 factors, such as community dynamics and drought stress, have been found to affect root production



### 32 under elevated O 3 . An earlier study at the AspenFACE experiment found that elevated O 3 reduced



### 33 coarse root and fine roots biomas s in young stands of paper birch and trembling aspen (King et al.,



### 34 2001, 041751 ) . However, this reduction disappeared several years later. Ozone significantly

increased fine

- root (<1.0 mm) in the aspen community (Pregitzer et al., 2008, 191677 ) . This increase



### 36 in fine root production was due to changes in community composition, such as better survival of the



### 37 O 3 - tolerant aspen genotype, birch, and maple, rather than changes in C allocation at the individual

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1 tree level (Pregitzer et al., 2 008, 191677 ) (Zak et al., 2007, 191239 ) . In an adult European



### 2 beech/Norway spruce forest in Germany, drought was found to nullify the O 3 - driven stimulation of



### 3 fine root growth. Ozone stimulated fine

- root production of beech during the humid year, but had no



### 4 significant impact on fine root production in the dry year (Nikolova et al., 2010, 626810 ) (Matyssek

et al., 2010, 628553 ).



### 6 Using a non

- destructive method, Vollsnes et al. (2010, 625576 ) studied the in vivo root



### 7 development of subterranean clover ( Trifolium subterraneum ) before, during and after short

- term O 3



### 8 exposure. It was found that O 3 reduced root tip formation, root elongation, the total root length, and



### 9 the ratios between below

- and above

- ground growth within one week after exposure. Those effects

persisted for up to three weeks; however, biomass and biomass ratios were not significantly altered



### 11 at the harvest five weeks after exposure.



### 12 Ozone has been shown to have negative (Jones e t al., 2010, 567354 ) , non -significant (Phillips



### 13 et al., 2009, 199802 ) (Andersen et al., 2010, 628559 ) and positive effects (Pregitzer et al., 2008,



### 14 191677 ) (Grebenc and Kraigher, 2007, 191265 ) on root biomass and root: shoot ratio. While the

findings of individual studies were mixed, several recent meta

- analyses have generally indicated that



### 16 O 3 reduced C allocated to roots. In one meta

- analysis, Grantz et al. (2006, 191545 ) estimated the



### 17 effect of O 3 on the root:shoot allometric coefficient (k), the ratio between the relative growth rate of



### 18 the root an d shoot. The results showed that O 3 reduced k by 5.6%, and the largest decline was



### 19 observed in slow

- growing plants. In another meta

- analysis including 263 publications, Wittig et al.

(2009, 191631 ) found that current O 3 exposure had no significant impacts on root biomass and



### 21 root:shoo t ratio when compared to pre

- industrial O 3 exposure. However, if O 3 concentrations rose to



### 22 81

- 101 ppb (projected O 3 levels in 2100), both root biomass and root:shoot ratio were found to



### 23 significantly decrease. Gymnosperms and angiosperms differed in their responses, with



### 24 gymnosperms being less sensitive to elevated O 3 . In two other meta

- analyses, Wang et al. (2010,

387478 ) found elevated O 3 reduced biomass allocation to roots by 8.3% at ambient CO 2 and 6.0% at



### 26 elevated CO 2 , and Morgan et al. (2003, 055527 ) found O 3 reduced root dry weight of soybean.



## 9.5.2.2. Reproduction

27 Studies during recent decades have demonstrated O 3 effects on different stages of plant



### 28 reproduction. The impacts of O 3 on reproductive development, as reviewed by Black et al. (2000,



### 29 036322 ) , can occur by influencing (1) age at which flowering occurs, particularly in long

- lived trees

that often have long juvenile periods of early growth without flower and seed production; (2) flower



### 31 bud initiation and development; (3) pollen germination and pollen tube growth; and (4) seed, fruit, or



### 32 cone yields and seed quality (Table 9

- 2) (U.S. EPA, 2006, 088089 )

. 

### 33 Several recent studies since the 2006 O 3 AQCD further demonstrate the effects of O 3 on



### 34 reproductive processes in herbaceous and woody plant species. Rämö et al. (2007, 191441 ) exposed

several meadow species to elevated O 3 (40

- 50 ppb) and CO 2 (+100 ppm), bot h individually and



### 36 combined, over three growing seasons in ground

- planted mesocosms, using OTC s. Elevated O 3

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24 delayed flowering of Campanula rotundifolia and Vicia cracca . Ozone also reduced the overall

number of produced flowers and decreased fresh weight of individual Fragaria vesca berries.

Black et al. (2007, 191558 ) exposed Brassica campes tris to 70 ppb for two days during late

vegetative growth or ten days during most of the vegetative phase. The two

- day exposure had no

effect on growth or reproductive characteristics, while the 10 day exposure reduced vegetative

growth and reproductive si te number on the terminal raceme, emphasizing the importance of

exposure duration and timing. Mature seed number and weight per pod were unaffected due to

reduced seed abortion, suggesting that, although O 3 affected reproductive processes, indeterminate

sp ecies such as B. campestris possess enough compensatory flexibility to avoid reduced seed

production (Black et al., 2007, 191558 )

. In the determinate species, Plantago major , Black et al. (2010, 625575 ) found that O 3 can

have direct effects on reproductive development in populations of differing sensitivity. Only the first

flowering spike was exposed to 120 ppb O 3 for 7 hour s per day on 9 successive days (corresponding

to flowe r development) while the leaves and second spike were exposed to charcoal

- filtered air.

Exposure of the first spike to O 3 affected seed number per capsule on both spikes even though spike

two was not exposed. The combined seed weight of spikes one and two was increased by 19% in the

two resistant populations, suggesting an overcompensation for injury; whereas, a decrease of 21%

was observed in the most sensitive population (Black et al., 2010, 625575 )

. A study by Darbah et al. (2007, 093288 ) (2008, 19689 0 ) of paper birch ( Betula papyrifera )

trees at the Aspen FACE site in Rhinelander, WI investigated the effects of elevated O 3 and/or CO 2 on reproductive fitness. Elevated O 3 increased flowering, but decreased seed weight and germination

success rate of s eeds from the exposed trees. These results suggest that O 3 can dramatically affect

flowering, seed production, and seed quality of paper birch, ultimately affecting its reproductive

fitness (Darbah et al., 20 07, 093288 ) (Darbah et al., 2008, 196890 ).

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### Table 9

- 2. Ozone effects on plant reproductive processes (derived from Table AX9

- 22 of the 2006

ozone AQCD)

Species Condition Measures References

Apocynun androsa emifolium Flowering time Bergweiler and Manning (1999, 036321 )

Buddleia davidii Flowering time Findley et al. (1997, 036337 )

Rubus cuneifolius Pollen germination Chappelka (2002, 052946 )

Plantago major Pollen tube elongation Stewart (1998, 094233 )

Fragaria × ananassa Fruit yield Drogoudi and Ashmore (2001, 052959 ) ; Drogoudi and Ashmore (2000, 042517 )

Plantago major Seed yield Lyons and Barnes (1998, 036354 ) ; Pearson et al. (1996, 053074 ) ; Reiling and Davison (1992, 043591 ) ; Whitfield et al. (1997, 036397 )

Understory herbs Seed yield Harward and Treshow (1975, 038366 )

Source: Adapted from 2006 O 3 AQCD (U.S. EPA, 2006, 088089 )



## 9.5.2.3. Vis ible Foliar Injury

1 Visible foliar injury resulting from exposure to O 3 has been well characterized and



### 2 documented over several decades on many tree, shrub, herbaceous, and crop species (U.S. EPA,



### 3 2006, 088089 ) (U.S. EPA, 1996, 080827 ) (U.S. EPA, 1984, 029711 ) (U.S. EPA, 1978, 040586 )

. 

### 4 Visible foliar injury symptoms are considered diagnostic as they have been verified experimentally



### 5 in exposure

- response studies, using exposure methodologies such as CSTRs, OTCs, and free

- air



### 6 fumigation (see Section 9.3 for mor e detail on exposure methodologies). Several pictorial atlases and



### 7 guides have been published, providing details on diagnosis and identification of O 3 - induced visible



### 8 foliar injury on many plant species throughout North America (Penn State, 1987, 626971 ) (Flagler,



### 9 1998, 025525 ) and Europe (Sánchez et al., 2001, 626980 ) (Sánchez et al., 2001, 626980 ) (Innes et al.,



### 10 2001, 048954 ) . Typical visible injury symptoms on broad

- leaved plants include: stippling, flecking,



### 11 surface bleaching, bifacial necrosis, pigmentation (e.g., bronzing), chlorosis, and/or premature



### 12 senescence. Typical visible injury symptoms for conifers include: chlorotic bandi ng, tip burn,



### 13 flecking, chlorotic mottling, and/or premature senescence of needles. Alt hough common patterns of



### 14 injury develop within a species, these foliar lesions can vary considerably between and within



### 15 taxonomic groups. Furthermore, the degree and ext ent of visible foliar injury development varies



### 16 from year to year and site to site (Chappelka et al., 2007, 093290 ) (Orendovici

- Best et al., 2008,



### 17 196940 ) (Smith et al., 2003, 044183 ) , even among co

- members of a population exposed to simil ar O 3



### 18 levels, due to the influence of co

- occurring environmental and genetic factors. Nevertheless,



### 19 Chappelka et al. (2007, 093290 ) reported that the average incidence of O 3 - induced foliar injury was



### 20 73% on milkweed in the Great Smokey Mountain National Park in the years 1992

- 1996.



### 21 Although the majority of O 3 - induced visible foliar injury occ urrence has been observed on



### 22 seedlings and small plants, many studies have reported visible injury of mature coniferous trees,



### 23 primarily in the western U.S. (Arbaugh et al., 1998, 040297 ) and to mature deciduous trees in eastern



### 24 North America (Chappelka et al., 1999, 041860 ; Chappelka et al., 1999, 052952 ; Hildebrand et al.,

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1 1996, 042670 ; Schaub et al., 2005, 191608 ; Somers et al., 1998, 044010 ; Vollenweider et al., 2003,



### 2 051157 )

. 

### 3 It is important to note that visible foliar injury occurs only when sensitive plants ar e exposed



### 4 to elevated O 3 concentrations in a predisposing environment. A major confounding factor for O 3 ­ induced visible foliar injury is the amount of soil moisture available to a plant during the year that



### 6 the visible foliar injury is being assessed. Thi s is because lack of soil moisture generally decreases



### 7 stomatal conductance of plants and, therefore, limits the amount of O 3 entering the leaf that can



### 8 cause injury (Grulke et al., 2003, 042637 ) (Matyssek et al., 2006, 191481 )( Panek, 2004,



### 9 079202 ) (Panek and Goldstein, 2001, 030190 ) (Temple et al ., 1992, 043349 ) (Temple et al., 1988,

043237 ) . Consequently, many studies have sh own that dry periods in local areas tend to decrease the



### 11 incidence and severity of O 3 - induced visible foliar injury; therefore, the incidence of visible foliar



### 12 injury is not always higher in years and areas with higher O 3 , especially with co

- occurring drou ght



### 13 (Smith et al., 2003, 044183 ) . Other factors such as leaf age influence the severity of symptom



### 14 expression with older leaves showing greater injury severity (Zhang et al., 2010, 628555 ).

Although visible injury is a valuable indicator of the presence of phytotoxic concentrations of



### 16 O 3 in ambient air, it is not always a reliable indicator of other negative effects on vegetation. The



### 17 significance of O 3 injury at the leaf and whole plant levels depends on how much of the total leaf



### 18 area of the plant has been affected, as well as the plant’s age, size, developmental stage, and degree



### 19 of functional redundancy among the existing leaf area. Previous O 3 AQCDs have noted the difficulty

i n relating visible foliar injury symptoms to other vegetation effects such as individual plant growth,



### 21 stand growth, or ecosystem characteristics (U.S. EPA, 2006, 088089 ) (U.S. EPA, 1996,



### 22 080827 ) (U.S. EPA, 1996, 080827 ) . As a result, it is not presently possible to determine, with



### 23 consistency across speci es and environments, what degree of injury at the leaf level has significance



### 24 to the vigor of the whole plant. However, in some cases, visible foliar symptoms have been

correlated with decreased vegetative growth (Benoit et al., 1982, 039778 ; Karnosky et al., 1996,



### 26 036347 ; Peterson et al., 1987, 042148 ; Somers et al., 1998, 044010 ) and with impaired reproductive



### 27 function (Black et al., 2000, 036322 ; Chappelka, 2002, 052946 ) . Conversely, the lack of visible



### 28 injury does not always indicate a lack of phytotoxic concentrations of O 3 or a lack of non

- visible O 3



### 29 effects (Gregg et al., 2003, 046996 ; Gregg et al., 2006, 186961 )

.



### Biomonitoring

The use of biological indicators to detect phytotoxic levels of O 3 is a longstanding and



### 31 effective methodology (Chappelka and Samuelson, 1998, 093687 ; Manning and Krupa, 1992,



### 32 044155 ) . A plant bioindicator can be defined as a vascular or nonvascular plant exhibiting a typical



### 33 and verifiable response when exposed to a plant stress such as an air pollutant (Manning, 2003,



### 34 053773 ) . To be considered a good indicator species, plants must (1) exhibit a distinct, verified

response ; (2) have few or no confounding disease or pest problems; and (3) exhibit genetic stability



### 36 (U.S. EPA, 2006, 088089 ) . Such sensitive plants can be used to detect the presence of a specific air



### 37 pollutant such as O 3 in the ambient air at a specific location or region and, as a result of the

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39 magnitude of their response, provide u nique information regarding specific ambient air quality.

Bioindicators can be either introduced sentinels, such as the widely used tobacco ( Nicotiana

tabacum ) variety Bel W3 (Calatayud et al., 2007, 191568 ; Gombert et al., 2006, 089309 ; Heggestad,

1991, 042533 ; Kostka

- Rick and Hahn, 2005, 199428 ; Laffray et al., 2007, 191384 ; Nali et al., 2007,

191478 ) or detectors, which are sensitive native plant species (e.g., tall milkweed [Asclepias

exaltata]; Chappelka et al., 2007, 093290 ; Souza et al., 2006, 191658 ) . The approach is especially

useful in areas where O 3 monitors are not operated (Manning , 2003, 053773 ) . For example, in

remote wilderness areas where instrument monitoring is generally not available, the use of

bioindicator surveys in conjuncti on with the use of passive samplers (Krupa e t al., 2001, 040430 )

may be a useful methodology (Manning, 2003, 053773 ) . However, the method requires expertise or

training in recognizing those signs and symptoms uniquely attributable to exposure to O 3 as well as

in their quantitative assessment.

Since the 2006 O 3 AQCD (U.S. EPA, 2006, 088089 ) , new sensitive plant species have been

identified from field surveys and verified in controlled e xposure studies (Kline et al., 2008, 191591 ;

Kline et al., 2009, 196918 ) . Several multiple

- year field surveys have also been conducted at National

Wildlife Refuges in Maine, Michigan, New Jersey, and South Carolina (Davis, 2007, 093291 ; Davis,

2007, 093292 ; Davis, 2009, 199287 ; Davis and Orendovici, 2006, 093293 )

. The USDA Forest Service through the Fores t Health Monitoring Program (FHM) (1990

­ 2001) and currently the Forest Inventory and Analysis (FIA) Program has been collecting data

regarding the incidence and severity of visible foliar injury on a variety of O 3 sensitive plant species

throughout the U .S. (Coulston et al., 2003, 041871 ) (Smith et al., 2003, 044183 ) . The plots where

these data are taken are known as biosites. These biosites are located throughout the country and

analysis of visible foliar injury within these sites follows a set of established protocols . F or more

details

, see http://www.nrs.fs.fed.us/fia/topics/ozone/ (USDA, 2011, 677550 ) . The network has

provided evidence of O 3 concentrations high enough to induce visible symptoms on sensitive

vegetation. F rom repeated observations and measurements made over a number of years, specific

patterns of areas experiencing visible O 3 injury symptoms can be identified. Coulston et al. (2003,

0 41871 ) used information gathered over a 6- year period (1994

- 1999) from the network to identify

several species that were sensitive to O 3 over a regional scale including sweetgum ( Liquidambar

styraciflua ), loblolly pine ( Pinus taeda ), and black cherry ( Pr unus serotina ). In a study of the west

coast of the U.S, Campbell et al. (2007, 602360 ) reported O 3 injury in 25

- 37% of biosites in

California forested ecosystems from 200 0- 2005.

A study by Kohut (2007, 093289 ) assessed the risk of O 3 - induced visible foliar injury on

bioindicator plants (NPS, 2006, 677536 ) in 244 national parks in support of the National Park

Service's Vital Signs Monitoring Network (NPS, 2007, 677537 ) . The risk assessment was based on a

simple model relating response to the interaction of t he plant, the level of O 3 exposure, and the

exposure environment. Kohut (2007, 093289 ) concluded that the risk of visible foliar injury was

high in 65 parks (27%), moderate in 46 parks (19%), and low in 131 parks (54%). Some of the well

known parks with a high risk of O 3 - induced visible foliar injury include Gettysburg, Valley Forge,

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1 Delaware Water Gap, Cape Cod, Fire Island, Antietam, Har pers Ferry, Manassas, Wolf Trap Farm



### 2 Park, Mammoth Cave, Shiloh, Sleeping Bear Dunes, Great Smoky Mountains, Joshua Tree, Sequoia



### 3 and Kings Canyon, and Yosemite.



## 9.5.2.4. Le a f Ga s Exc ha n ge

4 In general, there is strong experimental evidence over several decades of r esearch that

exposure to O 3 reduces photosynthesis and alters stomatal conductance in a wide variety of plant



### 6 species (Wittig et al., 2007, 191695 ) and these effects have been discussed in detail in previous O 3



### 7 AQCDs (U.S. EPA, 2006, 088089 ) (U.S. EPA, 1996, 080827 ) . Recent studies related to these effects



### 8 are discussed in several sections within this document; therefore, this section refers to those sections



### 9 for a more in depth discussion.

Ozone effects on photosynthesis were presented Section 9.4.5.1, with a focus on recent studies



### 11 on the effects of O 3 on light reactions. Changes in stomatal function in response to O 3 exposure were



### 12 discussed in Section 9.4.6. In addition, the implications of changes in stomatal control for water



### 13 cycling are discussed in Secti on 9.6.3. Leaf gas exchange as it relates to effects based air quality



### 14 exposure indices and dose modeling is discussed throughout Section 9.7.



## 9.5.3. Ag ric ultu ral Crops

The detrimental effect of O 3 on crop production has been recognized since the 1960’s and a



### 16 la rge body of research has stemmed from that recognition. Previous O 3 AQCDs have extensively



### 17 reviewed this body of literature (U.S. EPA, 2006, 088089 ) . Table 9

- 3 summarizes recent



### 18 experimental studies of O 3 effects on agricultural crops, exclusive of growth and yield. Growth and



### 19 yield results are summarized in Table 9 -1 6.

Ozone diffuses into the leaf apoplast via the stomata where it is rapidly converted into other



### 21 ROS that signal a diverse metabolic response (Kangasjarvi et al., 2005, 180341 ) (Long and Naidu,



### 22 2002, 038179 ) . The mechanism of O 3 sensing and cellular response is detailed in Section 9.4.3.1.



### 23 Ozone stress has been characterized as either acute or chronic, depending on the O 3 concentration



### 24 and the exposure duration (Fiscus et al., 2005, 079155 ) . While the actual concentration and duration

threshold for O 3 damage varies from species to species and sometimes even among genotypes of the



### 26 same species (Ariyaphanphita k et al., 2005, 191349 ) (Biswas et al., 2008, 191428 ) (Dalstein and Vas,



### 27 2005, 191570 ) (Guidi et al., 2009, 199825 ) (Keutgen et al., 2005, 191295 ) (Sawada and Kohno, 2009,



### 28 19942 6 ) , it is commonly accepted that acute damage results from a very high concentration of O 3



### 29 (>150 ppb) over a short period of time, and chronic O 3 damage results from a lower concentration of

exposure over a longer period of time. In general, acute O 3 dam age has been well characterized and



### 31 mimics the biochemical defense response of plants to pathogen attack (Kangasjarvi et al., 20 05,



### 32 180341 ) (Overmyer et al., 2003, 053537 ) . In contrast, the mechanism leading to chronic O 3 damage



### 33 is less well

- characterized but hallmark physiological symptoms include: decreased photosynthetic



### 34 productivity, decreased Rubisco activity and chlorophyll content, lower stomatal conductance, leaf

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1 chlorosis, accelerated senescence and a general decrease in gr een leaf area and plant productivity



### 2 (Ashmore et al., 2006, 191557 ) . Despite the knowledge gap pertaining to the mechanism of O 3



### 3 damage, a number of comprehens ive reviews and meta

- analyses have recently been published



### 4 discussing both the current understanding of the quantitative effects of O 3 concentration on a variety

of crop species and the potential focus areas for biotechnological improvement to a future gro wing



### 6 environment that will include higher O 3 concentrations (Ainsworth, 2008, 191646 ) (Booker et al.,



### 7 2009, 191569 ) (Feng et al., 2008, 191453 ) (Grantz et al., 2006, 191545 ) (Hayes et al., 2007,



### 8 196911 ) (Mills et al., 2007, 180221 ) (Morgan et al., 2003, 055527 ) (Van Dingenen et al., 2009,



### 9 199765 ) . Since the 2006 O 3 AQCD, exposure

- response indices for a variety of crops have been

suggested (Mills et al., 2007, 180221 ) and many reports have investigated the effects of O 3



### 11 concentration on seed or fruit quality to extend the knowledge base beyond yield quantity. This



### 12 section will outline the key findings from these papers as well as highlight some of the recent



### 13 research addressing the endpoints such as yields and crop quality.



### 14 Genetic vari ability is not the only factor that determines the crop response to O 3 - damage

.

Ozone concentrations throughout a growing

- season is not homogeneous and other environmental



### 16 conditions, such as elevated CO 2 concentrations, drought, cold or nutrient availability may alleviate



### 17 or exacerbate the oxidative stress response to a given O 3 concentration. This section will also



### 18 highlight recent literature that focuses on O 3 damage to crops as influenced by other environmental



### 19 factors.



## 9.5.3.1. Yield

It is well known that yield is negatively impacted in many crop species in response to high O 3



### 21 concentrations. However the threshold for damage varies from species to species. Reproductive



### 22 organs such as seeds may be particularly sensitive to injury or biomass reductions due to O 3 , a s



### 23 reviewed by Black et al. (2000, 036322 ) . Numerous analyses of experiments conducted in OTCs and



### 24 with naturally occurring gradients demonstrate that the effects of O 3 exposure vary depending on th e

growth stage of the plant. Plants grown for seed or grain are often most sensitive to exposure during



### 26 the seed or grain

- filling period (Lee et al., 1988, 594572 ) (Pleijel et al., 1998, 053021 ) (Soja et al.,



### 27 2000, 030388 ) (Younglove et al., 1994, 044162 )

. AX9.5.4.1 of the 2006 O 3 AQCD summarized many



### 28 previous studies on crop yield (U.S. EPA, 2006, 088089 )

. 

### 29 The effect of O 3 exposure on U.S. crops remains an important area of research and several

studies have been published on this topic since the 2006 O 3 AQCD (Table s 9- 3 and 9 -16 ). For



### 31 example, one study with cotton in a crop

- weed interaction study (Grantz and Shrestha, 2006,



### 32 191702 ) utilizing OTC s suggests that ambient O 3 concentrations (12 -h avg: 79.9 ppb) decreased



### 33 cotton biomass by 25% and 1 .5 × ambient O 3 concentration (12 -h avg: 122.7 ppb) decreased cotton



### 34 biomass by 75% compared to charcoal filtered control (12 -h avg: 12.8 ppb). Further, this study

suggests that the weed, yellow nutsedge, was less sensitive to increasing O 3 concentration w hich



### 36 would increase weed competition (Grantz and Shrestha, 2006, 191702 ) . In a study of peanuts in



### 37 North Carolina, near ambient and elevated exposures of O 3 reduced photosynthesis and yield

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38 compared to very low O 3 conditions (Booker et al., 2007, 191370 ) (Burkey et al., 2007, 191371 ) . In

another study, Grantz and Vu (2009, 19523 7 ) reported that sugarcane biomass growth significantly

declined under O 3 exposure.

The average yield loss reported across a number of meta

- analytic studies have been published

recently for soybean (Morgan et al., 2003, 055527 ) , wheat (Feng et al., 2008, 191453 ) , rice

(Ainsworth, 2008, 191646 ) , semi

- natural vegetation (Hayes et al., 2007, 196911 ) , potato, bean and

barley (Feng and Kobayashi, 2009, 199223 ) . The meta

- analytic technique allows for the objective

development of a quantitative consensus of the effects of a treatment across a wide body of literature

and therefore provides an average response ratio compiled from these sources. Further, this

technique allows for a compilation of data across a range of O 3 - fumigation techniques, durations and

concentrations in order to assemble the existing literature in a meaningful manner.

M organ et al. (2003, 055527 ) reported an average seed yield loss for soybean of 24%

compared to charcoal filtered air across all O 3 concentrations used in the 53 compil ed studies. The

decrease in seed yield appeared to be the product of nearly equal decreases (7

- 12%) in seed weight,

seed number and pod number. As would be expected, the lowest O 3 concentration (30

- 59 ppb)

resulted in the smallest yield losses, approximately 8%, while the highest O 3 concentration

(80

- 120 ppb ) resulted in the largest yield losses, approximately 35% (Morgan et al., 2003, 055527 )

. Further, the oil/prote in ratio within the soybean seed was altered due to growth at elevated O 3

concentrations, with a decrease in oil content. The studies included in this meta

- analysis all used

enclosed fumigation systems or growth chambers which may have altered the coupling of the

atmosphere to the lower plant canopy (McLeod and Long, 1999, 688834 ) . Utilizing the Soybean

Free Air gas Concentration Enrichment Facility (SoyFACE ; www.soyface.illinois.edu ), Morgan et al.

(2006, 079186 ) report a 20% seed yield loss due to a 23% increase in average daytime O 3
concentration (56

- 69 ppb) within a single soybean cultivar across two growing seasons in Illinois,

supporting the results from the meta

- analysis. A further breakdown of the effects of current O 3
concentrations (AOT40 of 4.7 ppm

- h) on bean seed quality ( Phaseolus vulgaris ) has identified that

growth at current O 3 concentrations compared to charcoal

- filtered air raised total lipids, total crude

protein and dietary fiber content (Iriti et al., 2009, 195635 ) . An increase in total phenolics was also

observed, however the individual phenolics compounds responded differently, with significant

decreases in anthocyanin content. The seeds from ambient O 3 exposed plants also displayed

increased total anti oxidant capacity compared to charcoal

- filtered air controls (Iriti et al., 2009,

195635 ) . Betzelberger et al. (2010, 644183 ) has recently utilized the SoyFACE facility to compare

the impact of elevated O 3 concentrations across 10 soybean cultivars to investigate intraspecific

variability of the O 3 resp onse to find physiological or biochemical markers for eventual O 3 tolerance

breeding efforts (Betzelberger et al ., 2010, 644183 ) . They report an average 17% decrease in yield

across all 10 cultivars across two growing seasons due to a doubling of ambient O 3 concentrations,

with a the individual cultivar responses ranging from

- 7% to

- 36%. The dose

- response functions

derived for these 10 modern cultivars were similar to the response functions derived from the

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inadvertent selection for more O 3 - tolerant cultivars in recent history.

A meta

- analysis has also been performed on studies investigating the effects of O 3 concentrations on wheat (Feng et al., 2008, 191453 ) . Across 23 studies included, elevated O 3 concentrations (ranging from a 7

- h daily average of 31

- 200 ppb) decreased grain yield by 29%.

Winter wheat and spring wheat did not differ in their responses; however the response in both

varieties to increasing O 3 concentrations resulted in successively larger decreases in yi eld, from a

20% decrease in 42 ppb to 60% in 153 ppb O 3 . These yield losses were mainly caused by a

combination of decreases in individual grain weight (

- 18%), ear number per plant (

- 16%), and grain

number per ear (

- 11%). Further, the grain starch concentration decreased by 8% and the grain protein

yield decreased by 18% due to growth at elevated O 3 concentrations as well. However, increases in

grain calcium and potassium levels were reported (Feng et al., 2008, 191453 ).

A recent meta

- analysis found that growth at elevated O 3 concentrations negatively impacts

nearly every aspect of rice pe rformance as well (Ainsworth, 2008, 191646 ) . While rice is not a major

crop in the U.S., it provides a staple food for over half of the global population (IRRI, 2002, 688833 )

and the effects of rising O 3 concentrations on rice yields merits consideration. On average, rice

yields decreased 14% in 62 ppb O 3 compared to charcoal

- filtered air. This yield loss was largely

driven by a 20% decrease in grain number (Ainsworth, 2008, 191646 )

. Feng and Kobayashi (2009, 199223 ) have recently compiled yield data for six major crop

species, potato, barley, wheat, r ice, bean and soybean and grouped the O 3 treatments used in those

studies into three categories: baseline O 3 concentrations (<26 ppb), current ambient 7

- or 12

- h daily

O 3 concentrations (31

- 50 ppb), and future ambient 7

- or 12

- h daily O 3 concentrations (51

- 75 ppb).

Using these categories, they have effectively characterized the effects of current O 3 concentrations

and the effects of future O 3 concentrations compared to the baseline O 3 concentrations. At current O 3 concentrations, which ranged from 41

- 49 ppb in the studies included, soybean (

- 7.7%), bean

(- 19.0%), barley (

- 8.9%), wheat (

- 9.7%), rice (

- 17.5%) and potato (

- 5.3%) all reported yield losses

compared to the baseline O 3 concentrations (<26 ppb). At future O 3 concentrations, averaging

63 ppb, soybean (- 21.6%), bean (

- 41.4%), barley (

- 14%), wheat (

- 28%), rice (

- 17.5%) and potato

(- 11.9%) all reported significantly larger yield losses compared to the losses at current O 3
concentrations (<26 ppb) (Feng and Kobaya shi, 2009, 199223 )

.
An extensive review of OTC literature has determined the AOT40 critical level that causes a

5% yield reduction across a variety of agri cultural and horticultural species (Mills et al., 2007,

1 80221 ) . They classify the species into three groups: sensitive, moderate and tolerant. The sensitive

crops, including watermelon, beans, cotton, wheat, turnip, onion, soybean, lettuce, and tomato,

respond with a 5% reduction in yield under a 3 -month AOT4 0 of 6 ppm

- h. Watermelon was the most

sensitive with a critical level of 1.6 ppm

- h. The moderately sensitive crops, including sugar beet,

oilseed rape, potato, tobacco, rice, maize, grape and broccoli, responded with a 5% reduction in yield

between 8.6 and 20 ppm

- h. The crops classified as tolerant, including strawberry, plum and barley,

responded with a 5% yield reduction between 62

- 83.3 ppm -h (Mills et al. , 2007, 180221 ).

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10

15

20

25

30

35

1 Feng and Kobayashi (2009, 199223 ) compared their response

- exposure results to those



### 2 published by Mills et al. (2007, 180221 ) and found the ranges of yield loss to be similar for soybean,



### 3 rice and bean. However, Feng and Kobayasi reported smaller yield losses for potato and wheat and



### 4 larger yield losses for barley compared to the dose

- response functions published by Mills et al.

(2007, 180221 ) , which they attributed to their more lenient criteria for literature inclusion.



### 6 While the studies investigating the impact of various O 3 concentrations on yield are important



### 7 and aid in determining the vulnerability of various crops to a variety of O 3 concentrations, there is



### 8 still uncertainty as to how these crops will respond under field conditions with interacting



### 9 environmental factors such as temperature, soil moisture, CO 2 concentration, and soil fertility

(Booker et al., 2009, 191569 ) . Further, there appears to be a distinct developmental and genotype



### 11 dependant influence on plant sensitivity to O 3 that has yet to be fully investigated across O 3



### 12 concentrations in a field setting.



### 13 Because O 3 is heterogeneous in both time and space and O 3 monitoring stations are



### 14 predominantly near urban areas, the O 3 impacts on current crop yields are difficult to estimate.

Fishman et al. (2010, 644259 ) have used satellite observations to estimate O 3 concent rations in the



### 16 contiguous tri

- state area of Iowa, Illinois and Indiana and have combined that information with other



### 17 measured environmental variables to model the historical impact of O 3 concentrations on soybean



### 18 yield across the 2002

- 2006 growing seasons. When soybean yield across Iowa, Indiana and Illinois



### 19 was modeled as a function of seasonal temperature, soil moisture and O 3 concentrations, O 3 had the

largest contribution to the variability in yield for the southern

- most latitudes included in the datase t.



### 21 Fishman et al. (2010, 644259 ) determined that O 3 concentrations significantly reduced soybean yield



### 22 by - 0.38 to

- 1.63% ppb / v across the 5 years. This value is consistent with previous chamber studies



### 23 (NCLAN; Heagle, 1989, 093985 ) and results from SoyFACE (Morgan et al., 2006, 079186 )

. 

### 24 Satellite estimates of tropospheric O 3 concentrations exist globally (Fishman et a l., 2008, 193927 )

, therefore utilizing this historical modeling approach is feasible across a wider geographical area,



### 26 longer time

- span and perhaps for more crop species.



## 9.5.3.2. Crop Quality

27 In general, it appears that increasing O 3 concentrations above current ambient concentrations



### 28 can cause species dependant biomass losses, decreases in root biomass and nutritive quality,



### 29 accelerated senescence and shifts in biod iversity. A study conducted with highbush blackberry has

demonstrated decreased nutritive quality with increasing O 3 concentration despite no change in



### 31 biomass among a charcoal

- filtered control, an ambient O 3 and a 2 × ambient O 3 treatment (Di tchkoff



### 32 et al., 2009, 192230 ) . A study conducted with sedge using control (30 ppb), low (55 ppb), medium



### 33 (80 ppb) and high (105 ppb) O 3 treatments has demon strated decreased root biomass and accelerated



### 34 senescence in the medium and high O 3 treatments (Jones et al., 2010, 56735 4 ) . Alfalfa showed no

biomass changes across two years of double ambient O 3 concentrations (AOT40 of 13.9 ppm -h)



### 36 using FACE fumigation (Maggio et al., 2009, 191645 ) . However a modeling study has demonstrated



### 37 that 84% of the relative feed value in high

- yielding alfalfa was due to the variability in mean O 3

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### 1

2

3

4

5

6

7

8

9

10

11

12

13 concentration from 1998

- 2002 (Lin et al., 2007, 196925 ) . Further, in a managed grassland FACE

system, the reduction in total biomass harvest over five years decr eased twice as fast in the elevated

treatment (AOT40 of 13

- 59 ppm

- h) compared to ambient (AOT40 of 1

- 20.7 ppm

- h). Compared with

the ambient control, loss in annual dry matter yield was 23% after 5 year . Further, there was

significant changes in the functio nal categories growing in each plot with legumes showing the

strongest negative response (Volk et al., 2006, 191434 ) . This study suggests a shift in biodiversity

away from nitrogen

- fixers in managed grasslands. An OTC study conducted with Trifolium

subterraneum exposed to filtered ( <15 ppb), ambient, and 40 ppb above ambient O 3 demonstrates

decreases in biomass in the highest O 3 treatment as well as 10

- 20% decreased nutritive quality which

was mainly attributed to accelerated senescence (Sanz et al., 2005, 196963 ) . A study conducted with

Eastern gamagrass and big bluestem in OTCs suggests that big bluestem is not sensitive to O 3 , but

gamagrass displayed decreased nutritive quality, due to higher ligni n content and decreased N, in the

2 × ambient O 3 treatment (Lewis et al., 2006, 191542 ).

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### Table 9

- 3. S ummary of recent studies of ozone effects on crops (exclusive of growth and yield)

Species Facility

Location Exposure

Duration Ozone Exposure a (Additional treatment) Variable(s) measured percent change from

CF b
( percent change from ambient) Reference

Alfa lfa ( Medicago

sativa cv. Beaver)

Growth chambers 1, 2 or 4 days 3, 5 or 7 h/day 85 ppb

( Exposure duration ) Relative feed value n.s *high variability among

tr eat m ent groups (N/A) Muntifering et al. (2006, 191270 )

Bean ( Phaseolus vulgaris I.

cv Bor lotto)

OTC, ground

- p lanted

Curno, Italy 4 mo nths Seasonal AOT40:
CF = 0.5 ppm

- h;

Ambient = 4.6 ppm -h

(N/A) Seed lipid,

Protein content

Fiber content +28.5 (N/A)

+7.88 (N/A)

+14.54 (N/A) Iriti et al. (2009,

195635 )

Big Blue Stem ( Andropogon gerardii )

OTC 4 mo nths 12

- h avg:
CF = 14 ppb;

Ambient = 29 p pb;

Elevated = 71 ppb Relative feed value n.s

. (n.s.) Lewis et al. (2006,

191542 )

Alabama, U.S. (N/A)

Brassica napu s

Growth chambers

Belgium 4 days CF & 176 ppb
for 4 h/day

(N/A) Glucosinolates −41 (N/A) Gielen et al. (2006,

191271 )

Brassica napus

cv. W estar

Growth chambers

Finland 17

- 26 days 8- h avg:
CF & 100 ppb

(Bt/non

- Bt; herbivory) VOC emissions −30.7 (N/A);

−34 (N/A) Himanen et al. (2009, 191338 )

Eastern Gamagrass ( Tripsacum dactyloides )

OTC 4 mo nths 12

- h avg:
CF = 14 ppb;

Ambient = 29 ppb;

Elevated = 71 ppb Relative feed value −17 (−12) Lewis et al. (2006,

191542 )

Alabama, U.S. (N/A)

Lettuce ( Lactuca sativa )

OTC

Carcaixent 30 days 12

- h mean:
CF = 10.2 ppb;

NF = 30.1 ppb;
NF+O 3 = 62.7 ppb Lipid peroxidation;

Root length +77 (+38)

−22 (−14) Calatayud et. al. (2002, 684222 )

Experimental Station, Spain (4 cultivars)

Peanut ( Arachis hypogaea )

OTC 3 yr 12

- h avg:
CF = 22 ppb;

Ambient = 46 ppb;

Elevated = 75 ppb Harvest biomass −40 (−10) Booker et al. (2007,

191370 )

Raleigh, NC; U.S. (CO 2 : 375 ppm; 548 ppm; 730 ppm)

Poa pratensis

OTC

Braunschweig, Germany 3 yr;

4- 5 wk

in the spring 8- h avg:
CF+25 = 21.7 ppb;

NF+5 0 = 73.1 ppb

(Competition) Relative feed value N/A (n.s.; −8) Bender et al. (2006,

191437 )

Potato ( Solanum tuberosum

cv. Bintje

OTC

Sweden & Finland 2 yr CF = 10 ppb;

Ambient = 25 ppb); Ambient(+) = (36 ppb); Ambient(++) = (47 ppb)

(N/A) [K], [Ca], [Mg], [P], [N] per dry weight

of tubers *dose

- response regression, report significant positive or negative slope with increasing [O 3 ] [N] [P] [Ca] n.s.; [K] & [ Mg] sig +

(N/A) Piikki et al. (2007,

191451 )

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Location Exposure

Duration Ozone Exposure a (Additional treatment) Variable(s) measured percent change from
CF b
( percent change from ambient) Reference

Potato (Solanum tuberosum

cv. Indir a)

Climate chambers

Germany 8 wk CF = 10 ppb;

Ambient = 50 ppb;

2×Ambient = 100 ppb

(CO 2 : 400 ppm &

700 ppm) Pathogen infestation using %

necrosis +52 (n.s.) Plessl et al. (20 07,

196952 )

AOT40: Soybean

OTC 3 yr CF = 0 ppm

- h;

Ambient = 3.4 ppm

- h;

Elevated = 9.0 ppm -h Daily evapotransp iration −28 (−14) Jaude et al. (2008,

191222 )

Italy (Well

- watered &

water

- stressed)

Soybean ( Glycine max cv. 93B15)

SoyFACE

Urbana, IL; U.S. 3 yr May

- Oct AOT40:

Ambient = 5- 22 ppm

- h;

Elevated = 20

- 43 ppm -h

(CO 2 : 550 ppm; environmental variability) Photosynthesis in new leaves

, N/A (n.s.) Bernacchi et al. (2006, 158001 )

Soybean ( Glycine max cv. 93B15)

SoyFACE

Urbana, IL; U.S. 4 mo nths 8- h avg:

Ambient = 38.5 ppb;

Elevated = 52 ppb

(Herbivory) Herbivory
defense

- related

genes N/A (N/A) Casteel et al. (2008, 191696 )

Soybean ( Glycine max

c v. Essex)

OTC, ground

- p lanted

Raleigh, NC; U.S. 2 yr 12

- h avg:
CF = 21 ppb;

1.5×Ambient = 74 ppb

(CO 2 : 370 ppm &

714 ppm) Post

- harvest res idue N/A ( −15.46) Booker et al. (2005,

079151 )

Soybean ( Glycine max cv. Essex)

OTCs, 21 L pots

Raleigh, NC; U.S. 2×3

mo nths 12

- h avg:
CF = 18 ppb);

Elevated = 72 ppb)

(CO 2 : 367 & 718) Water

- use eff icien cy n.s. (N/A) Booker et al. (Booker et al., 2004, 079138 )

Soybean ( Glycine max ) 10 cultivars)

SoyFACE

Urbana, IL; U.S. 2 yr 8- h avg (ppb):

Ambient = 46.3 & 37.9;

Elevated = 82.5 & 61.3

(Cultivar comparisons) Total antioxidant capacity N/A (+19) Betzelberger et al. (20 10, 644183 )

Spring Wheat ( Triticum aestivum

cv. Minaret; Satu; Drabant; Dragon)

OTCs

Belgium, Finland,
& Sweden 7 yr Seasonal AOT40s ranged from

0 t o16 ppm -h

(N/A) Seed protein content;

1,000

- seed weight regressed across all experiments N/A (Significant negative correlation)

N/A (Significant negative correlation) Piikki et al. (2008,

199812 )

Strawberry ( Fragaria x ananassa

Duch. Cv. Korona & Elsanta)

Growth chambers 2 mo nths 8- h avg:
CF = 0 ppb;

E levated = 78 ppb

(N/A) Total leaf area −16 (N/A) Keutgen et al. (2005, 191295 )

Bonn, Germany

Sweet Potato

Growth Chambers 4 wk 8

- h avg:
CF = 0 ppb;

Ambient < 40 ppb;

Elevated = 255 ppb Tuber weight −14 (−11.5) Keutgen et al. (2008, 191690 )

Bonn, Germany (N/A)

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Location Exposure

Duration Ozone Exposure a (Additional treatment) Variable(s) measured percent change from
CF b
( percent change from ambient) Reference

Tomato ( Lycopersicon

esculentum )

OTC

Valencia, Spain 133 days 8

- mean: CF = 16.3 ppb;

NF = 30.1 ppb; NF(+) = 83.2 ppb

(Various cultivars; early & late harvest) Brix degree −7.2 (− 3.6) Calvo, et al. (2005,

191570 )

Trifolium repens & Trifolium pretense

Aspen FACE

Rhinelander, WI; U.S. 3 mo nths 3- mo daylight avg:

Ambient = 34.8 ppb;

1.2×Ambient = 42.23 ppb

(CO 2 ; 560 ppm) Lignin;

Dry

- matter digestibility N/A (+19.3)

N/A ( −4.2) Muntifering et al. (2006, 191 270 )

a Ozone exposure in ppb unless otherwise noted.

b CF = Carbon

- filtered air.

NF = Non

- filtered air.



## 9.5.4. Fa c to rs th at Mo dify Fu nc tio n al a nd Gro wth Re s p on s e

1 Many biotic and abiotic factors, including insects, pathogens, root microbes and fungi,



### 2 temperatu re, water and nutrient availability, and other air pollutants, as well as elevated CO 2 , 

### 3 influence or alter plant response to O 3 . These modifying factors we re comprehensively reviewed in



### 4 AX 9.3 of the 2006 O 3 AQCD (U.S. EPA, 2006, 088089 ) and thus, this section serves mainly as a



### 5 brief summary of the previous findings. A limited number of new studies published since the 2006



### 6 O 3 AQCD add to our understanding of the role of these interactions in modifying O 3 - induced plant



### 7 responses. Many of these modifying factors and interactions are integrated into discussions



### 8 elsewhere in this ch apter and the reader is directed to those sections.



## 9.5.4.1. Genetics

9 It is well known that species vary greatly in their responsiveness to O 3 . Even within a given



### 10 species, individual genotypes or populations can also vary significantly with respect to O 3 sensitivi ty



### 11 (see section AX 9.3.2 of the 2006 AQCD; U.S. EPA, 2006, 088089 ) . Therefore, caution should be



### 12 taken when considering a species ' degree of sensitivity to O 3 . Plant response to O 3 is determined by



### 13 genes that are directly related to oxidant stress and to an unknown number of genes that are not



### 14 specifically related to oxidants, but instead control leaf and cell wall thickness, stomatal



### 15 conductance, and the internal architecture of the air spaces. It is rarely the case that single genes are



### 16 responsible for O 3 tolerance. Studies using molecular biological tools and transgenic plants have



### 17 positively verified the role of various genes and gene products in O 3 tolerance and are continuing to



### 18 increase the understanding of O 3 toxicity and differences in O 3 sensitivity. See Section 9.4.3.2 of this



### 19 document for a discussion of recent studies related to gene expression changes in response to O 3

.

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### 5

10

15

20

25

30

35 

## 9.5.4.2. Environme ntal Biological Factors

1 As stated in the 2006 O 3 AQCD, the biological factors within the plant’s environment that may



### 2 directly or indirectly in fluence its response to O 3 in a positive or negative manner encompass insects



### 3 and other animal pests, diseases, weeds, and other competing plant species. Ozone may influence the



### 4 severity of a disease or infestation by a pest or weed, either by direct effec ts on the causal species, or

indirectly by affecting the host, or both. In addition, the interaction between O 3 , a plant, and a pest,



### 6 pathogen, or weed may influence the response of the target host species to O 3 (U.S. EPA, 2006,



### 7 088089 ) . Several recent studies on the effects of O 3 on insects via their interactions with plants a re



### 8 discussed in S ection 9.6.6.1. In addition, O 3 has also been shown to alter soil fauna communities



### 9 (S ection 9.6.6.2 ).

In contrast to detrimental biological interactions, there are mutually beneficial relationships or



### 11 symbioses involving higher plants and bacteria or fungi. These include (1) the nitrogen

- fixing



### 12 species Rhizobium and Frankia that nodulate the roots of legumes and alder and (2) the mycorrhizae



### 13 that infect the roots of many crop and tree species, all of which may be affected by exposure of th e



### 14 host plants to O 3 . Some discussion of mycorrhizae can be found in S ection 9.6.4.

In addition to the interactions involving animal pests, O 3 also has indirect effects on higher



### 16 herbivorous animals, e.g., livestock, due to O 3 - induced changes in feed qualit y. Recent studies on the



### 17 effects of O 3 on nutritive quality of plants are discussed in S ections 9.5.3 and 9.6.6.3.



### 18 Intra- and interspecific competition are also important factors in determining vegetation



### 19 response to O 3 . Plant competition involves the abi lity of individual plants to acquire the

environmental resources needed for growth and development: light, water, nutrients, and space.



### 21 Intraspecific competition involves individuals of the same species, typically in monoculture crop



### 22 situations, while inte rspecific competition refers to the interference exerted by individuals of



### 23 different species on each other when they are in a mixed culture. This topic was previously reviewed



### 24 in AX 9.3.3.4 of the 2006 O 3 AQCD (U.S. EPA, 2006, 088089 ) . Recent studies on competition and its

implications for community composition are discussed in Section 9.6.5.



## 9.5.4.3. Phys ical Factors

26 Physical or abiotic factors play a large role in modifying plant response to O 3 , and have been



### 27 extensively discussed in previous O 3 AQCDs (U.S. EPA, 1996, 080828 ) (U.S. EPA, 2006, 088089 )

. 

### 28 This section summarizes those findings as well as recent studies published since the last review.



### 29 Although some studies have indicated that O 3 impact significantly increases with increased

ambient temperature (Ball et al., 2000, 026354 ) (Mills et al., 2000, 030098 ) , other studies have



### 31 indicated that temperature has little effect (Balls et al., 1996, 026370 ) (Fredericksen et al., 1996,



### 32 026653 ) . A recent study by Riikonen et al. (2009, 195664 ) at the Ruohoniemi open air exposure field



### 33 in Kuopio, Finland found that the ef fects of temperature and O 3 on total leaf area and photosynthesis



### 34 of Betula pendula were counteractive. Elevated O 3 reduced the saplings’ ability to utilize the warmer

growth environment by increasing the stomatal limitation for photosynthesis and by reduc ing the

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### 1 redox state of ascorbate in the apoplast in the combination treatment as compared to temperature



### 2 alone (Riikonen et al., 2009, 195664 )

. 

### 3 Temperature affects the rates of all physiological processes based on enzyme catalysis and



### 4 diffusion; each process and overall growth (the integral of all processes) has a distinct optimal



### 5 te mperature range. It is important to note that a plant’s response to changes in temperature will



### 6 depend on whether it is growing near its optimum temperature for growth or near its maximum



### 7 temperature (Rowland

- Bamford, 2000, 030257 ) . However, temperature is very likely an important



### 8 variable affecting plant O 3 response in the presence of the elevated CO 2 levels contributing to global



### 9 climate change. In contrast, some evidence suggests that O 3 exposure sensitizes plants to low



### 10 temperature stress (Colls and Unsworth, 1992, 026469 ) and, also, that O 3 decreases below -ground



### 11 carbohydrate reserves, which may lead to responses in perennial species ranging from rapid demise



### 12 to impaired growth in subsequent seasons (i.e., carry

- over effects) (Andersen et al., 1997, 052923 ).



### 13 Light, a component of the plant’s physical environment, is an essential “resource” of energy



### 14 content that drives photosynthesis and C assimilation. It has been suggest ed that increased light



### 15 intensity may increase the O 3 sensitivity of light

- tolerant species while decreasing that of shade


### 16 tolerant species, but this appears to be an oversimplification with many exceptions. Several studies



### 17 suggest that the interaction bet ween O 3 sensitivity and light environment is complicated by the



### 18 developmental stage as well as the light environment of individual leaves in the canopy (Chappelka



### 19 and Samuelson, 1998, 093687 ) (Topa et al., 2001, 035398 ) (Kitao et al ., 2009, 191331 )

. 

### 20 Although the relative humidity of the ambient air has generally been found to increase the



### 21 adverse effects of O 3 by increasing stomatal conductance (thereby increasing O 3 flux into the leaves ),



### 22 abundant evidence also indicates that the ready availability of soil moisture results in greater O 3



### 23 sensitivity (Mills, 2002, 035322 ) . The partial “protection” against the adverse effects of O 3 afforded



### 24 by drought has been observed in field experiments (Low et al., 2006, 191396 ) and modeled in



### 25 computer simulations (Broadmeadow and Jackson, 2000, 021325 ). Conversely, O 3 may enhance the



### 26 negative effects of O 3 on plants (Grulke et al., 2003, 052984 ) (Pollastrini et al., 2010, 644392 ). There



### 27 is also some evidence that O 3 can predispose plants to drought stress (Maier

- Maercker, 1998,



### 28 029961 ) . Hence, the nature of the response is larg ely species

- specific and will depend to some extent



### 29 upon the sequence in which the stressors occur

.



## 9.5.4.4. Inte ra c tio ns with o the r Polluta nts

Ozone

- Nitrogen Interactions

30 Elevated O 3 exposure and N deposition often co

- occur as major pollutant types. However, the



### 31 i nteractions of O 3 exposure and N deposition on vegetation are complex and less well understood



### 32 compared to their independent effects. Consistent with the conclusion of the 2006 O 3 AQCD



### 33 (U.S. EPA, 2006, 088089 ) , studies published since the last review indicated that the interactive



### 34 effects of N and O 3 varied among species and ec osystems (Table 9

- 4). This section will focus on O 3



### 35 and N interactions at the plant scale. Responses at the ecosystem scale are discussed in Section 9.6.

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### 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20 Nitrogen deposition could stimulate relative growth rate (RGR), and lead to increased stomatal

conduct ance. Therefore, plants might become more susceptible to O 3 exposure. Alternatively, N

deposition may increase the availability of photosynthates for use in detoxification and plants could

become more tolerant to O 3 (Bassin et al., 2007, 196879 ) . Only a few recent studies have

investigated the interactive effects of O 3 and N in the U.S. Grulke et al. (2005, 199433 ) measured

stomatal conductance of California black oak ( Quercus kelloggii ) at a long

- term N

- enrichment site

located in the San Bernardino Mountains, which is accompanied by high O 3 exposure (80 ppb,

24 -h avg . over a six month growing season). The authors found that N amendment led to poor

stomatal control in full sun in midsummer of the average precipitation years, but enhanced stomatal

contro l in shade leaves of California black oak . In an OTC study

, Handley and Grulke (2008,

191485 ) found that O 3 lowered p hotosynthetic ability and water

- use efficiency, and increased leaf

chlorosis and necrosis of California black oak. N itrogen fertilization tended to reduce plant

sensitivity to O 3 exposure; however

, the interaction was not statistically signifi cant.

Studies conducted outside the U.S. are also summarized in Table 9

- 4. Generally, the responses

were species specific. The O 3 - induced reduction in photosynthetic rate and biomass loss were greater

in the relatively high N treatment for watermelon ( Citr illus lanants ) (Calatayud et al., 2006, 191482 )

and Japanese beech ( Fagus crenata ) seedlings (Yamaguchi et al., 2007, 191438 ) . However, there

was no significant interactive effect of O 3 and N on biomass production for Quercus serrata

seedlings (Watanabe et al., 2007, 191474 ) , young Norway spruce ( Picea abies ) trees (Thomas et al.,

2005, 075930 ) , and young European beech ( Fagus sylvatica ) trees (Thomas et al., 2006, 191362 ).

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### Table 9 -4. R esponse of plants to the interactive effects of elevated ozone exposure and N enrichment

Site Species Ozone exposure N addition Responses R eferences

N

- amended trees had lower late summer
San C gain and greater foliar chlorosis in the California black Bernardino 0, and 50 kg N/ drought year, and poor stomatal control Grulke et al. (2005, oak ( Quercus 80 ppb Mountains, ha/yr and lower leaf water use efficiency and in 199433 ) kelloggii ) U.S. midsummer of the average precipitation

year.

San California black N fertilization tended to reduce p lant Bernardino 0, and 50 kg N/ Handley and Grulke oak ( Quercus 0, 75, and 150 ppb sensitivity to O 3 expo sure; however the Mountains, ha/yr (2008, 191485 ) kelloggii ) interaction was not statistically significant. U.S.

Higher nitrogen levels alleviated the spruce trees Filtered (19.4

- 28.1 ppb); 0, 20, 40 and 80 Thomas et al. (2005, Swit zerland negative impact of O 3 on root starch ( Picea abies ) ambient (37.6

- 47.4 ppb) kg N/ ha/yr 075930 ) concentrations

Nitrogen addition amplified the negative beech trees Filtered (19.4

- 28.1 ppb); 0, 2 0, 40 and 80 Thomas et al. (2006, Switzerland effects of O 3 on leaf area and shoot ( Fagus sylvatica ) ambient (37.6

- 47.4 ppb) kg N/ ha/yr 191362 ) elongation.

ambient ( AOT40 of 11.1

­ The positive effects of N addition on 12.6 ppm

- h); 1.2 ambient 0, 5, 10’ 25, 50 canopy greenness were counteracted by Bassin et al. (2007, Switzerland Alpine pasture (AOT40 of 15.2

- 29.5 ppm

- h) kg N/ ha/yr accelerated leaf senescence in the highest 191534 ) and 1.6 ambient (28.4

- 64.9 O 3 treatment. ppm

- h)

ambient ( AOT40 of 11.1

- Only a small number of species showed

12.6 ppm

- h); 1.2 ambient significant O 3 and N interactive effects on 0, 5, 10, 25, 50 Bassin et al. (2009, Switzerland Alpine pasture (AOT40 of 15.2

- 29.5 ppm

- h) leaf chlorophyll concentration, leaf weight kg N/ha/yr 191333 ) and 1.6 ambient (28.4

- 64.9 and chang e in 18 O, and the patterns were

ppm

- h) not consistent.

ambient ( AOT40 of 11.1

­ The positive effects of N addition on 12.6 ppm

- h); 1.2 ambient 0, 5, 10’ 25, 50 canopy greenness were counteracted by Bassin et al. (2007, Switzerland Alpine pasture (AOT40 of 15.2

- 29.5 pp m- h) kg N/ ha/yr accelerated leaf senescence in the highest 191534 ) and 1.6 ambient (28.4

- 64.9 O 3 treatment. ppm

- h)

O 3 free (AOT40 of 0 ppm

- h), High N concentration enhanced the

watermelon ambient (AOT40 of 5.1

- 6.3 140, 280, and detrimental effects of O 3 on Chl a Calatayud et al. (2006, Spain ( Citrillus lanants ) ppm

- h) and elevated O 3 436 kg N/ ha/yr fluorescence parameters, lipid 191482 ) (AOT40 of 32.5

- 35.6 ppm

- h) peroxidation, and the total yield.

Ozone reduced total aerial biomass. N Filtered (24 -h avg

. of 8

- 22 fertilization counterbalanced O 3 - induced 10, 30, and 60 Sanz et al. (2007, Spain Trifolium striatum ppb); ambient (29

- 34 ppb), effects only when plants were exposed to kg N/ ha/yr 199245 ) elevated O 3 (35

- 56 ppb) moderate O 3 levels (ambient) but not

under elevat ed O 3 concentrations.

Filtered (24

- h avg

. of 10.3

­ The O 3 - induced reduction in net Japanese beech 13.2 ppb); ambient (42.0

­ 0, 20 and 50 kg photosynthesis and whole

- plant dry mass Yamaguchi et al. (2007, Japan seedlings ( Fagus 43.3 ppb), 1.5 ambient (62.6

­ N/ ha/yr were greater in the relativel y high N 191438 )
cren ata ) 63.9 ppb) and 2.0 ambient treatment than that in the low N treatment. (82.7

- 84.7 ppb)

Filtered (24

- h avg

. of 10.3

­ 13.2 ppb); ambient (42.0

- N o significant interactive effects of O 3 and Quercus serrata 0, 20 and 50 kg Watanabe et al. (2007, Japan 43.3 ppb), 1.5 ambient (62.6

- N load on the growth and net seedlings N/ ha/yr 191474 ) 63.9 ppb) and 2.0 ambient photosynthetic rate were detected

. (82.7

- 84.7 ppb)



### Ozone

- Carbon Dioxide Interactions

1 Several decades of research has shown that exposure to elevated CO 2 increases photosynthetic



### 2 rates (Tissue et al., 1999, 029011 ) (Bernacchi et al., 2006, 158001 ) (Wi ll and Ceulemans, 1997,



### 3 679719 ) (Tissue et al., 1997, 679718 ) (Bernacchi et al., 2005, 679713 ), decrease s stomatal



### 4 conductance (Bernacchi et al., 2006, 158001 ) (Ainsworth and Rogers, 2007, 092940 ) (Paoletti et al.,



### 5 2007, 199365 ) (Medlyn et al., 2001, 679716 ) (Leakey et al., 2006, 679714 ) and generally increases



### 6 the growth of plants (McCarthy et al., 2010, 679715 ) (Norby et al., 2005, 679717 ) . This is in contrast



### 7 to the decrease on photosynthesis and growth in many plants that are exposed to elevated O 3 . The

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39 interactive effects on vegetation have been the subject of research i n the past two decades because

the implications on productivity and water use of ecosystems. This area of research wa s covered

thoroughly in AX9.3.8.1 of the 2006 O 3 AQCD and much of the conclusions made then are still

relevant (U.S. EPA, 2006, 088089 ).

The bulk of the available evidence shows that, under the various experimental conditions used

(which almost exclusively employed abrupt or “step” increases in CO 2 concentration, as discussed

below), increased CO 2 levels (ambient + 200 to 400 ppm ) may protect plants from the adverse

effects of O 3 on growth. This protection may be afforded in part by CO 2 acting together with O 3 in

inducing stomatal closure, thereby reducing O 3 uptake, and in part by CO 2 reducing the negative

effects of O 3 on Rubisco and its activity in CO 2 - fixation. Although both CO 2 - induced and

O 3 - induced decreases in stomatal conductance have been observed primarily in short

- term studies,

recent data show a long

- term and sustained reduction in stomatal conductance under elevated CO 2 for a number of species (Ainsworth and Long, 2005, 042647 )( Ellsworth et al., 2004,

080092 ) (Gunder son et al., 2002, 080097 ) . Instances of increased stomatal conductance have also

been observed in response to O 3 exposure, suggesting partial stomatal dysfunction after extended

periods of exposure (Maier

- Maercker, 1998, 029961 ) (Grulke et al., 2007, 186963 ) (Paoletti and

Grulke, 2010, 628561 ).

I mportant caveats must be raised with r egard to the findings presented in published research.

The first caveat concerns the distinctly different natures of the exposures to O 3 and CO 2 experienced

by plants in the field. Changes in the ambient concentrations of these gases have very different

dy namics. In the context of climate change, CO 2 levels increase relatively slowly (globally

2 ppm/ year ) and may change little over several seasons of growth. On the other hand, O 3 presents a

fluctuating stressor with considerable hour

- to

- hour, day

- to -day and regional variability (Polle and

Pell, 1999, 093689 ) . Almost all of the evidence presented comes from experimentation involving

p lants subjected to an abrupt step increase to a higher , steady CO 2 concentration. In contrast, the O 3 exposure concentrations usually varied from day to day. Luo and Reynolds (1999, 03531 9 ) , Hui e t

al. (2002, 035288 ) , and Luo (2001, 035318 ) noted the difficulties in predicting the likely effects of a

gradual CO 2 increase from experiments involving a step increase or those using a range of CO 2

concentrations. It is also important to note that the levels of elevated CO 2 in many of the studies will

not be experienced in the field for 30 or 40 years, but elevated levels of O 3 can occur in several areas

of the U.S. Therefore, the CO 2 × O 3 interaction studies may be less policy relevant for current

ambient conditions.

Another caveat concerns the interactions of O 3 and CO 2 with other c limatic variables, such as

temperature and precipitation. In light of the key role played by temperature in regulating

physiological processes and modifying plant response to increased CO 2 levels (Long, 1991,

029710 ) (Morison and Lawlor, 1999, 094194 ) and the knowledge that rela tively modest increases in

temperature may lead to dramatic consequences in terms of plant development (Lawlor, 1998,

0 29015 ) , it is important to consider that studying CO 2 and O 3 interactions alone may not create a

complete understanding of effects on plants under future climate change.

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# 9.6. Effects of Ozone on Ecos ys tems and Services

9.6.1. Ecos ys tem Scale, Function, and Structure

Information presented in this section was collected at multiple scales, ranging from the

physiology of a given species to population, community, and ecosystem

- level investigations. For this

assessment, “ecosystem” is defined as a functional entity consist ing of interacting groups of living

organisms and their abiotic (chemical and physical) environment. Ecosystems cover a hierarchy of

spatial scales and can comprise the entire globe, biomes at the continental scale, or small, well

circumscribed systems suc h as a small pond.

Ecosystems have both structure and function. Structure may refer to a variety of measurements

including the species richness, abundance, community composition and biodiversity as well as

landscape attributes. Competition among and withi n species and their tolerance to environmental

stress or s are key elements of survivorship. When environmental conditions are shifted, for example,

by the presence of anthropogenic air pollution, these competitive relationships may change and

tolerance to s tress may be exceeded. “Function” refers to the suite of processes and interactions

among the ecosystem components and their environment that involve nutrient and energy flow as

well as other attributes including water dynamics and the flux of trace gases. Plant processes

including photosynthesis, respiration, C allocation, nutrient uptake and evaporation, are directly

related to functions of energy flow and C , nutrient and water cycling. The energy accumulated and

stored by vegetation (via photosynthetic C capture) is available to other organisms. Energy moves

from one organism to another through food webs, until it is ultimately released as heat. Nutrients and

water can be recycled. Air pollution alters the function of ecosystems when elemental cycles or t he

energy flow are altered. This alteration can also be manifested in changes in the biotic composition

of ecosystems.

There are at least three levels of ecosystem response to pollutants: (1) the individual organism

and its environment; (2) the population and its environment; and (3) the biological community

composed of many species and their environment (Billings, 1978, 034165 ) . Individual organisms

within a population vary in their ability to withstand the stress of environmental change. The

response of individual organisms within a population is based on their genetic constitution, stage of

growth at time of exposure to stress, and the microhabitat in which they are growing (Levine and

Pinto, 1998, 029599 ) . The stress ra nge within which organisms can exist and function determines the

ability of the population to survive. Those best able to cope with environmental stressors survive and

reproduce. Competition among different species results in succession (community change o ver time)

and, ultimately, sensitive species may be progress ively replaced and communities shift to favor those

species that may have the capability to tolera te stressors such as O 3 (Guderian, 1985,

019325 ) (Rapport and Whitford, 1999, 004595 ) . In the sections that follow, available information on

individual, population and community response to O 3 will be discussed. Effects of O 3 on productivity

and C sequestration, water cycling, below

- ground processes, competition and biodiversity, and

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### 1 insects and wildlife are considered below and in the context of ecosystem services where



### 2 appropriate.



## 9.6.1.1. Ecos ys te m Services

3 Ecosystem structure and function may be translated into ecosystem services. Ecosystem



### 4 services identify the varied and numerous ways that ecosystems are important to human welfare.



### 5 Ecosystems provide many goods and services that are of vital importance for the functioning of the



### 6 biosphere and provide the basis for the delivery of tangible benefits to human society. Hassan et al.



### 7 (2005, 092759 ) define these benefits to include supporting, provisioning, regulating, and cultural



### 8 services:



### 9  Supporting se rvices are necessary for the production of all other ecosystem services.



### 10 Some examples include biomass production, production of atmospheric O 2 , soil



### 11 formation and retention, nutrient cycling, water cycling, and provisioning of habitat.



### 12 Biodiversity is a supporting service that is increasingly recognized to sustain many of the



### 13 goods and services that humans enjoy from ecosystems. These provide a basis for three



### 14 higher

- level categories of services.



### 15  Provisioning services, such as products (Gitay et al., 2001, 092761 ) , i.e., food (including



### 16 game, roots, seeds, nuts and other fruit, spices, fodder), fiber (including wood, textiles),



### 17 and medicinal and cosmetic products ( such as aromatic plants, pigments).



### 18  Regulating services that are of paramount importance for human society such as



### 19 (1) C sequestration, ( 2 ) climate and water regulation, ( 3 ) protection from natural hazards



### 20 such as floods, avalanches, or rock

- fall, ( 4 ) water and air purification, and ( 5 ) disease and



### 21 pest regulation.



### 22  Cultural services that satisfy human spiritual and aesthetic appreciation of ecosystems



### 23 and their components.



## 9.6.1.2. As s e s s ing Ozone Effe c ts a t La rge r S pa tia l Sc a le s

24 Ozone effects at large spatial scales start as effects on plants at smaller spatial scales. Ozone enters



### 25 leaves through stomata, and has been shown to alter stomatal conductance and reduce the activity



### 26 and concentration of Rubisco (S ection 9.4.6). Th ose change s alter the rates of CO 2 uptake and water



### 27 loss from leaves, and therefore the rates of photosynthesis and transpiration at th e plant level



### 28 (Section 9.5.2). Those O 3 - induced effects could translate from the plant level to the ecosystem level,



### 29 and cause changes in ecosystem services, such as C storage, water production, nutrient cycling, and

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1 community composition (Figure 9 -1 ). Cha nges at the ecosystem level are difficult to evaluate



### 2 directly due to the complexity and the large spatial and temporal scale of ecosystems. These



### 3 assessments generally involve the extrapolation of laboratory or field results by ecological models or



### 4 long -t erm field experiments as discussed below.



## 9.6.2. Productivity and Carbon Seques tration

During the previous NAAQS review, there were very few studies that investigated the effect of



### 6 O 3 exposure on ecosystem productivity and C sequestration. Recent studies from lon g- term FACE



### 7 experiments provide new evidence of the association of O 3 exposure and changes in productivity at



### 8 the ecosystem level. In addition to experimental studies, several model studies also assessed the



### 9 impact of O 3 exposure on productivity and C sequ estration from stand to global scales. Three types

of models are most often used to study the ecological consequences of O 3 exposure: ( 1) regression



### 11 models such as dose

- response function derived from the Nation Crop Loss Assessment Network



### 12 (NCLAN) (Wang and Mauzerall, 2004, 179978 ) (Tong and Mauzerall, 2008, 621169 ) ; ( 2) tree growth



### 13 models such as TREGRO and ECOPHYS (Hogsett et al., 2008, 191229 ) (Martin et al., 2 001,



### 14 043678 ) ; and ( 3) process

- based ecosystem models such as PnET, Dynamic Land Ecosystem Model

(DLEM) and Terrestrial Ecosystem Model (TEM) (Ollinger et al., 2002, 180189 ) (Ren et al., 2007,



### 16 191366 ) (Felzer et al., 2009, 191460 ) . The experimental and model studies on ecosystem productivity



### 17 and C sequestration, at the stand scale as well as regionally and globally, are reviewed in the



### 18 fo llowing section.



## 9.6.2.1. Stand Scale

19 The above

- and below

- ground biomass and net primary production (NPP) were measured at

the Aspen FACE site after 7

- year O 3 exposure. Elevated O 3 caused 23, 13 and 14% reductions in



### 21 total biomass relative to the control in the a spen, aspen

– birch and aspen

– maple communities,



### 22 respectively (King et al., 20 05, 191701 ) . At the Kranzberg Forest FACE experiment, O 3 reduced



### 23 annual volume growth by 9.5 m 3 /ha in a mixed mature stand of Norway spruce and European bee ch



### 24 (Pretzsch et al., 2010, 580435 ) . Ozone also altered C accumulation and turnover in soil, and the

det ails of these studies are discussed in Section 9.6.4.



### 26 Changes in stand productivity under elevated O 3 were assessed by several model studies.



### 27 TREGRO is a process

- based, single tree growth model and has been widely used to simulate the



### 28 effects of O 3 on the growth of several species in different regions in the U.S. Ozone acts within the



### 29 model by reducing the maximum potential photosynthetic rate as a function of the cumulative uptake

of O 3 (Weinstein et al., 1991, 043993 ) (Tingey et al., 2004, 042385 ) . Hogsett et al. (2008, 191229 )



### 31 used TREGRO to evaluate the effectiveness of various forms and levels of a ir q uality s tandards for



### 32 protecting tree growth in the San Bernardino Mountains of California. They found that O 3 exposures



### 33 at the Crestline site resulted in a mean 20.9% biomass reduction from 1980 to 1985 and 10.3%



### 34 biomass reduction from 1995 to 2000, compared to the ‘‘backgrou nd’’ O 3 concentrations (O 3

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### 5

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1 concentration in Crook County, Oregon). The level of vegetation protection projected was different



### 2 depending on the air quality scenarios under consideration. Specifically, when air quality was



### 3 simulated to just meet t he California 8 h average maximum of 70 ppb and the maximum 3 months



### 4 12 -h SUM06 of 25 ppm -h

, annual growth reductions were limited to 1% or less, while air quality

that just met a previous NAAQS (the seco nd highest 1

- h max [125 ppb]) resulted in 6

- 7% annual



### 6 reduction in growth , resulting in the least protection relative to background O 3 (Hogsett et al., 2008,



### 7 191229 ).



### 8 Combining TREGRO with ZELIG, Weinstein et al. (2005, 179965 ) simulated the effects of



### 9 different O 3 levels ( 0.5, 1.5, 1.75, and 2 times ambient) on the growth and competitive interactions

of white fir and ponderosa pine at three sites in California: Lassen National Park, Yosemite Nat ional



### 11 Park, and Crestline. Their results suggested that O 3 had little impact on white fir, but greatly reduced



### 12 the growth of ponderosa pine. If current O 3 concentrations continue over the next century, ambient



### 13 O 3 exposure (SUM06 of 110 ppm

- h) at Crestline was predicted to decrease individual tree C budget



### 14 by 10% and decrease ponderosa pine abundance by 16%. Effects at Lassen National Park and

Yosemite National Park sites were found to be smaller because of lower O 3 exposure levels



### 16 (Weinstein et al., 2005, 179965 ).



### 17 The effects of O 3 on stand productivity and dynamics were also studied by other tree growth or



### 18 stand models, such as ECOPHYS, INTRAST and LINKAGES. ECOPHYS is a functional

- structural



### 19 tree growth model. The model used the linear relationship between the maximum capacity of

carboxylation and O 3 dose to predict the relative effect of O 3 on leaf photosynthesis (Martin et al.,



### 21 2001, 043678 ) . Simulations with ECOPHYS found that O 3 decreased stem dry matter production,



### 22 stem diameter and leaf dry matter production, induced earlier leaf abscission, and inhibited root



### 23 growth (Martin et al., 2001, 043678 ) . Simulation with INTRAST and LINKAGES showed similar



### 24 adverse effects on stand growth. Link ing INTRAST with LINKAGES, Hanson et al

. (2005, 191461 )

found that a simulated increase O 3 concentration in 2100 (a mean 20

- ppb increase over the current



### 26 O 3 concentration) yields a 35% loss of net ecosystem C exchange (NEE) with respect to th e current



### 27 conditions (174 g C/m 2 / year ).



## 9.6.2.2. Regional and Global Scales

28 Since the publication of the 2006 O 3 AQCD, there is additional evidence suggesting that O 3



### 29 exposure alters ecosystem productivity and biogeochemical cycling at the regional and continental

scale. Most of those studies were conducted by using process

- based ecosystem models (Table 9 -5 )



### 31 and are briefly reviewed in the following sections.



### Carbon Dynamics in Natural Ecosystems

32 Results of Reich (1987, 019314 ) and Tjoelker et al. (1995, 035394 ) indicated that O 3 effects



### 33 on photosynthesis can be determined as a function of O 3 uptake to internal leaf surface. Pooling data



### 34 from Reich (1987, 019314 ) and Tjoelker et al. (1995, 035394 ) , Ollinger et al

. (1997, 040707 )

derived an equation to simulate the O 3 effects on photosynthesis by cumulative O 3 dose (AOT40)

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38 and stomatal conductance. They incorporated this equation into the PnET

- II model and studied the

effect of O 3 on hardwood forest productivity of 64 hardw ood sites in northeastern U.S. Their model

indicated that O 3 caused a 3

- 16% reduction in NPP from 1987 to 1992 (Table 9 -5 ). Ollinger et al.

(2002, 180189 ) assessed the interactive effects of O 3 , N deposition, elevated CO 2 and land use

history on C dynamics by PnET

- CN. Their results indicated that O 3 offset the increase in net

C exchange caused by elevated CO 2 and N deposition by 13% (25.0 g C/m 2 / year ) under agriculture

site history, and 23% (33.6 g C/m 2 / year ) under timber harvest site history. PnET

- CN was also used to

assess changes in C sequestration of U.S

. Mid

- Atlantic temperate forest. Pan et a l. (2009, 596032 )

designed a factorial modeling experiment to separate the effects of changes in atmospheric

composition, historical climatic variability and land

- disturbances on the C cycle. They also found O 3 acted as a negative factor, partially offsetting the growth stimulation caused by elevated CO 2 and N

deposition of U. S. Mid

- Atlantic temperate forest. Ozone decreased NPP of most forest types by 7

­ 8%. Among all the forest types, spruce

- fir forest was most resistant to O 3 damage, and NPP

decreased by only 1% (Pan et al., 2009, 596032 )

.
Felzer et al. (2004, 186927 ) developed TEM 4.3 to simulate the effects of O 3 on plant growth.

The effects of O 3 on NPP and C sequestration of deciduous trees, conifers and crops in the

conterm inous U.S. were estimated by TEM. The results indicated that O 3 reduced NPP and

C sequestration in the U.S. (Table 9 -5 ) and the largest decreases (over 13% in some locations) in

NPP occurred in the Midwest agricultural lands during the mid

- summer. TEM was also used to

evaluate the magnitude of O 3 damage at a global scale (Table 9 -5 ) (Felzer et al., 2005, 186928 )

. Simulations for the historical period (1860

- 1995) show that the largest reductions in NPP and net

C exchange occurred in the mid w estern U.S., e astern Europe, and e astern China (Felzer et al., 2005,

186928 ) . DLEM was developed to simulate the detrimental e ffect of O 3 on ecosystems, and has been

used to examine the O 3 damage on NPP and C sequestration in Great Smoky Mountains National

Park (Zhang et al., 2007, 196983 ) , grassland ecosystems and terrestrial ecosystems in China (Ren et

al., 2007, 581541 ) (Ren et al., 2007, 191366 ) . Results of those simulations are listed in Table 9 -5

. Instead of using AOT40 as their O 3 exposur e metric as PnET, TEM and DLEM did, Sitch et al.

(2007, 093294 ) incorporated a different O 3 metric named CUOt (cumulative stomatal upt ake of O 3 ),

derived from Pleijel et al. (2004, 056608 ) , into th e MOSES

- TRIFFID coupled model. In the CUOt

metric, the fractional reduction of plant production is dependent on O 3 uptake by stomata over a

critical threshold for damage and this threshold level is different for different plant functional types.

Consistent with previous studies, their model simulation indicated that O 3 reduced global gross

primary production (GPP), C exchange rate and C sequestration (Table 9 -5 ). The largest reductions

in GPP and land -C storage were projected over North America, Europe, Chi na and India. In the

model, reduced ecosystem C uptake due to O 3 damage, results in additional CO 2 accumulation in the

atmosphere and an indirect radiative forcing of climate change. Their simulations indicated that the

indirect radiative forcing caused by O 3 ( 0.62- 1.09 W/m 2 ) could have even greater impact on global

warming that the direct radiative forcing of O 3 (0.89 W/m 2 ) (Sitch et al., 2007, 093294 ).

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### Crop Yield Loss

Two large scale field studies were conducted in the U.S

. (NCLAN) and in Europe (European

Open Top Chamber Programme, EOTCP) to assess the impact of O 3 on crop production. Ozone

exposure

- response regression models derived from the two programs have been widely used to

estimate crop yield loss (Wang and Mauzerall, 2004, 179978 ) (Tong and Mauzerall, 2008,

621169 ) (Van Dingenen et al., 2009, 199765 ) . Those studies found that O 3 generally reduced crop

yi eld and different crops showed different sensitivity to O 3 pollution (Table 9 -5 ). Ozone was

calculated to induce a possible 45

- 82 million metric tons loss for wheat globally. Production losses

for rice, maize and soybean were on the order of 17

- 23 million metric tons globally (Van Dingenen

et al., 2009, 199765 ) . The la rgest yield losses occur in high

- production areas exposed to high O 3 concentrations, such the Midwest and the Mississippi Valley regions in the U.S

. , Europe, China and

India (Van Dingenen et al., 2009, 199765 ) (Tong et al., 2007, 107431 ).

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### Table 9 -5 . The effects of ozone on primary production, C exchange, C sequestration and yield loss

Scale Model Index Ozone Impacts Reference

GPP Global MOSES

- CUOta D ecreased by 14

- 23% over the period 1901

- 2100 Sitch et al. (2007,

TRIFFID 093294 )

NPP Global TEM AOT40 Decreased by 0.8% without agricultural manageme nt and a Felzer et al. (2005, decrease of 2.9% with optimal agricultural management 186928 )

U.S. TEM AOT40 Reduced by 2.3% without optimal N fertilization and 7.2% Felzer et al. (2005, with optimal N fertilization from 1983

- 1993 186928 )

U.S. TEM AOT40 Reduced by 2.6

– 6.8% during the late 1980s

- early 1990s. Felzer et al. (2004,

186927 )

north eastern PnET AOT40 A reduction of 3

- 16% from 1987

- 1992 Ollinger et al.
U.S. (1997, 040707 )

U.S. Mid

- PnET AOT40 Decreased NPP of most forest types by 7

- 8% Pan et al. (2009,

Atlantic 596032 )

China DLEM AOT40 Reduced NPP of grassland in China by 8.5 Tg C from Ren et al. (2007,

1960s to 1990s 581541 )

C exchange Global TEM AOT40 Reduced net C exchange (1950

– 1995) by 0.1 Pg C/yr Felzer et al. (2005,

with out agricultural management and 0.3 Pg C/yr with 186928 ) optimal agricultural management

Global MOSES

- CUOt Decreased global mean land

– atmosphere C fluxes by 1.3 Sitch et al. (2007,

TRIFFID Pg C/yr and 1.7 Pg C/yr for the ‘high’ and ‘low’ plant O 3 093294 ) sensitivity models, respectively

C sequestration Global MOSES

- CUOt Reduced land

- C storage accumulation by between 143 Pg C /yr Sit ch et al. (2007,

TRIFFID and 263 Pg C/yr from 1900

– 2100 093294 )

U.S. TEM AOT40 Reduced C sequestration by 18

– 38 Tg C/yr from 1950 to Felzer et al. (2004,

1995 186927 )

GSM National DLEM AOT40 Decreased the ecosystem C storage of deciduous forests Zhang et al. (2007,

Park by 2. 5% and pine forest by 1.4% from 1971 to 2001 196983 )

China DLEM AOT40 Reduced total C storage by 0.06% in 1960s and 1.6% in Ren et al. (2007, 1990s in China’s terrestrial ecosystems 191366 )

Crop yield loss Global Dose
– M7b; Reduced by 7.3% to 12.3% for wheat, 5.4% to 15.6% for Van Dingenen et al. response M12c; soybean, 2.8 % to 3.7% for rice, and 2.4% to 4.1% for maize (2009, 199765 )

function AOT40 in year 2000.

U.S. Dose
– M7; M12; Reduced by 4.1% to 4.4% for wheat, 7.1% to 17.7% for Van Dingenen et al.

response AOT40 soybean, 2.6% to 3.2% for rice, and 2.2% to 3.6% for maize (2009, 199765 )

function in year 2000.

U.S. Dose
– SUM06 Caused a loss of 53.8 million to 438 million bush els in Tong et al. (2007,

response soybean production, which account for 1.7

– 14.2% of total 107431 )

function U.S. soybean production in 2005

East Asia Dose
– M7; M12 Reduced the yield of wheat, rice and corn by 1

– 9% and Wang and

response soybean by 23

– 27% in China, Japan and South Korea in Mauzerall (20 04,

function 1990 179978 )

a CUOt is defined as the cumulative stomatal uptake of O 3 , using a constant O 3 - uptake rate threshold of t nmo l/m 2 /s

. b M7 is defined as 7

- h mean O 3 concentration (ppb)

. c M12 is defined as 12

- h mean O 3 concentration (ppb)

. d Pg equals 1 × 10 15 grams.



## 9.6.3. Wa te r Cyclin g

1 Ozone has been shown to alter stomatal performance, which affects plant and stand



### 2 transpiration and therefore hydrological cycling. However, there is not a clear consensus on stomatal



### 3 response to O 3 exposure. A meta

- analysis found that O 3 reduced stomatal conductance by 11%



### 4 (Wittig et al., 2007, 191695 ) . Stomatal closure could help protect the plant from water loss. On the



### 5 other hand, a number of studies suggested that O 3 exposure could impair stomatal fun ction, which



### 6 leads to greater stomatal apertures, delays stomatal closure at night and results in higher transpiration



### 7 (Grulke et al., 2004, 042646 ) (McLaughlin et al., 2007, 090348 ) (McLaughlin et al., 2007,

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### 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

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20

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22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39 090347 ) (Mills et al., 2009, 191272 ) (Wilkinson and Davies, 2009, 199758 ) (Wilkinson and Davies,

2010, 598245 ) . This O 3 - induced impairment of stomatal control may be more pronounced for plants

growing under drought stress (McLaughlin et a l., 2007, 090348 ) (McLaughlin et al., 2007,

090347 ) (Wilkinson and Davies, 2010, 598245 ) . The discrepancy regarding stomatal function has

lead to furt her debate on the effects of O 3 on ecosystem water production.

Felzer et al. (2009, 191460 ) used TEM

- Hydro to assess the interactions of O 3 , climate,

elevated CO 2 and N limitation on the hydrological cycle in the eastern U.S . They found that elevated

CO 2 decreased evapotranspiration by 2

- 4% and increased runoff by 3

- 7%, as compared to the e ffects

of climate alone. When O 3 damage and N limitation w ere included, evapotranspiration was reduced

by an additional 4

- 7% and runoff was increased by an additional 6

- 11% (Felzer et al., 2009,

191460 ) . Based upon simulation with INTRAST and LINKAGES, Hanson et al. (2005, 191461 )

found that increasing O 3 concentration by 20 ppb above the current ambient level yields a modest

3% reduction in water use. Those ecological models were generally built on the assumption that O 3 induces stomatal closure. Therefore, results of those models normally found that O 3 reduced water

use.

I n contrast to the model simulations (Hanson et al., 2005, 191461 ) (Felzer et al., 2009,

19146 0 ) , field studies conducted by McLaughlin et al. (2007, 090348 ) (2007, 090347 ) indicated that

O 3 increases water use in a mixed deciduous forest in eastern Tennessee. McLaughlin et al. (2007,

090348 ) (2007, 090347 ) found that O 3 , with daily max imum levels ranging from 69.2 to 82.9 ppb,

reduced stem growth by 30

- 50% in the high -O 3 year 2002. The decrease in growth rate was caused

in part by amplification of diurnal cycles of water loss and recovery. Peak hourly O 3 exposure

increased the rate of w ater loss through transpiration as indicated by the increased stem sap flow.

The increased canopy conductance resulted in higher water uptake as reflected in the reduced soil

moisture in the rooting zone. The change in tree water use led to further impacts on the hydrological

cycle at the landscape level. Increased water use under high O 3 exposure was reported to reduce late

season modeled streamflow in three forested watersheds in eastern Tennessee (McLaughlin e t al.,

2007, 090347 ).

In addition to the impacts on stomatal performance, O 3 - induced physiological changes, such as

reduced leaf area index and accelerated l eaf senescence, could alter water use efficiency. At the

Aspen FACE experiment, stand

- level water use, as indicated by sap flux per unit ground area, was

not significantly affected by elevated O 3 despite a 22% decrease in leaf area index and 20% decrease

i n basal area (Uddling et al., 2008, 191655 ) . Several factors could contribute to the lack of negative

effect of elevated O 3 on stand water use. The maximum sap flux per unit total leaf area was

substantially increased by elevated O 3 , suggesting that whole

- plant hydraulic conductance per unit

leaf area was increased (Uddling et al., 2009, 596219 ) . Other potential contributing factors included

the higher proportion of sun leaves, and similar or ev en increased fine root biomass under elevated

O 3 (Uddling et al., 2008, 191655 ) . Elevated O 3 could also affec t evapotranspiration by altering tree

crown interception of precipitation . Ozone has been shown to change branch architectural

parameters, and the effects were species specific at the Aspen FACE experiment (Rhea et al., 2010,

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### 1 647084 )

. The authors found that there was a significant correlation between canopy architecture



### 2 parameters and stem flow for birch but not aspen.



## 9.6.4. Below

- Ground Proces s es

3 Above

- ground and below

- ground processes are tightly interconnected. Because roots and soil



### 4 organisms are not exposed directly to O 3 , below

- ground processes are affected by O 3 more through



### 5 alteri ng the quality and quantity of C supply from photosynthates and litterfall (Andersen, 2003,



### 6 041673 ) . Ozone can decrease leaf C uptake by altering stomatal function, reducing the activity and



### 7 concentration of Rubisco, a nd accelerating leaf senescence ( S ection 9.4) . Ozone can also increase



### 8 the metabolic costs by stimulating the production of chemical compounds for defense and repair



### 9 processes, and increasing the synthesis of antioxidants to neutralize free radicals (see S ection 9.4),



### 10 which increase the consumption of carbon for above

- ground processes. Therefore, O 3 could



### 11 significantly reduced the amount of C available for allocation to below

- ground by decreasing



### 12 C uptake but increasing C consumption of above

- ground process es (Andersen, 2003, 041673 )

. 

### 13 Since the 2006 O 3 AQCD, there is additional evidence for O 3 effects on below -ground



### 14 processes. Ozone has been found to alter root growth, soil food web structure, decomposer activities,



### 15 C turnover and nutrient flow (Figure 9

- 7). Ozone effects on root development and root biomass



### 16 production (Section 9.5.2.1) and soil food web structure (Section 9.6.5.3) are reviewed in other



### 17 sections. The focus in this section is on the response of litter in put, decomposer activities, soil



### 18 respiration, soil C formation and nutrient cycling.

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### Figure 9

- 7. Conceptual diagram showing where ozone disrupts C flow in a t ree

- soil system, including transfer between biotic and abiotic components below ground that
Arrows denote C flux pathways that ﷒ influence soil physical and chemical properties. are affected by ozone . Dashed lines indicate where the impact of ozone is susp ected

but unknown.



## 9.6.4.1. Litter Carbo n Che mis try, Litter Nu trien t an d Their Ecos ys tem Bu dge ts

1 Consistent with previous findings, recent studies show that, although the responses are often



### 2 species specific, O 3 tends to alter litter chemistry (U.S. EPA, 2006, 088089 ) .Alterations in chemical



### 3 parameters, such as changes in C chemistry an d nutrient concentrations, were observed in both leaf



### 4 and root litter (Table 9 -6 ).



### 5 At the Aspen FACE site, several studies investigated litter chemistry changes (Chapman et al.,



### 6 2005, 191345 ) (Liu et al., 2005, 187005 ) (Johnson and Pregitzer, 2007, 191287 ) (Parsons et al., 2008,



### 7 191853 ) . In both aspen and birch leaf litter, elevated O 3 increased the concentrations of soluble



### 8 sugars, soluble phenolics and condensed tannins (L iu et al., 2005, 187005 ) (Parsons et al., 2008,



### 9 191853 ) . Compared to other treatments, aspen litter under elevated O 3 had the highest fiber



### 10 concentration, with the lowest concentration associated with the birch litter under the same



### 11 conditions (Parsons et al., 2008, 191853 ) . Chapman et al. (2005, 191345 ) measured chemical



### 12 changes in fine root litter and found that elevated O 3 decreased lignin concentration. The O 3 -induced

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### 1 chemistry changes were also reported from other experimental sites. Results from an OTC study in



### 2 Finland su ggested that elevated O 3 increased the concentration of acid

- soluble lignin, but had no



### 3 significant impact on other chemicals such as total sugars, hemicelluloses, cellulose or total lignin in



### 4 the litter of silver birch (Kasurinen et al., 2006, 191269 ) . Results from the free air canopy O 3



### 5 exposure experiment at Kranzberg Forest, showed tha t O 3 increased starch concentrations but had no



### 6 impact on cellulose and lignin in beech and spruce leaf litter (Aneja et al., 2007, 191472 ) . The effect



### 7 of O 3 on three antioxidants (ascorbate, glutathione and **α**

- tocopherol) in fine roots of beech was also



### 8 assessed at Kranzberg Forest

. The re sults indicated that O 3 had no significant effect on **α** -tocopherol



### 9 and ascorbate concentrations, but decreased glutathione concentrations in fine roots (Haberer et al.,



### 10 2008, 191334 ). In addition to changing C chemistry, O 3 also altered nutrient concentrations in green



### 11 leaves and litter (Table 9 -6 ).



### 12 The combined effects of O 3 on bio mass productivity and chemistry changes could alter



### 13 C chemicals and nutrient contents at the canopy or ecosystem level. For example, although O 3 had



### 14 different impacts on their concentrations, annual fluxes of C chemicals (soluble sugar, soluble



### 15 phenolics, condensed tannins, lipid and hemicelluloses), macro nutrients (N, P, K and S) and micro



### 16 nutrients (Mg, B, Cu and Zn) to soil were all reduced due to lower litter biomass productivity at



### 17 Aspen FACE (Liu et al., 2005, 187005 ) (Liu et al., 2007, 093286 ) . At the Kranzberg Forest, N



### 18 content of spruce canopy in a mixed culture and Ca 2+ content of beech canopy in a monoculture



### 19 increased due to elevated O 3 increased leaf concentrations of those nutrients although leaf production



### 20 was not significantly altered by O 3 (Rodenkirchen et al., 2009, 1 91540 )

.



### Table 9 -6. The effect of elevated ozone on leaf/litter nutrient concentrations

Study Site Species Ozone Concentration Response Reference

Suonenjoki Research Silver birch Ambient: 10

- 60 ppb Decreased the con centration of P, Mn, Zn Kasurinen et al. Station, Finland Elevated: 2×ambient and B in leaf litter (2006, 191269 )

Aspen FAC E Aspen and birch Ambient: 50

- 60 ppb Decreased the concentrations of P, S, Ca Liu et al. (2007,

Elevated: 1.5×ambient and Zn, but had no impact on the 093286 ) concentrations of N, K, Mg, Mn, B and Cu in

leaf litter.

Aspen FACE Birch Ambient: 50

- 60 ppb Increase N concentration in birch litter Parsons et al. (2008,

Elevated: 1.5×ambient 191853 )

Kranzberg Forest, Germany Beech and spruce Ambient: 9

- 41 ppb Increased N concentration in beach leaf, but Kozovit s et al.

Elevated: 2×ambient not in spruce needle (2005, 191282 )

Kranzberg Forest, Germany Beech and spruce Ambient: 9

- 41 ppb 1) Had no significant effects on spruce Rodenkirchen et al.

Elevated: 2×ambient needle chemistry; 2) increased Ca (2009, 191540 )

concentration in beech leaves in monoculture, but had no impacts on other

nutrient s



## 9.6.4.2. De c o mp o s er Meta bolis m a nd Litter Decompos ition

21 The above

- and below

- ground physiological changes caused by O 3 exposure cascade through



### 22 the ecosystem and affect soil food webs. In the 2006 O 3 AQCD, there were very few studies on the



### 23 effect of O 3 on the structure a nd function of soil food webs, except two studies conducted by Larson



### 24 et al. (2002, 053015 ) and Phillips et al. (2002, 041768 ) . Since the last O 3 AQCD (U.S. EPA, 2006,

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### 5

10

15

20

25

30

35

1 088089 ) , new studies have provided more information on how O 3 affects the metabolism of soil



### 2 microbes and soil fauna.



### 3 Chung et al. (2006, 191729 ) found that the activity of the cellulose

- degrading enzyme 1,4

- **β**



### 4 glucosidase was reduced by 25% under elevated O 3 at AspenFACE. The decrease in cellulose

degrading enzymatic activity was associated wit h the lower cellulose availability under elevated O 3



### 6 (Chung et al., 2006, 191729 ) . The suppression of soil enzyme activities was also found in the



### 7 rhizo sphere of beech trees ( Fagus sylvatica ) in a lysimeter study in Germany (Esperschutz et al.,



### 8 2009, 595669 ) (Pritsch et al., 2009, 626808 ) . Except for xylosidase, enzyme activities involved in



### 9 plant cell wall degradation (cellobiohydrolase, beta -gluco sidase and glucuronidase) were decreased

in rhizosphere soil samples under elevated O 3 (2 × ambient level) (Pritsch et al. , 2009, 626808 )

. 

### 11 Similarly, Chen et al. (2009, 191452 ) found O 3 exposure, with a 3

- month AOT40 of



### 12 21.4-44.1 ppm

- h, decreased the microbial metabolic capability in the rhizosphere and bulk soil of



### 13 wheat, although the observed reduction in bulk soil was not significant.



### 14 Ozone

- ind uced change in soil organisms’ activities could affect litter decomposition rates.

However, no general decomposition pattern has been identified. The responses varied among



### 16 species, sites and exposure length. Parsons et al. (2008, 191853 ) collected litter from aspen and birch



### 17 seedlings at AspenFACE site, and conducted a 23

- mo nth field litter incubation starting in 1999. Th ey



### 18 found that elevated O 3 had different impacts on the decomposition of aspen and birch litter. Elevated



### 19 O 3 was found to reduce aspen litter decomposition. However, O 3 accelerated birch litter

decomposition under ambient CO 2 , but reduced it under elevated CO 2 (Parsons et al., 2008, 191853 )

. 

### 21 Liu et al. (2009, 191470 ) conducted another litter decomposition study at Aspen FACE from 2003 to



### 22 2006, when stand leaf area index (LAI) reached its maximum. During the 935

- day field incubation,



### 23 elevated O 3 was shown to reduce litter mass loss in the first year, but not in the second year. They



### 24 suggested that higher initial tannin and phenolic concentrations under eleva ted O 3 reduced microbial

activity in the first year (Liu et al., 2009, 191470 ) . In an OTC experiment, Kasurinen et al. (2006,



### 26 191269 ) collected silver birch leaf litter from three consecutive growing seasons and conducted three



### 27 separate litter

- bag incubation experiments. Litter decomposition was not affected by O 3 exposure in



### 28 the first two incubations, but a slower decomposition rate was found in the third incubation. Their



### 29 principle component analysis indicated that the litter chemistry changes caused by O 3 (decreased

Mn, P, B and increased C:N) might be partially responsible for the decreased mass loss of their third



### 31 incub ation.



## 9.6.4.3. Soil re s pira tio n a n d c a rb on forma tion

32 Ozone could reduce the availability of photosynthates for export to roots, and increase root



### 33 mortality and turnover rates. Ozone has also been shown to reduce above

- ground litter productivity



### 34 and alter litter c hemistry, which would affect the quality and quantity of the C supply to soil

organisms (Section 9.6.6.1). The complex interactions among those changes make it difficult to



### 36 predict the response of soil C cycling under elevated O 3 . The 2006 O 3 AQCD conclude d that O 3 had



### 37 no consistent impact on soil respiration (U.S. EPA, 2006, 088089 ) . Ozone could increase or decrease

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### 1 soil respiration, depending on the approach and timing of the measurements. Ozone may also alter



### 2 soil C formation. However, very few experiments directly measured changes in soil organic matter



### 3 content under O 3 fumi gation (U.S. EPA, 2006, 088089 ) . Recent studies on soil respiration and s oil



### 4 C content also found mixed responses. Most importantly, new publications derived from long

- term



### 5 fumigation experiments, such as the Aspen FACE experiment, suggest that ecosystem response to O 3



### 6 exposure can change over time. Observations made during the late exposure years can be



### 7 inconsistent with those during the early years, highlighting the need for caution to assess O 3 effects



### 8 based on short

- term studies (Table 9 -7 ).



### Table 9 -7 . The temporal variation of ecosystem responses to ozone exposure at Aspen FACE site

The Time of the Endpoint Response Reference Measurement

Litter decomposition 1999

- 2001 O 3 reduced aspen litter decomposition. However, O 3 accelerated birch Parsons et al. (2008, 191853 ) litter decomposition under ambient CO 2 , but reduced it under elevated
CO 2

2003

- 2006 O 3 reduced litter mass loss in the first year, but not in the second year. Liu et al. (2009, 191470 )

Fine root production 199 9 O 3 had no significant impact on fine root biomass King et al. (2001, 041751 )

2002, 2005 O 3 increased fine root biomass Pregitzer et al. (2008, 191677 )

Soil respiration 1998

- 1999 Soil respiration under +CO 2 +O 3 treatment was lower than that under King et al. (2001, 041751 )
+CO 2 treatment

2003

- 2007 Soil respiration under +CO 2 +O 3 treatment was 5

- 25% higher than Pregitzer et al. (2006,
under elevated CO 2 t reatment. 191676 ) , Pregitzer et al. (2008, 191677 )

Soil C formation 1998

- 2001 O 3 reduced the formation rates of total soil C by 51% and acid

- Loya et al. (2003, 074380 )

insolubl e soil C by 48%

2004

- 2008 No significant effect of O 3 on the new C formed under elevated CO 2 Talhelm et al. (2009, 596189 )



### Soil Respiration

9 Ozone has shown inconsistent impacts on soil respiration. A sun

- lit controlled

- env ironment



### 10 chamber study found that O 3 had no significant effects on soil respiration, fine root biomass or any



### 11 of the soil organisms in a reconstructed ponderosa pine/soil

- litter system (Tingey et al., 2006,



### 12 191341 ) . In an adult European beech/Norway spruce forest at Kranzberg Forest, the free air O 3



### 13 fumigation (AOT40 of 10.2- 117 ppm

- h) increased soil respiration under both beech and spruce



### 14 during a humid year (Nikolova et al., 2010, 626810 ) . The increased soil respiration under beech has



### 15 been accompanied by the increase in fine root biomass and ectomycorrhizal fungi diversity and



### 16 turnover (Grebenc and Kraigher, 2007, 191265 ) . The stimulating effect on soil respiration



### 17 disap peared under spruce in a dry year, which was associated with a decrease in fine root production



### 18 in spruce under drought. This finding suggested that drought was a more dominant stress than O 3 for



### 19 spruce (Nikolova et al., 2010, 626810 ) . Andersen et al. (2010, 628559 ) labeled the canopies of



### 20 European beech and Norway spruce with CO 2 depleted in 13 C at the same site. They did not observe



### 21 any significant changes in soil respiration for either species.



### 22 The nearly 10 year long studies at AspenFACE indicated that the response of soil respiration to



### 23 O 3 interacted with CO 2 exposure and varied temporal ly (Table 9 -7 ) (King et al., 2001,



### 24 041751 ) (Pregitzer et al., 2006, 191676 ) (Pregitzer et al., 2008, 191677 ) . Ozone treatment alone

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### 5

10

15

20

25

30

1 generally had the lowest mean soil respiration rates, although those differences between control and



### 2 elevated O 3 were usually not significant. However, soil respiration rates were different with O 3 alone



### 3 and when acting in combination with elevated CO 2 . In the first five years (1998

- 2002), soil



### 4 respiration under +CO 2 +O 3 treatment was similar to that under control and lower than that under

+CO 2 treatment (King et al., 2001, 041751 ) (Pregitzer et al., 2006, 191676 ) . Since 2003, +CO 2 +O 3



### 6 treatmen t started to show the greatest impact on soil respiration. Compared to elevated CO 2 , soil



### 7 respiration rate under +CO 2 +O 3 treatment was 15

- 25% higher from 2003

- 2004, and 5

- 10% higher



### 8 from 2005

- 2007 (Pregitzer et al., 2006, 191676 ) (Pregitzer et al., 2008, 191677 ) . Soil respiration was



### 9 highly correlated with the biomass of roots with diameters of <2 mm and <1 mm

, across plant

community and atmospheric treatments. The authors suggested that the increase in soil respiration



### 11 rate may be due to +CO 2 +O 3 increased fine root (<1.0 mm) biomass production (Pregitzer et al.,



### 12 2008, 191677 ).



### Soil Carbon Formation

13 Ozone

- induced reductions in plant growth can result in reduced C input to soil and therefore



### 14 soil C content (Andersen, 2003, 041673 ) . The simulations of most ecosystem models support this

prediction (Felzer et al., 2004, 186927 ) (Zhang et al., 2007, 196983 ) (Ren et al., 2007, 191366 )

. 

### 16 However, very few studies have directly measured soil C dynamics under elevated O 3 . After the first



### 17 four years of fumigation (from 1998 to 2001) at the Aspen FACE site, Loya et al. (2003, 074380 )



### 18 found that forest stands exposed to both elevated O 3 and CO 2 accumulated 51% less total soil C, and



### 19 48% less acid

- insoluble soil C compared to stands exposed only to elevated CO 2 . Soil o rganic carbon

(SOC) was continuously monitored at the Aspen FACE site, and the later data showed that the initial



### 21 reduction in new C formation (soil C derived from plant litter since the start of the experiment) by



### 22 O 3 under elevated CO 2 is only a temporary effect (Table 9 -7 ) (Talhelm et al., 2009, 596189 ) . The



### 23 amount of new soil C in the elevated CO 2 and the combined elevated CO 2 and O 3 treatments has



### 24 converged since 2002. There was no significant effect of O 3 on the new C formed under elevated

CO 2 over the last four years of the study (2004

- 2008). Talhelm et al. (2009, 596189 ) suggested the



### 26 observed reduction in the early years of the experiment might be driven by a suppression of



### 27 C allocated to fine roo t biomass. During the early exposure years, O 3 had no significant impact on



### 28 fine root production (King et al., 2001, 041751 ) . However, the effect of O 3 on fine root biomass was



### 29 observed later in the experiment. Ozone increased fine root production and the highest fine root

biomass was observed under the combined elevated CO 2 and O 3 treatment in the late exposure years



### 31 (Table 9 -7 ) (Pregitzer et al., 2006, 191676 )

. This increase in fine root production was due to changes



### 32 in community composition, such as better survival of O 3 - tolerant aspen genotype, birch and maple,



### 33 rather than changes in C allocation at the individual tree level (Pregitzer et al., 2008, 191677 ) (Zak et



### 34 al., 2007, 191239 )

.

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30

35 

## 9.6.4.4. Nutrient cycling

1 Nutrient cycling is important for sustaining ecosystem production. Ozone can affect nutrient



### 2 cycling by changing nutrient release from litter and uptake by plants. Nitrogen is the limiting



### 3 nutrien t for most ecosystems, and several studies examined N dynamics under elevated O 3 . Holmes



### 4 et al. (2006, 191372 ) found that elevated O 3 decreased gross N mineralization at the Aspen FACE

site, indicating that O 3 may reduce N availability. However, other N cycling processes, such as NH 4 +



### 6 immobilization, gross nitrification, microbial biomass N and soil or ganic N, were not affected by



### 7 elevated O 3 (Holmes et al., 2006, 191372 ) . Similarly, Kanerva et a l. (2006, 191747 ) found total N,



### 8 NO 3 - , microbial biomass N, potential nitrification and denitrifi cation in their meadow mesocosms



### 9 were not affected by elevated O 3 (40

- 50 ppb). Ozone also showed small impact on other micro and

macro nutrients. Liu et al. (2007, 093286 ) assessed N, P, K, S, Ca, Mg, Mn, B, Zn and Cu release



### 11 dynamics at Aspen FACE, and they found that O 3 had no effects on most nutrients, except to



### 12 decrease N and Ca release from litter.



### 13 Using the SImple NItrogen Cycle model (SINIC), Hong et al. (2006, 186989 ) evaluated the



### 14 impacts of O 3 exposure on soil N dynamics and streamflow nitrate flux. The detrimental ef fect of O 3 on plant growth was found to reduce plant uptake of N and therefore increase nitrate leaching. Their



### 16 model simulation indicated that ambient O 3 exposure increased the mean annual stream fl ow nitrate



### 17 export by 12% (0.042 g N/m 2 / year ) at the Hubbard Brook Experimental Watershed from 1964

- 1994



### 18 (Hong et al., 2006, 186989 ).



## 9.6.4.5. Dis s olve d Orga nic Ca rb on a nd Bio genic Trac e Gas es Emis s io n

19 The O 3 -induced changes in plant growth, C and N fluxes to soil and microbial metabolism can

alter other biogeochemical cycling processes, such as soil dissolved organic carbon (DOC) turnover



### 21 and trace gases emission.



### 22 Jones et al. (2009, 199881 ) collected fen cores from two peatlands in North Wales, UK and



### 23 exposed them to one of four levels of O 3 (AOT40 of 0, 3.69, 5.87 and 13.80 ppm

- h for 41 days).



### 24 Th ey found the concentration of porewater DOC in fen cores was significantly decreased by

increased O 3 exposure. A reduction of the low molecular weight fraction of DOC was concurrent



### 26 with the observed decrease in DOC concentration. Their results suggested t hat O 3 damage to



### 27 overlying vegetation may decrease utilizable C flux to soil. Microbes, therefore, have to use labile C



### 28 in the soil to maintain their metabolism, which, the authors hypothesized, leads to a decreased DOC



### 29 concentration with a shift of the DO C composition to more aromatic, higher molecular weight

organic compounds.



### 31 Several studies since the 2006 O 3 AQCD have examined the impacts of O 3 on nitrous oxide



### 32 (N 2 O) and methane (CH 4 ) emission. Kanerva et al

. (2007, 191405 ) measured the fluxes of N 2 O and



### 33 CH 4 in meadow mesocosms, which were exposed to elevated CO 2 and O 3 in OTCs in south

- western



### 34 Finland. They found that the daily N 2 O fluxes were decreased in the NF+O 3 (non

- filtered air +

elevated O 3 , 40

- 50 ppb) after three seasons of exposure. Elevated O 3 alone or combined with CO 2

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### 5

10

15

20

25

30

1 did not have any significant effect on the daily fluxes o f CH 4 (Kanerva et al., 2007, 191405 ) . In



### 2 another study conducted in central Finland, the 4 y ea r open air O 3 fumigation (AOT40 of 20.8

­



### 3 35.5 ppm -h for growing season) also did not affect the rate of potential CH 4 production, but it



### 4 slightly increased potential CH 4 oxidation by 15% in the peatland microcosms (Morsky et al., 2008,

191507 )

. However, O 3 has no overall effects on CH 4 emissions, which is the net result of the



### 6 potential CH4 production and oxidation (Morsky et al., 2008, 191507 ).



## 9.6.5. Competition and biodivers ity

7 The effects of O 3 on species competition ( AX 9.3.3.4) and community comp osition ( AX 9.6.4)



### 8 were summarized in the 2006 O 3 AQCD (U.S. EPA, 2006, 088089 ) . Plant species differ in their



### 9 sensitivity to O 3 . Fast growing plants with high stomatal conductance and high specific leaf area

(SLA) were more likely to be sensitive to O 3 exposure. Further, different genotypes of a given



### 11 species also vary in their sensitivity. This differential sensitivity could change the competitive



### 12 interactions that lead to loss in O 3 sensitive species or genotypes. A shift in community composition



### 13 in forest and grassland ecosystems noted in the 2006 O 3 AQCD (U.S. EPA, 2006, 088089 ) has



### 14 continued to be observed from experimental and gradient studies. Additionally, research since the

last review has shown that O 3 can alter community composition and diversity of soil microbial



### 16 communities.



## 9.6.5.1. Fores t

17 In the San Bernardino Mountains in southern California, O 3 pollution caused a significant



### 18 decline in ponderosa pine ( Pinus ponderosa ) and Jeffrey pine ( Pinus jeffreyi ) (U.S. EPA, 2006,



### 19 088089 ) . Pine trees in the young mature age class group exhibited higher mortality rates compared

with mature trees at a site with severe O 3 visible foliar injury. The vulnerability of young mature



### 21 pines was most likely caused by the fact that trees i n this age class were emerging into the canopy

, 

### 22 where higher O 3 concentrations were encountered (McBride and Laven, 1999, 053050 ) . Because of



### 23 the loss of O 3 - sensitive pines, mixed forests of ponderosa pine, Jeffery Pine and white fir ( Abies



### 24 concolor ) shifted to predominantly white fir (Miller, 1973, 039165 ) . Ozone may have indirectly

caused the decline in understory diversity in conif erous forests in the San Bernardino Mountains



### 26 through an increase in pine litterfall. This increase in litterfall from O 3 exposure results in an



### 27 understory layer that may prohibit the establishment of native herbs, but not exotic annual Galium



### 28 aparine (Allen et al., 2007, 196876 )

. 

### 29 Ozone damage to conifer forests has also been observed in several other regions. In the Valley

of Mexico, a widespread mortality of sacred fir ( Abies religiosa ) was observed in the heavily



### 31 polluted area of the Desierto de los Leones National Park in the early 1980s (de Lourdes de Bauer



### 32 and Hernand ez

- Tejeda, 2007, 196891 ) (Fenn et al., 2002, 626806 ) . Ozone damage was widely



### 33 believed to be an important causal factor in the dramatic decline of sacred fir. In alpine regions of



### 34 southern France and the Carpathians Mountains, O 3 was also considered as the major cause of th e

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1 observed decline in c embran pine ( Pinus cembra ) (Wieser et al., 2006, 191391 )

. However, f or those



### 2 pollu tion gradient studies, several possible factors, such as drought, insect outbreak and forest



### 3 management, may also contribute to the mortality of trees (de Lourdes de Bauer and Hernandez



### 4 Tejeda, 2007, 196891 ) (Wieser et al., 2006, 191391 ).

New evidence from long

- term fr ee O 3 fumigation experiments provided additional support for



### 6 the potential impacts of O 3 on species competition and community composition changes in forest



### 7 ecosystems. At the Aspen FACE site, community composition at both the genetic and species levels



### 8 was altered after seven years of fumigation with O 3 (Kubiske et al., 2007, 191336 ) . In the pure aspen



### 9 community, O 3 fumigation reduced growth and increased mortality of sensitive clone 259, while the

O 3 tolerant clone 8L emerged as the dominant clone. Growth of clone 8L was even greater under



### 11 elevated O 3 compared to controls, probably due to O 3 alle viated competitive pressure on clone 8L by



### 12 reducing growth of other clones. In the mixed aspen

- birch and aspen

- maple communities, O 3



### 13 reduced the competitive capacity of aspen compared to birch and maple (Kubiske et al., 2007,



### 14 191336 ) . In a phytotron study, O 3 fumigation reduced growth of beech but not spruce in mixed

culture, suggesting a higher susceptibility of beech to O 3 under interspecific competition (Kozovits



### 16 et al., 2005, 191282 ).



## 9.6.5.2. Gra s s la nd a nd Ag ric ultura l La n d

17 The response of managed pasture, often cultivated as a mixture of grasses and clover, to O 3



### 18 pollution has been studied for many years. The tendency for O 3 - exposure to shift the biomass of



### 19 grass- legume mixtures in favor of grass species, reported in the previous O 3 AQCD (U.S. EPA, 2006,

088089 ) has been generally confirmed by recent studies. In a mesocosm study, Trifolium repens and



### 21 Lolium perenne mixtures were exposed to an episodic rural O 3 regime within so lardomes for



### 22 12 weeks. T. repens showed significant changes in biomass but not L. perenne , and the proportion of



### 23 T. repens decreased in O 3 - exposed mixtures compared to the control (Hayes et al., 2 009, 191360 )

. 

### 24 After 5

- year O 3 fumigation ( AOT40 of 13.3-59.5 ppm -h ) at the Le Mouret FACE experiment,

Switzerland, legumes in fumigated plots declined from their initial over

- representation (128%) to a



### 26 mere 59% in control plots (Volk et al., 2006, 191434 ) . However, Stampfl i and Fuhrer (2010, 102180 )



### 27 re

- analyzed the species and soil data and suggested that Volk et al. (2006, 191434 ) overestimated the



### 28 O 3 effect. Stampfli and Fuhrer (2010, 102180 ) found that the difference in the species dynamics



### 29 between control and O 3 treatment was more caused by heterogeneous initial conditions than O 3

exposure

. Several studies also suggested the mature/species

- rich ecosystems were more resilient to



### 31 O 3 exposure. At another FACE experiment, located at Alp Flix, Switzerland, O 3 fumigation ( AOT40



### 32 of 15.2-64.9 ppm -h ) showed no significant impact on community composition of this species -rich



### 33 pasture (Bassin et al., 2007, 191534 ) . Pfleeger et al. (2010, 644281 ) collected seed bank soil from an



### 34 agricultural field and examined how the plant community responded over several generations to

elevated O 3 exposures. Sixty plant species from 22 families emerged in the chambers ove r their



### 36 four year study. Overall, they found that O 3 appeared to have small impacts on seed germination and



### 37 only a minor effect on species richness of pioneer plant communities.

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1 Several review papers have discussed the physiological and ecological characte ristics of O 3 ­



### 2 sensitive herbaceous plants. Hayes et al. (2007, 196911 ) assessed species traits associated with O 3



### 3 sensitivi ty by the changes in biomass caused by O 3 exposure. Plants of the therophyte (e.g., annual)



### 4 life form were particularly sensitive to O 3 . Species with higher mature leaf N concentration tended to

be more sensitive than those with lower leaf N concentration. Plants growing under high oxidative



### 6 stress environments, such as high light or high saline, were more sensitive to O 3 . Using the same



### 7 dataset from Hayes et al. (2007, 196911 ) , Mills et al. (2007, 196934 ) identi fied the O 3 sensitive



### 8 communities. They found that the largest number of these O 3 sensitive communities were associated



### 9 with grassland ecosystems. Among grassland ecosystems, alpine grassland, sub

- alpine grassland,

woodland fringe, and dry grassland were i dentified as the most sensitive communities.



## 9.6.5.3. Mic ro be s

11 Several methods have been used to study microbial composition changes associated with



### 12 elevated O 3 . Phospholipid fatty acid (PLFA) analysis is widely used to determine whether O 3 elicits



### 13 an overall effec t on microbial community composition. However, since PLFA markers cover a broad



### 14 range of different fungi, resolution of this method may be not fine enough to detect small changes in

the composition of fungal communities. Methods, such as microscopic analys es and polymerase



### 16 chain reaction

– denaturing gradient gel electrophoresis (PCR

– DGGE), have better resolution to



### 17 specifically analyze the fungal community composition. The resolution differences among those



### 18 methods needs to be considered when assessing the O 3 impact on microbial community composition.



### 19 Kanerva et al. (2008, 191264 ) found that elevated O 3 (40

- 50 ppb) decreas ed total, bacterial,

actinobacterial and fungal PLFA biomass values as well as fungal:bacterial PLFA biomass ratio in



### 21 their meadow mesocosms in south

- western Finland. The relative proportions of individual PLFAs



### 22 between the control and elevated O 3 treatmen ts were significantly different, suggesting that O 3



### 23 modified the structure of the microbial community. Morsky et al. (2008, 191507 ) exposed boreal



### 24 peatland microcosms to elevated O 3 , with growing season AOT40 of 20.8 -35.3 ppm

- h, in an open

- air

O 3 exposure field in Central Finland. They also found that microbial composition was altered after



### 26 three growing seasons with O 3 fumigation, as measured by PLFA. Ozone tended to increase the



### 27 presence of Gram

- positive bacteria and the biomass of fungi in the peatland m icrocosms. Ozone also



### 28 resulted in higher microbial biomass, which co -oc curred with the increases in concentrations of



### 29 organic acids and leaf density of sedges (Morsky et al., 2008, 191507 ) . In a lysimeter study in

Germany, O 3 was found to alter the PLFA profiles in the upper 0

- 20 cm rhizosphere soil of European



### 31 beech. Elevated O 3 reduced bacterial abundance but had no detectable effect on fungal abundance



### 32 (Pritsch et al., 2009, 626808 ) . Using microscopic analyses, Kasurinen et al. (2005, 191245 ) found



### 33 that elevated O 3 , with 5 or 6 months of AOT40 of 2 0.6 -30.9 ppb

- h, decreased the proportions of



### 34 black and liver

- brown mycorrhizas and increased that of light brown/orange mycorrhizas. In an

herbaceous plant study, SSCP (single

- strand conformation polymorphism) profiles indicated that O 3



### 36 stress (about 75 pp b) had a very small effect on the structural diversity of the bacterial community in



### 37 rhizospheres (Dohrmann and Tebbe, 2005, 191320 ) . At the A spen FACE site, O 3 had no significant

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### 1 effect on fungal relative abundance, as indicated by PLFA profile. However, elevated O 3 altered



### 2 fungal community composition, according to the identification of 39 fungal taxonomic units from



### 3 soil using polymerase chain reaction

– denaturing gradient gel electrophoresis (PCR

- DGGE) (Chung



### 4 et al., 2006, 191729 ) . Ozone was found to change microbial community composition in an



### 5 agricultural system. Chen et al. (2010, 644257 ) found elevated O 3 (100

- 150 ppb) had significant



### 6 effects on soil microbial composition expressed as PLFA percentage in a rice paddy in China.



## 9.6.6. Ins ec ts an d Wildlife

9.6.6.1. Ins e c ts

7 Insects may respond indirectly to changes to plants (i.e., increased reactive oxygen species,



### 8 altered phytochemistry, altered nutrient content) that occur under elevated O 3 conditions

, or O 3 can



### 9 have a direct effect on insect performance (Menendez et al., 2009, 191430 ) . Effects of O 3 on insects



### 10 occur at the species level (i.e., growth, survival, reproduction, development, feeding behavior) and at



### 11 the population and community

- lev el (i.e., population growth rate, community composition). In



### 12 general, effects of O 3 on insects are highly context

- and species

- specific (Bidart

- Bouzat and Imeh



### 13 Nathaniel, 2008, 191431 ; Lindroth, 2010, 596479 ) . Furthermore, plant responses to O 3 exposure and



### 14 herbivore attack have been demonst rated to share signaling pathways, complicating characterization



### 15 of these stressors (Lindroth, 2010, 596479 ; Menendez et al., 2009, 191430 ; Menendez et al., 2010,



### 16 384046 )



### Species-Level Responses

17 In considering insect growth, survival and reproduction in elevated O 3 conditions, several



### 18 studies have indicated an effect while others have found no correlation. The performance of fiv e



### 19 herbivore species (three moths and two weevils) was assessed in an OTC experiment at 2 × ambient



### 20 concentration (Peltonen et al., 2010, 596482 ) . Growth of larvae of the Autumnal moth, Epirrita



### 21 autumna , was significantly decreased in the O 3 treatment while no effects were observed in the other



### 22 species. In a n aphid oviposition preference study using birch buds grown in a three year OTC



### 23 experiment, O 3 had neither a stimulatory or deterring effect on egg -laying (Peltonen et al., 2006,



### 24 196946 ) . Furthermore, changes in birch bud phenolic compounds associated with the doubled



### 25 ambient concentrations of O 3 did not correlate with changes in aphid oviposition (Peltonen et al.,



### 26 2006, 196946 ) . Rep roduction in Popillia japonica , that were fed soybeans and grown under elevated



### 27 O 3 , appeared to be unaffected (O'Neill et al., 2008, 195653 ) . In a meta

- analysis of effects of elevated



### 28 O 3 on 22 species of trees and 10 species of insects, the rates of survival, reproduction and food



### 29 consumption were typically unaffect ed while development times were reduced and pupal masses



### 30 were increased (Valkama et al., 2007, 191348 ).



### 31 At the Aspen FACE site insect performance under elevated (50

- 60 ppb) O 3 conditions



### 32 (approximately 1.5 × background ambient levels of 30

- 40 ppb O 3 ) have been considered for several

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1 species. Cumulative fecundity of aphid s ( Cepe gillettea betulaefoliae ) , that were reared on O 3 ­



### 2 exposed paper birch ( Betula papyrifera ) trees

, was lower than aphids from control plots (Awmack et



### 3 al., 2004, 05292 6 ) . No effects on growth, development, adult weight, embryo number and birth



### 4 weight of newborn nymphs were observed. In a study conducted using three aspen genotypes,

performance of the aspen beetle ( Chrysomela crochi ) decreased across all parameters mea sured



### 6 (development time, adult mass and survivorship) under elevated O 3 (Vigue and Lindroth, 2010,



### 7 644185 ) . There was an increase in the development time of male and female aspen beetle larvae



### 8 although the percentages varied across genotypes. Decreased beetle adult mass and survivorship was



### 9 observed across all genotypes under elevated O 3 conditions. Another study from the Aspen FACE

site, did not find any significant effects of elevated O 3 on performance (longevity, fecundity,



### 11 abundance) of the invasive weevil ( Polydrusus sericeus ) (Hillstrom et al., 2010, 644679 )

. 

### 12 Since the 2006 O 3 AQCD, several studies have considered the e ffect of elevated O 3 on feeding



### 13 behavior of insects. In a feeding preference study, the common leaf weevil ( Phyllobius pyri )



### 14 consumed significantly more leaf discs from one aspen clone when compared to a second clone

under ambient air conditions (Freiwald et al., 2008, 196903 ) . In a moderately elevated O 3



### 16 environment (1.5 × ambient), this preference for a certain aspen clone was less evident, however,



### 17 leaves from O 3 - exposed trees were significantly preferred to leaves grown under ambient conditions.



### 18 Other plant -h erbivore interactions have shown no effects of elevated O 3 on feeding. Feeding



### 19 behavior of Japanese beetles ( P. japonica ) appeared to be unchanged when beetles were fed soybean

leaves grown under elevated O 3 conditions (O'Neill et al., 2008, 195653 ) . At the Aspen FACE site,



### 21 feeding by the invasive weevil ( Polydrusu s sericeus ), as measured by leaf area consumption

, was not



### 22 significantly different between foliage that was grown under elevated O 3 versus ambient conditions



### 23 (Hillstrom et al., 2010, 644679 )

.



### Population

- Level and Community -Level Responses

24 Recent data on insects provide evidence of population

- level and c ommunity

- level responses to

O 3 . Elevated levels of O 3 can affect plant phytochemistry and nutrient content which in turn can alter



### 26 population density and structure of the associated herbivorous insect communities and impact



### 27 ecosystem processes (Lindroth, 2010, 596479 ) . In a long

- term study of elevated O 3 on herbivore



### 28 performance at the Aspen FACE site, individual performance and population -l evel effects of the



### 29 aphid C. betulaefoliae were assessed. Elevated O 3 levels had a strong positive effect on the

population growth rates of the aphids ; although effects were not detected by measuring growth,



### 31 development, adult weight, embryo number or birth weight of newborn nymphs (Awmack et al.,



### 32 2004, 052926 ) . Conversely, a lower rate of population growth was observed in aphids previously



### 33 exposed to O 3 in an OTC (Menendez et al., 2010, 384046 ) . No direct effects of O 3 were observed ;



### 34 howe ver

, nymphs born from adults exposed to and feeding on O 3 exposed plants were less capable of

infesting new plants when compared to nymphs in the control plots (Menendez et al. , 2010, 384046 )

. 

### 36 Elevated O 3 reduced arthropod abundance by 17% at Aspen FACE, largely as a result of the negative



### 37 effects on parasitoids, although phloem -f eeding insects may benefit (Hillstrom and Lindroth, 2008,

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### 1 191367 ) . Herbivore communities effected by O 3 and N were sampled along an air pollution gradient



### 2 in the Los Angeles basin (Jones and Paine, 2006, 191301 ) . Abundance, diversity, and ric hness of



### 3 herbivores were not affected. However, a shift in community structure

, from phloem

- feeding to



### 4 chewing dominated communities

, was observed along the gradient. No consistent effect of elevated



### 5 O 3 on herbivory or insect population size was detected a t SoyFACE (Dermody et al., 2008, 191850 ).



### 6 Evidence of modification of insect populations and communities in response t o elevated O 3



### 7 includes genotypic and phenotypic changes. In a study conducted at the Aspen FACE site, elevated



### 8 O 3 altered the genotype frequencies of the pea aphid ( Acyrthosiphon pisum ) grown on red clover



### 9 ( Trifolium pratense ) over multiple generations (Mondor et al., 2005, 191217 ) . Aphid color was used



### 10 to distinguish between the two genotypes . Ozone increased the genotypic frequencies of



### 11 pink

- mo rph:green

- mo rph aphids from 2:1 to 9:1, and depressed wing

- induction responses more



### 12 strongly in the pink than the green genotype (Mondor et al., 2005, 191217 ) . Growth and



### 13 development of individual green and pink aphids reared as a single genotype or mixed genotypes



### 14 were un affected by elevated O 3 (Mondor et al., 2010, 644271 ) . Furthermore, growth of pea aph id



### 15 populations is not readily predictable using individual growth rates.



## 9.6.6.2. Wildlife

Herpatofauna

16 Since the 2006 O 3 AQCD , direct effects of O 3 exposure including physiological changes and



### 17 alterations of ecologically important behaviors such as feeding and th ermoregulation have been



### 18 observed in wildlife. These studies have been conducted in limited laboratory exposures, and the



### 19 levels of O 3 treatment (e.g. 0.2

- 0.8 ppm ) were often unrealistically higher than the ambient levels.



### 20 Amphibians may be especially vuln erable to airborne oxidants due to the significant gas exchange



### 21 that occurs across the skin (Andrews et al., 2008, 645771 ) (Dohm et al., 2008, 604584 ) . Exposure to



### 22 0.2 ppm to 0.8 ppm O 3 for 4 h resulted in a decrease of oxygen consumption and depressed lung



### 23 ventilation in the California tree frog Pseudacris cadaverina (Mautz and Dohm, 2004, 644188 )

. 

### 24 Following a s ingle 4 -h exposure to O 3 , reduced pulmonary macrophage phagocytosis was observed



### 25 at 1 and 24 hour s postexposure in the marine toad ( Bufo marinus ) indicating an effect on immune



### 26 system function (Dohm et al., 2005, 180452 ) . There was no difference in macrophage function at



### 27 48 hour s postexposure in exposed and control individuals.



### 28 Behavioral effects of O 3 observed in amphibians include responses to minimize the surface



### 29 area of the body exposed to the air and a decrease in feeding rates (Dohm et al., 2008, 604584 ;



### 30 Mautz and Dohm, 2004, 644188 ) . The adoption of a low

- profile “water conservation posture” during



### 31 O 3 exposure was observed in experiments with the California tree frog (Mautz and Dohm, 2004,



### 32 644188 ) . Toads, Bufo marinus , e xposed to 0.06 μL /L O 3 for 4 hour s ate significantly fewer



### 33 mealworms at 1 h our and 48 h ours postexposure than control toads (Dohm et al., 2008, 604584 ) . In



### 34 the same study, escape/exploratory behavior as measured by total distance moved was not adversely



### 35 affected in the O 3 - exposed individuals as compared to the controls (Dohm et al., 2008, 604584 )

.

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1 Water balance and thermal preference in herpatofauna are altered with elevated O 3 . Toads



### 2 exposed to 0.8 ppm O 3 for 4 hours exhibited behavioral hypothermia when preferred body



### 3 temperatures were measured at 1, 24 and 48 hours postexposure (Dohm et al., 2001, 017086 )

. 

### 4 Ozone

- exposed individuals lost almost 5g more body mass on average than controls due to

evaporative water loss. At 24 hours after exposure, the individuals that had lost significant body



### 6 mass had lower preferred body temperatures (Dohm et al., 2001, 017086 ) . Behavioral hypothermia



### 7 was also observed in reptiles following 4

- h exposures to 0.6 ppm O 3 . Exposure of the Western Fence



### 8 Lizard ( Sceloporus occidentalis ) at 25 o C induced behavioral hypothermia that recovered to control



### 9 temperatures by 24 hours (Mautz and Dohm, 2004, 644188 ) . The behavioral hypothermic response

persisted in lizards exposed to O 3 at 35 o C at 24 hours postexposure resulting in a mean body



### 11 temperature 3.3 o C over controls.



### Soil Fauna Communities

12 Ozone has also been shown to alter soil fauna communities (Kasurinen et al., 2007, 199826 ;



### 13 Loranger et al., 2004, 072712 ; Meehan et al., 2010, 644184 ) . Abundance of Acari (mites and ticks)



### 14 decreased by 47% under elevated O 3 at AspenFACE site, probably due to the higher secondary

metabolites and lower N concentrations in litter and foliage under elevated O 3 (Loranger et al., 2004,



### 16 072712 ) . In another study from the AspenFACE site, leaf litter collected from aspen grown under



### 17 elevated O 3 conditions were higher in fiber and lignin concentrations than trees grown under ambient



### 18 conditions. These chemical characteristics of the leaves were associated with an increased springtail



### 19 population growth following 10 weeks in a laboratory microcosm (Meehan et al., 2010, 644184 )

. Consumption rates of earthworms fed on leaf litter for 6 weeks from trees grown under elevated O 3



### 21 conditions and ambient air did not vary significantly between treatments (Meehan et al., 2010,



### 22 644184 ) . In another study on juvenile earthworms Lumbricus terrestris , individual growth was



### 23 reduced when worm s were fed high -O 3 birch litter from trees exposed for three years to elevated O 3



### 24 in an OTC system (Kasurinen et al., 2007, 199826 ) . In the same study no significant growth or

mortality effects were observed in isopods.



## 9.6.6.3. Indire c t Effe c ts on Wildlife

26 In addition to the direct effects of O 3 exposure on physiological and behavioral endpoints



### 27 observed in the laboratory, there are indirect effects to wildlife. These effects include changes in



### 28 biomass and nutritive quality of O 3 - exposed plants (reviewed in Section 9.5) that are consumed by



### 29 wildlife. Reduced digestibility of O 3 - exposed plants may alter dietary intake and foraging strategies

in herbivores. In a study using native highbush blackberry ( Rubus argutus ) relative feed value of the



### 31 plants decreased in bushes exposed to double ambient concentrations of O 3 (Ditchkoff et al., 2009,



### 32 192230 ) . Indirect effects of elevated O 3 on wildlife include changes in chemical signaling impor tant



### 33 in ecological interactions reviewed below.

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### Chemical Signaling in Ecological Interactions

1 Ozone has been shown to degrade or alter biogenic VOC signals important to ecological



### 2 interactions including; ( 1) attraction of pollinators and seed dispersers ; ( 2) defense against herbivory ;



### 3 and ( 3) predator

- prey interactions (McFrederick et al., 2009, 191329 ; Pinto et al., 2007, 196949 ;



### 4 Pinto et al., 2007, 196950 ; Pinto et al., 2010, 596490 ; Yuan et al ., 2009, 199779 ) . Each signal

released by emitters has an atmospheric lifetime and a unique chemical signature comprised of



### 6 different ratios of individual h ydrocarbons that is susceptible to atmospheric oxidants such as O 3



### 7 (Wright et al., 2005, 626463 ; Yuan et al., 2009, 199779 ) . Under elevated O 3 conditions, these



### 8 olfactory cues may travel shorter distances befor e losing their specificity (McFrederick et al., 2008,



### 9 196931 ; McFrederick et al., 2009, 191329 ) . Additional non

- phytogenic VOC

- mediated

interrelationships with the potential to be modified by O 3 include territorial marking, pheromones for



### 11 attraction of mates and various social interactions includi ng scent trails, nestmate recognition and



### 12 signals involved in aggregation behaviors (McFrederick et al., 2009, 191329 ) . In general, effects of



### 13 O 3 on scent

- mediated ecological interactions are highly context

- and species

- specific (Bidart -Bouzat



### 14 and Imeh

- Nathaniel, 2008, 191431 ; Lindroth, 2010, 596479 ).



### Pollination and Seed Dispersal

Phytogenic VOC’s attract pollinators and seed dispersers to fl owers and fruits (Dudareva et al.,



### 16 2006, 626458 ; Theis and Raguso, 2005, 626461 ) . These floral scent trails in plant

- insect interactions



### 17 may be destroyed or transformed by O 3 (McFrederick et al., 2008, 196931 ) . Using a Lagrangian



### 18 model, the rate of destruction of phytogenic VOC’s was estimated in air parcels at increasing



### 19 distance from a source in response to increased regional levels of O 3 , hydroxyl and nitrate radicals

(McFrederick et al., 2008, 196931 ) . Based on the model, the ability of po llinato rs to locate highly



### 21 reactive VOC s from emitting flowers may have decreased from kilometers during pre

- industrial



### 22 times to <200 m at current ambient conditions (McFrederick et al., 2008, 196931 ) . Scents that travel



### 23 shorter distances (0

- 10 m) are less susceptible to air pollutants, while highly reactive scents that



### 24 travel longer distances (10 to 100’s of meters), are at a higher risk for degradation (McFrederick et

al., 2009, 191329 ) . For example, male euglossine bees can detect bait stations from a di stance of at



### 26 least one kilometer (Dobson, 1994, 626466 ) . The alcohols, ketones and aldehydes comprising sex



### 27 pheromones in moths could be especially vulnerable to degradation by O 3 , since some males travel



### 28 >100 m to find mates (Carde and Haynes, 2004, 626467 ).



### Defense Against Herbivory

29 Ozone can alter the chemical signature of VOCs emitted by plants and these VOCs are

su bsequently detected by herbivores (Blande et al., 2010, 643928 ; Cannon, 1990, 626460 ; Iriti and



### 31 Faoro, 2009, 199313 ; Jackson et al., 1999, 026877 ; Pin to et al., 2007, 196949 ; Vuorinen et al., 2004,



### 32 626462 )

. These modifications can make the plant either more attractive or repellant to phytophagous



### 33 insects (Pinto et al., 2010, 596490 ) . For example, under elevated O 3 , the host plant preference by



### 34 forest tent caterpillars increased for birch compared to aspen (Agrell et al., 2005, 074324 ) . Ozone

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1 induced emissions from red spruce needles were found to repel spruce budworm larvae (Cannon,



### 2 1990, 626460 ) . Transcriptional profiles of field grown soybean ( Glycine max ) grown in elevated O 3



### 3 conditions were altered due to herbivory by Japanese beetles. The herbivory resulted in a higher



### 4 number of transcripts in the leaves of O 3 - exposed plants and up

- regulation of antioxidant metabolism

associated with plant defense (Casteel et al., 2 008, 191696 )

. 

### 6 Ozone may modify signals involved in plant

- to

- plant interactions and plant defense against



### 7 pathogens (Blande et al., 2010, 643928 ; McFrederick et al., 2009, 191329 ; Pinto et al., 2010,



### 8 596490 ; Yuan et al., 2009, 199779 ) . In a recent study with lima beans, 80 ppb O 3 degraded several



### 9 herbivore

- induced VOC’s, reducing the distance over which plant

- to

- plant signaling occurred

(Blande et al., 2010, 643928 ).



### Predator

- Prey Interactions

11 Elevated O 3 conditions are associated with disruption of pheromone

- mediated interactions at



### 12 higher trophic levels (e.g., predators and parasitoids of herbivores). In a study from the Aspen FACE



### 13 site, predator escape behaviors of the aphid ( Chatophorus stevensis ) were enhanced on O 3 -fumigated



### 14 aspen trees although the mechanism of this response remains unknown (Mondor et al., 2004,

074334 ) . The predatory mite Phytoseiulus persimilis can distinguish between the VOC signature of



### 16 ozonated l ima bean plants and ozonated plants simultaneously damaged by T. urticae (Vuorine n et



### 17 al., 2004, 626462 ) however, other tritrophic interactions have shown no effect (Pinto et al. , 2007,



### 18 196950 )

. 

### 19 There are few studies that consider host location behaviors of parasites under elevated O 3 . In

closed chambers fumigated with O 3 , the sear ching efficiency and proportion of the host larval fruit



### 21 flies parasitized by Asobara tabida , declined when compared to filtered air controls (Gate et al.,



### 22 1995, 026655 ) . The host loca tion behavior and rate of parasitism of the wasp ( Coesia plutellae ) on



### 23 Plutella xylostella

- infested potted cabbage plants was tested under ambient and doubled O 3



### 24 conditions in an open

- air fumigation system (Pinto et al., 2008, 616554 ) . The number of wasps

found in the field and the percentages of parasitized larvae were not significantly different from



### 26 controls under elevated O 3

. 

### 27 Elevated O 3 has the potential to perturb specialized food

- web communication in transgenic



### 28 crops. In insect

- resistant oilseed rape Brassica napus grown under 100 ppb O 3 in a growth chamber,



### 29 reduced feeding damage by Putella xylostella led to deceased attraction of the endoparasitoid

( Costesia vestalis ) (Himanen et al., 2009, 191338 ) . Under chronic O 3 - exposure, the insect resistance



### 31 trait BT cry1Ac in transgenic B. napus was higher than the control (Himanen et al., 2009, 191369 )

. 

### 32 There was a negative relative growth rate of the Bt target herbivore, P. xylostella

, in all O 3



### 33 treatments.

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# 9.7. Effects

- Ba s e d Air Qu a lity Exp o s u re In d i ces and Dos e Mo d e lin g

9.7.1. Introd uc tion

1 Exposure indices are metrics that quantify exposure as it relates to measured plant damage



### 2 (i.e., reduced growth). They are summary measures of monitored ambient O 3 concentrations over



### 3 time

, intended to provide a consistent metric for reviewing and comparing exposure

- response effects



### 4 obtained from various studies. Such indices may also provide a basis for developing a biologically


### 5 relevant air quality standard for protecting vegetation and ecosystems. Effects on plant grow th and/or



### 6 yield have been a major focus of the characterization of O 3 impacts on plants for purposes of the air



### 7 quality standard setting process (U.S. EPA, 1986, 017607 ) (U.S. EPA, 1996, 039046 ) (U.S. EPA,



### 8 2007, 090207 ) . The quantitative characterization of the relationship of O 3 and pla nt responses has



### 9 been referred to as “dose

- response” and “exposure

- response” alternatively. The distinction is in how



### 10 the pollutant concentration is expressed: “dose” is the pollutant concentration absorbed by the leaf



### 11 over some time period, and is very di fficult to measure directly, whereas “exposure” is the ambient



### 12 air concentration measured near the plant over some time period, and summarized for that period



### 13 using an index. Exposure indices have been most useful in considering the form of secondary O 3



### 14 NA AQS standard, in large part because they only require ambient air quality data rather than more



### 15 complex indirect calculations of dose to the plant. The attributes of exposure indices that most



### 16 require consideration are the weighting of O 3 concentrations, a nd the daily and seasonal time

- periods



### 17 that are most relevant to plant damage. Several different types of exposure indices are discussed in



### 18 Section 9.7.2.



### 19 Theoretically, a measure of plant O 3 uptake or dose from ambient air (either rate of uptake or



### 20 cumula tive seasonal uptake) might be a more ideal predictor of O 3 damage to plants than an



### 21 exposure index and may be more useful in improving risk assessment. An uptake measure would



### 22 have to integrate all those environmental factors that influence stomatal conductance, including but



### 23 not limited to temperature, humidity, and soil water status (Section 9.7.4). Even when integrating



### 24 those environmental factors, a direct measure of the internal leaf concentration of O 3 , however, is



### 25 technically difficult. Therefore, u ptake values are generally obtained with simulation models that



### 26 require knowledge of species

- and site

- specific values for the variables mentioned. In addition, it has



### 27 also been recognized that O 3 detoxification processes and the temporal dynamics of detox ification



### 28 must be taken into account in dose modeling (Heath et al., 2009, 196783 ) (Section 9.7.4). Because of



### 29 this, research has focused historically on predictors of O 3 damage to plants based only on exposure



### 30 as a summary measure of monitored ambient pollutant concentration over some integral of time,



### 31 rather than dose (Lee et al., 1988, 042136 ) (Lefohn and Benedict, 1982, 039395 ) (O'Gara, 1922,



### 32 015018 ) (U.S. EPA, 1986, 017607 ) (U.S. EPA, 1992, 042599 ) (U.S. EPA, 1996, 080828 ).

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## 9.7.2. De s cription o f Exp o s u re Indice s Availa b le in the Lite ratu re

Mathematical approaches for summarizing ambient air quality information in biologically

meaningful forms for O 3 vegetation effects assessment purposes have been explored for more than

80 years (O'Gara, 1922, 015018 ) (U.S. EPA, 1996, 080827 ) . In the context of broad

- scale national

NAAQS standards that protect for "known or anticipated" effects on many plant species in a variety

of habitats, exposure indices provide a numerical summary of very large numbers of ambient

observations of concentration over extended periods. Like any summary statistic, exposure indices

retain information on some characteristics of the original observations, at the exclusion of others. It

is expected that indi ces that incorporate the characteristics that are most relevant to plant response

will lead to more reliable protection. Several indices have attempted to incorporate some of the

biological, environmental, and exposure factors (directly or indirectly) that influence the magnitude

of the biological response and contribute to observed variability (Hogsett et al., 1988, 042128 ) . In

the 1996 O 3 AQCD (U.S. EPA, 1996, 080828 ) , the exposure indices were arranged into five

categories; (1) One e vent, (2) Mean, (3) Cumulative, (4) Concentration w eighted, and (5)

Multicomponent, and were discussed in detail (Lee et al., 1989, 042137 ) . Figure 9

- 8 illustrates how

several of the indices weight concentration and accumulate exposure.

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### 1

Source: Used with permission from Air and Waste Manage ment Association, Tingey et al. (1991, 042623 )



### Figure 9

- 8. Diagrammatic representation of several exposure indices, illustrating how they (a) SUM06: the upper graphic ﷒ weight concentration and accumulate exposure. 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 to 1) function. The lower portion of the graphic illustrates how concentration is

accu mulated 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 sigm oid concentration weighting function. This is similar to the W126 function. The lower portion of the graphic illustrates how concentration is accumulated over the exposure period. (c) seco nd HDM and M

- 7: the upper graphic illustrates an episodic exposure p rofile. The lower portion of the graphic illustrates

that the seco nd HDM considers only a single exposure peak, while the M

- 7 (average
of 7

- h daily means) applies a constant exposure value over the exposure period.

2 Various components of the exposure

- respon se relationship, including concentration, time of



### 3 day, respite time, frequency of peak occurrence, plant phenology, predisposition, etc., were weighted



### 4 with various functions and evaluated on their ability in ordering the regression of exposure versus



### 5 grow th or yield response (Lee et al., 1989, 042137 ) . The statistical evalua tions for each of these



### 6 indices were completed using growth or yield response data from many earlier exposure studies



### 7 (e.g., NCLAN). This retrospective approach was necessary because there were no studies



### 8 specifically designed to test the goodness of fit of the various indices. The goodness of fit of a set of



### 9 linear and nonlinear models for exposure

- response was ranked as various proposed indices were



### 10 used in turn to quantify exposure. This approach provided evidence for the best indices.

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39 Most of the early retrospective studies reporting regression approaches used data from the

NCLAN program or data from Corvallis, Oregon or California (Lee et al., 1987, 042135 ) (Lee et al.,

1988, 042136 ) (Lefohn et al., 1988, 042138 ) (Musselman et al., 1988, 042144 ) (U.S. EPA, 1992,

042599 ) (U.S. EPA, 1986, 017607 ) . These studies were previously reviewed by the EPA (U.S. EPA,

1992, 042599 ) (U.S. EPA, 1996, 080828 ) and were in general agreement that the best fit of the data

were cumulative concentration

- weighted exposure indices. Lee et al. (1987, 042135 ) suggested that

exposure indices that included all the 24

- h data performed better than those that used only 7 hour s of

data; this was consistent with the conclus ions of Heagle et al. (1987, 042124 ) that plants receiving

exposures for an additional 5 h/day showed 10% greater yield loss than those exposed for 7 h/day. In

an earlier analysis using the National Crop Loss Assessment Network (NCLAN) data, Lee et al.

(1988, 042138 ) found the “best” exposure index was a phenologically weighted cumulative index,

with sigmoid weighting on concentration and a gamma weighting function as a surrogate for plant

growth stage. This index provided the best statistical fit when used in the models under

consideration, but it required data on species and site conditions

, making specification of weighting

functions difficult for general use. The next best fits were the several indices which only cumulated

and weighted higher co ncentrations (e.g., W126, SUM06, SUM08, and AOT40). Amongst this group

no index lead to consistently better fits across all studies and species (Heagle et al., 1994,

042656 ) (Lefohn et al., 1988, 042138 ) (Musselman et al., 1988, 042144 ).

Other factors, including predisposition time (Hogsett et al., 1988, 042128 ) (McCool et al.,

1988, 041877 ) and crop development stage (Heagle et al., 1991, 042530 ) (Tingey et al., 2002,

040896 ) contributed to variation in the biological response and suggested the need for weighting O 3

concentrations to a ccount for predisposition time and phenology. However, the roles of

predisposition and phenology in plant response vary considerably with species and environmental

conditions; therefore, specification of a weighting function for general use in characterizi ng plant

exposure was not possible.

European scientists took a similar approach in developing indices describing growth and yield

loss in crops and tree seedlings, using OTCs with modified ambient exposures, but many fewer

species and study locations were employed in the European studies. There is evidence from some

European studies that a lower (Pleijel et al., 1997, 04374 7 ) or higher (Finnan et al., 1996,

042545 ) (Finnan et al., 1997, 083315 ) cutoff value in indices with a threshold may provide a better

statistical fit to the experimental data. Finnan et al. (1997, 083315 ) used seven exposure studies of

spring wheat to confirm that cumulative exposure indices emphasizing higher O 3 concentrations

were best related to plant response and that cumulative exposure indices using weighting functions,

including cutoff concentrations, allometric and sigmoidal, provided a better fit and that the ranking

of these indices differed depending on the exposure

- response model used. Weighting those

concentrations associated with sunshine hours in an attempt to incorporate an element of plant

uptake did not improve the index performance (Finnan et al., 1997, 083315 ) . A more recent study

using data from several European studies of Norway sp ruce, analyzed the relationship between

relative biomass accumulation and several cumulative, weighted indices, including the AOT40 and

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### 1 the SUM06 (Skarby et al., 2004, 080375 ) . All the indices performed relatively well in regressing



### 2 biomass and exposure index, with the AOT20 and AOT30 doing slightly better than others (r 2
=



### 3 0.46- 0.47 ). In another comparative study o f four independent data sets of potato yield and different



### 4 cumulative uptake indices with different cutoff values, a similarly narrow range of r 2 was observed



### 5 (r 2 = 0.3

- 0.4) (Pleijel et al., 2004, 036662 ).



### 6 In both the U.S. and Europe, the adequacy of these numerical summaries of exposure in



### 7 relating biomass and yield changes have, for the most part, all been evaluated using data from



### 8 studies not necessarily designed to compare one index to another (Lefohn et al., 1988, 042138 ) (Lee



### 9 et al., 1989, 042137 ) (Skarby et al., 2004, 080375 ) . Very few studies in the U.S. have addressed this



### 10 issue since the 2006 O 3 AQCD. McLaughlin et al. (2007, 090348 ) reported that the cumulative



### 11 exposure index of AOT60 related well t o reductions in growth rates at forest sites in the southern



### 12 Appalachian Mountains. However, the authors did not report an analysis to compare multiple



### 13 indices. Overall, given the available data from previous O 3 AQCDs and the few recent studies, the



### 14 cumula tive, concentration

- weighted indices perform better than the peak or mean indices. It is still



### 15 not possible, however, to distinguish the differences in performance among the cumulative,



### 16 concentration

- weighted indices.



### 17 The main conclusions from the 1996 and 2006 O 3 AQCDs (U.S. EPA, 1996,



### 18 080828 ) (U.S

. EPA, 2006, 088089 ) regarding an index based on ambient exposure are still valid. No



### 19 information has come forth since the 2006 O 3 AQCD to alter those conclusions significantly. These



### 20 key conclusions can be restated as follows:



### 21  O 3 effects in plants are cumulative;



### 22  higher O 3 concentrations appear to be more important than lower concentrations in



### 23 eliciting a response;



### 24  plant sensitivity to O 3 varies with time of day and plant development stage; and



### 25  exposure indices that accumulate the O 3 hourly concentrations and preferentially weight



### 26 the higher concentrations have better statistical fits to growth/yield response than do the



### 27 mean and peak indices.



### 28 Following the 20 06 criteria review process (U.S. EPA, 2006, 088089 ) , the EPA proposed an



### 29 alternative form of the secondary NAAQS for O 3 using a cumulative, concentration -weighted



### 30 exposure index to protect vegetation from damage (72 FR 37818 (2007, 684055 ) , 75 FR 2938 (2010,



### 31 684211 ) , p. 3003 ). The EPA considered two specific concentration

- weighted indices: the cutoff



### 32 concentration weighted SUM06 and the sigmoid

- weighted W126 exposure index (U.S. EPA, 2007,



### 33 090207 ) . These two indices performed equally well in predicting the exposure -r esponse relationships



### 34 observed in the crop and tree seedlings studies (Lee et al., 1989, 042137 ) . At a workshop convened

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### 1 to consider the science supporting these indices (Heck and Cowling, 1997, 084484 ) the participants



### 2 agreed that these cumulative concentration

- weigh ted indices being considered were equally capable



### 3 of predicting plant response. A short

- term daily 8

- h avg was also proposed in the review that ended



### 4 in 2008 (73 FR 16436

, (2008, 684051 ) ), but at the t ime there were no scientific studies supporting



### 5 the utility of this index for vegetation. Below are the definitions of the three index forms considered



### 6 in the previous staff paper review (U.S. EPA, 2007, 090207 ):



### 7  8-h average form : 4th

- highest daily max 8 -h avg over the O 3 season.



### 8  SUM06: Sum of all hourly O 3 concentrations greater than or equal to 0.06 ppm observed



### 9 during a specified daily and seasonal time window (Figure 9

- 8a).



### 10  W126: Sigmoidally weighted sum of all hourly O 3 concentrations observed during a



### 11 specified daily and seasonal time window ( Similar to Figure 9

- 8b). The sigmoidal



### 12 weighting of hourly O 3 concentration is given in the equation below, where C is the



### 13 hourly O 3 concentration in ppm:

C c 

## e w 126 

## 44031 1

**−** 

## + **=**

Equation 9 -1



### 14 The SUM06 and W126 indices have a variety of relevant time windows that may be applied and a re



### 15 discussed in Section 9.7.3.



### 16 Other exposure indices are discussed in the literature and are currently used outside the U.S. In



### 17 Europe, the cutoff concentration

- weighted index AOT40 was selected in developing exposure



### 18 response relationships based on OTC s tudies of a limited number of crops and trees (Grunhage and



### 19 Jager, 2003, 052972 ) . The United Nations Economic Commission for Europe (United Nations



### 20 Economic Commission for Europe UNECE, 1988, 055354 ) adopted the critical levels approach for



### 21 assessment of O 3 risk to vegetation across Europe. As used by the UNECE, the critical levels are not



### 22 like the air quality regulatory standards used in the U.S., but rather planning targets for reductio ns in



### 23 pollutant emissions to protect ecological resources. Critical levels for O 3 are intended to prevent



### 24 long

- term deleterious effects on the most sensitive plant species under the most sensitive



### 25 environmental conditions, but not intended to quantify O 3 e ffects. A critical level was defined as “the



### 26 concentration of pollutant in the atmosphere above which direct adverse effects on receptors, such as



### 27 plants, ecosystems, or materials may occur according to present knowledge” (United Nations



### 28 Economic Commission for Europe UNECE, 1988, 055354 ) . The nature of the “adverse effects” was



### 29 not specified in the original definition, which provided for different levels for different types of



### 30 harmful effect (e.g., visible injury or loss of crop yield). There are also different critical levels for



### 31 crops, forests, and semi

- natural vegetation. The caveat, “according to present knowle dge” is

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1 important because critical levels are not rigid; they are revised periodically as new scientific



### 2 information becomes available. For example, the original critical level for O 3 specified



### 3 concentrations for three averaging times, but further research and debate led to the current critical



### 4 level being stated as the cumulative exposure (concentration × hours) over a cutoff concentration of

40 ppb (AOT40) (Fuhrer et al., 1997, 030380 )

. 

### 6 In Europe, a decision wa s made to work towards a flux

- based approach for the critical levels



### 7 (“Level II”), with the goal of modeling O 3 flux

- effect relationships for three vegetation types: crops,



### 8 forests, and semi

- natural vegetation (Grunhage and Jager, 2003, 052972 ) . Progress has been made in



### 9 modeling flux (see section AX9.4.5; U.S. EPA, 2006, 088089 ) and the Mapping Manual is being

revised (Ashmore et al., 2004, 056623 ) (Ashmore MEmberson et al., 2004, 056624 ) (Grennfelt, 2004,



### 11 056625 ) (Karlsson et al., 2003, 055331 ) . The revisions may include a flux

- based approach for three



### 12 crops: wheat, potatoes, and cotton. However, because of a lack of flux

- response data, a cumulative,



### 13 cutoff concentration

- based (AOTx) exposure index will remain in use for the near future for most



### 14 crops and for forests and semi

- natural herbaceous vegetation (As hmore et al., 2004, 056623 ).



## 9.7.3. Impo rta n t Co mp o ne nts o f Expo s ure In dice s

The efficacy of exposure indices in predicting biological responses requires that rese archers



### 16 identify a relationship between measured growth and/or yield effects and important components of



### 17 exposure indices. In the previous O 3 AQCDs it was established that higher hourly concentrations



### 18 have greater effects on vegetation than lower concentra tions (U.S. EPA, 1996, 080828 ) (U.S. EPA,



### 19 200 6, 088089 ) . Further, it was determined that the diurnal and seasonal duration of exposure is

important for plant response. Weighting of hourly concentrations and the diurnal and seasonal time



### 21 window of exposure are the most important variables in a cumulative exposure index and will be



### 22 discussed below. However, these variables must be taken in the context of plant phenology, diurnal



### 23 conductance rates, plant ca nopy structure, and detoxification mechanisms of vegetation as well as



### 24 the climate and meteorology, all of which are determinants of plant response. These more specific

factors will be discussed in the uptake and dose modeling section (Section 9.7.4).



## 9.7.3.1. Rol e of Co nc e n tra tio n

26 The significant role of higher O 3 concentrations was established based on several experimental



### 27 studies (U.S. EPA, 1996, 080828 ) . Several studies (Nussbaum et al., 1995, 030141 ) (Oksanen and



### 28 Holopainen, 2001, 019538 ) (Yun and Laurence, 1999, 044165 ) have added support for the important



### 29 role that peak co ncentrations, as well as the pattern of occurrence, plays in plant response to O 3

. Oksanen and Holopainen (2001, 019538 ) found that the peak concentrations and the shape of the O 3



### 31 exposure (i.e., duration of the event) were important determinants of foliar injury in European white



### 32 birch saplings, but growth reductions were found to be more related to total cumulative exposure.



### 33 Based on air quality data from 10 U.S. c ities, three 4

- week exposure treatments having the same



### 34 SUM06 value were constructed by Yun and Laurence (1999, 044165 ) . The authors used differe nt

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during the exposure period. The authors reported that the variable peak exposures were important in

causing injury, and that the different exposure treatments, although having the same SUM06,

resulted in very different patterns of foliar injury. Nussbaum et al. (1995, 030141 ) also found peak

concentrat ions and the pattern of occurrence to be critical in determining the measured response. The

authors recommended that to describe the effect on total forage yield, peak concentrations

>0.11 ppm must be emphasized by using an AOT with higher threshold concen trations.

A greater role for higher concentrations affecting plant growth might be inferred based on air

quality analyses for the s outhern California area (Lee et al., 2003, 053031 ) (Tingey et al., 2004,

042385 ) . In the late 1960s and 1970s, extremely high O 3 concentrations had impacted the San

Bernardi no National Forest. However, over the past 20+ y ears , significant reductions in O 3 exposure

have occurred (Davidson, 1993, 043366 ) (Lee et al., 2003, 053031 ) (Lefohn and Shadwick, 2000,

040483 ) (Bytnerowicz et al., 2008, 196881 ) . An i llustration of this improvement in air quality is

shown by the 37

- year history of O 3 air quality at the Crestline site in the San Bernardino Mountains

(Figure 9

- 9) (Lee et al., 2003, 053031 ) . Ozone exposure increased from 1963 to 1979 concurrent

with increased population and vehicular miles, followed by a decline to the present mirroring

decreases in precursor emissions. The pattern in exposure was evident in various exposure indices

including the cumulative concentration weighted (SUM06), as well as maximum peak event (1 h

peak), and the number of days having hourly averaged O 3 concentrations greater than or equal to

95 ppb. The number of days having hourly averaged O 3 concentrations greater than or equal to

95 ppb declined significantly from 163 days in 1978 to 103 days in 1997. The changes in ambient O 3 air quality for the Crestline site were reflected in the changes in frequency and magnitude of the peak

hourly concentration and the duration of exposure (Figure 9

- 9). Considering the role of exposure

patterns in determining response, the seasonal and diurnal patterns in hourly O 3 concentration did not

vary appreciably from year to year over the 37

- year peri od (Lee et al., 2003, 053031 ).

The inference for a role of higher concentrations comes both from results of measures of tree

conditions on established plo ts and from results of model simulations. Across a broad area of the San

Bernardino National Forest, the Forest Pest Management (FPM) method of injury assessment

indicated an improvement in crown condition from 1974 to 1988; and the area of improvement in

injury assessment is coincident with an improvement in O 3 air quality (Miller and Rechel, 199 9,

040702 ) . A more recent analysis of forest changes in the San Bernardino National Forest using an

expanded network of monitoring sites has verified signifi cant changes in growth, mortality rates,

basal area, and species composition throughout the area since 1974 (Arbaugh et al., 2003, 052925 )

. A model simulation of ponderosa pine growth over the 40

- year period in the San Bernardino

National Forest showed a significant impact of O 3 exposure on tree growth and indicates improved

growth with improving O 3 air quality. This area has also experienced elevated N deposition and

based on a number of environmental indicators, it appears that this area is experiencing N saturation

(Fenn et al., 1996, 083540 ) . To account for this potential interaction, the model simulations were

conducted under conditions of unlimited soil N. The actual interactions are not known. The

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was made regarding the relative role of mid

- range and higher hourly concentrations, only that

improved growth tracked decreasing SUM06, maximum peak concentration, and number of days of

hourly O 3 > 95 ppb (Tingey et al., 2004, 042385 ) . A summary of air quality data from 1980 to 2000

for the San Bernardino National Forest area of the number of “m id

- range” hourly concentrations

indicated no dramatic changes over this 20

- year period, ranging from about 1

, 500 to 2

, 000 hours per

year (Figure 9

- 10). There was a slow increase in the number of mid

- range concentrations from 1980

to 1986, which corresponds to the period after implementation of the air quality standard. Another

sharper increase was observed in the late 1990s. This pattern of occurrence of mid

- range hourly

concentrations suggests a lesser role for these concentration ranges compared to the hi gher values in

either of the ground

- level tree injury observations of the model simulation of growth over the

40

- year period.

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### Figure 9

- 9. Trends in May to September 12

- h SUM06 , peak 1

- h ozone concentration and number of daily exceedances of 95 ppb for the Crestline site in 1963 to 1999 in relation to trends in mean daily maximum temperature for Crestline and daily reactive organic

Annual ROG ﷒ ) for San Bernardino C ounty

. X 

### gases (ROG) and oxides of nitrogen (NO
and NO X emissions data for San Bernardino County were obtained from Alexis et al. (2001, 079886 ) and the California Air Resource Board’s emission inventory available

at http://www.arb.ca.g ov/aqd/aqdpage.htm (Cal EPA, 2009, 677487 )

.

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### Figure 9

- 10. The number of hourly average concentrations between 50 and 89 ppb for the period

1980

- 2000 for the Crestline, San Bernardino County , CA, monitoring site.



## 9.7.3.2. Diurna l a n d Se a s o na l Exp os ure

Diurnal Exposure

1 The diurnal patterns of maximal leaf/needle conductance and occurrence of higher ambient



### 2 concentrations can help determine which hours during the day over a season should be cumulated.



### 3 Stomatal conductance is species and phenology dependent and is linked to both diurnal and seasonal



### 4 meteorological activity as well as to soil/site conditions (e.g., soil moisture). Daily patterns of



### 5 leaf/needle conductance are often highest in midmorning, whereas higher ambient O 3 concentrat ions



### 6 generally occurred in early to late afternoon when stomata were often partially closed and



### 7 conductances were lower. Total O 3 flux depends on atmospheric and boundary layer resistances, both



### 8 of which exhibit variability throughout the day. Experimental studies with tree species demonstrated



### 9 the decoupling of ambient O 3 exposure, peak occurrence, and gas exchange, particularly in areas of



### 10 drought (Panek, 2004, 079202 ) . Several studies have suggested that ponderosa pine trees in the



### 11 southern and northern Sierra Nevada Mountains may not be as susceptible to high O 3 concentrations



### 12 as to lower concentrations, due to reduced needle conductance and O 3 u ptake during the period when



### 13 the highest concentrations occur (Arbaugh et al., 1998, 040297 ) (Bauer et al., 2000, 040315 ) (Panek et



### 14 al., 2002, 040712 ) (Panek and Goldstein, 2001, 030190 ) . Panek et al. (2002, 040712 ) compared direct



### 15 O 3 flux measurements into a canopy of ponderosa pine and demonstrated a lack of correlation of



### 16 daily patterns of conductance and O 3 occurrence, especially in the late season drought period; the

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role of soil moisture and conductance/uptake. In contrast, Grulke et al. (2002, 035283 ) reported high

conductance when O 3 concentrations were high in the same species, but under different growing site

conditions. The decoupling of conductance and higher ambient O 3 concentration would hold true for

more me sic environments as well as xeric landscapes. The longer

- term biological responses reported

by Miller and Rechel (1999, 040702 ) for ponderosa pine in the same region, and the general

reduction in recent years in ambient O 3 concentrations, suggest that stomatal conductance alone may

not be a sufficient indicator of potential vegetation injury or damage. Another consideration for the

effect of O 3 uptake is the diurnal pattern of detoxification capacity of the plant. The detoxification

capacity may not fol low the same pattern as stomatal conductance (Heath et al., 2009, 196783 ).

A 12

- h (8 :00 a.m. to 8 :00 p.m. ) daylight period for a W126 cumulating exposure was proposed

in 2007 (72 FR 37818

, (2007, 684055 ) ) and 2010 (75 FR 2938, (2010, 684211 ) , p. 3003 ) following

the release of the 2006 O 3 AQCD, based primarily on evidence that the conditions for uptake of O 3 into the plant occur mainly during the daytime hours. In general, plants have the highest stomatal

conductance during the daytime and in many areas atmospheric turbulent mixing is greatest during

the day as well (U.S. EPA, 2006, 088089 ) (Uddling et al., 2010, 387073 ) . However, notable

exceptions to maximum daytime conductance are cacti and other plants with crassulacean acid

metabolism (CAM photosynthesis) which only open their stomata at night. This section will focus on

plants with C3 and C4 photosynthesis, w hich generally have maximum stomatal conductance during

the daytime.

Recent reviews of the literature reported that a large number of species had varying degrees of

nocturnal stomatal conductance (Caird et al., 2007, 199337 ) (Dawson et al., 2007,

670381 ) (Musselm an and Minnick, 2000, 011612 ) . This night

- time conductance can also be

enhanced by O 3 damage during the day that could result in loss of stomatal control

, an d less

complete closure of stomata

, than under low O 3 conditions (Grulke et al., 2007, 199376 ) . In general,

the rate o f stomatal conductance at night is much lower than during the day (Caird et al., 2007,

199337 ) . Atmospheric turbulence at night is also often low , which results in stable boundary layers

and unfavorable conditions for O 3 uptake into vegetation (Finkelstein et al., 2000, 024029 )

. Nevertheless, nocturnal turbulence does intermittently occur and may result in nonnegligible O 3 flux

into the plants. In addition, plants might be more susceptible to O 3 exposure at night than during the

daytime, because of potentially lower plant defenses (Musselm an and Minnick, 2000,

011612 ) (Musselman et al., 2006, 121678 ) (Loreto and Fares, 2007, 180259 ) (Heath et al., 2009,

196783 ) . For significant nocturnal stomatal flux and O 3 effects to occur, specific conditions must

exist. A suscep tible plant with nocturnal stomatal conductance and low defense must be growing in

an area with relatively high night

- time O 3 concentrations and appreciable nocturnal atmospheric

turbulence. It is unclear how many areas there are in the U.S. where these co nditions occur. It may

be possible that these conditions exist in mountainous areas of southern California, front

- range of

Colorado (Turnipseed et al., 2009, 588752 ) and the Great Smoky Mountains of North Carolina and

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21 Tennessee. More information is needed in these locations to assess the local O 3 patterns,

micrometeorology and responses of potentially vulnerable plant species.

Several field studies have attempted to quantify night

- time O 3 uptake with a variety of

methods. However, many of these studies have not linked the night

- time flux to measured effects on

plants. Grulke et al. (2004, 042646 ) showed that the stomatal conductance at night for ponderosa

pine in the San Bernardino National Forest (CA) ranged from one tenth to one fourth that of

maximum daytime stomatal conductance. In June, at a high

- elevation site, it was calculated that 11%

of the total daily O 3 uptake of pole

- sized trees occurred at night. In late summer, however, O 3 uptake

at night was negligible. However, thi s study did not consider the turbulent conditions at night.

Finklestein et al. (2000, 024029 ) investiga ted O 3 deposition velocity to forest canopies at three

different sites. The authors found the total flux (stomatal and non

- stomatal) to the canopy to be very

low during night

- time hours as compared to day

- time hours. However, the authors did note that

high er nocturnal deposition velocities at conifer sites may be due to some degree of stomatal opening

at night (Finkelstein et al., 2000, 024029 ) . Work by Mereu et al. (2009, 102168 ) in Italy on

m editerranean species indicated that nocturnal uptake was from 10 to 18% of total daily uptake

during a weak drought and up to 24% as the drought became more pronounced. The proportion of

night- time uptake was greater during the drought due to decreases in daytime stomatal conductance

(Mereu et al., 2009, 102168 ) . In a recent study at the AspenFACE site in Wisconsin, calculated leaf

level stomatal O 3 flux was near zero from the night

- time hours of 8 :00 p.m. to 5 :00 a.m. (Uddling et

al., 2010, 387073 ) . This was likely due to low horizontal wind speed (>1 m/s) and low O 3 concentrations (<25 ppb) during those same night

- time hours (Figure 9

- 11).

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### Figure 9

- 11. Mean diurnal

.

(a) conductance through boundary layer and stomata (g bs 

### ), (b) O zone concentration, and leaf

level stomatal ozone flux wi thout flux cut

- off threshold (Fst0 l 

### ) in control plots from

mid

- June through August in (c) 2004 and (d) 2005 in the Aspen FACE experiment. Subscripts “max” and “min” refer to stomatal fluxes calculated neglecting and accounting for potential non

- stomatal ozone flux, respectively.

1 A few studies have tested the biological effect s of night

- time O 3 exposure on vegetation in



### 2 controlled chambers. Biomass of ponderosa pine seedlings was significantly reduced when seedlings



### 3 were exposed to either daytime or nightti me episodic profiles (Lee and Hogsett, 1999, 040451 )

. 

### 4 However, the biomass reductions were much greater with daytime peak concentrations than with



### 5 nighttime peak concentrations. Similarly, birch cuttings grown in field chambers that were exposed



### 6 to O 3 at night only, daytime only

, and 24 hours showed similar reductions in biomass in night only



### 7 and day only treatments. Birch seedling showed greater reductions in growth in 2 4- h exposures than



### 8 those exposed to O 3 at night or day only (Matyssek et al., 1995, 040700 ) . Field mustard ( Brassica



### 9 rapa ) plants exposed to O 3 during the day or night showed little significant difference in the amounts



### 10 of injury or reduced growth response to O 3 treatment, although the stomatal conductance was



### 11 70

- 80% lower at night (Winner et al., 1989, 043403 ) . These studies show that effects can be seen

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1 with night

- time exposures to O 3 but if atmospheric conditions are stable at night, it is uncertain how



### 2 these exposures may af fect plants and trees with complex canopies in the field.



### Seasonal Exposure

3 Vegetation across the U.S

. has widely varying periods of physiological activity during the year



### 4 due to variability in climate and phenology. In order for a particular plant to be v ulnerable to O 3 pollution, it must have foliage and be physiologically active. Annual crops are typically grown for



### 6 periods of two to three months. In contrast, perennial species may be photosynthetically active



### 7 longer (up to 12 mon ths each year for some s pecies) depending on the species and where it is grown.



### 8 In general, the period of maximum physiological activity and thus, potential O 3 uptake for vegetation



### 9 coincides with some or all of the intra

- annual period defined as the O 3 season, which varies on a

state

- by

- state basis (Figure 3

- 18). This is because the high temperature and high light conditions that



### 11 typically promote the formation of tropospheric O 3 also promote physiological activity in vegetation.



### 12 There are very limited exceptions to this pattern where O 3 can form in the winter in areas in the



### 13 western U.S. with intense natural gas exploration (P into, 2009, 187038 ) , but this is typically when



### 14 plants are dormant and there is little chance of O 3 uptake. The selection of any single window of

time for a national standard to consider hourly O 3 concentrations represents a compromise, given the



### 16 significant variability in growth patterns and lengths of growing season among the wide range of



### 17 vegetation species that may experience adverse effects associated wi th O 3 exposure.



### 18 Various intra

- annual averaging and accumulation time periods have been considered for the



### 19 protection of vegetation. The 2010 proposal for secondary O 3 standard (75 FR 2938, (2010, 684 211 )

, p. 3003 ) proposed to use the maximum consecutive 3

- mo nth period within the O 3 season. The U.S.



### 21 Forest Service and federal land managers have used a 24

- h W126 accumulated for 6 mo nths from



### 22 April through September (see FLAG report; Federal land managers' air quality related values



### 23 workgroup (FLAG) phase I report, 2000, 088923 ) . However, some monitors in the U.S. are



### 24 operati onal for as little as four mon ths and would not have enough data for a 6- mo nth seasonal

window. The exposure period in the vast majority of O 3 exposure studies conducted in the U.S. has



### 26 been much shorter than 6 months. Most of the crop studies done through NCLAN had exposures less



### 27 than three mo nths with an average of 77 days. Open

- top chamber studies of tree seedlings

, compiled



### 28 by the EPA

, had an average exposure of just over three months or 99 days. In more recent FACE



### 29 experiments, SoyFACE exposed soybeans for an average of approximately 120 days per year and the

Aspen FACE experiment exposed trees to an average of approximately 145 days per year of elevated



### 31 O 3 , which included the entire growing season at those particular sites. Despite the possibility that



### 32 plants may be exposed to ambient O 3 longer than 3 mo nths in some locations, there is a lack of



### 33 exposure experiments conducted for longer than 3 mo nths.



### 34 In an analysis of the 3

- and 6

- mo nth max imum W126 values calculated for over 1

, 200 AQS

(Air Quality Sys tem) and CASTNET (Clean Air Status and Trend Network) EPA monitoring s ites for



### 36 the years 200 8- 2009

, it was found that these 2 accumulation periods resulted in highly correlated



### 37 metrics (Figure 9

- 12). The two cumulation periods were centered on the yearly m aximum for each

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7 monitoring site, and it is possible that this correlation would be weaker if the two periods were not

temporally aligned. In the U.S., W126 cumulated over 3 month s , and W126 cumulated over 6

months are proxies of one another, as long as the period in which daily W126 is accumulated

corresponds to the seasonal maximum. Therefore, it is expected that either statistic will predict

vegetation response equally well. In other words, the strength of the correlation between maximum

3- mo n th W126 and maximum 6

- mo n th W126 is such that there is no material difference in their

predictive value for vegetation response.

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100

90

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60

50

40

30

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0

0 10 20 30 40 50 60 70

Highest 3 month W126

A

110

100

90

80

70

60

50

40

30

20

10

0

0 10 20 30 40 50 60 70

Highest 3 month W126

B



### ﷒ Figure 9

- 12. Maximum 3

- mo n th , 12

- h W126 plotted against maximum 6

- mo n th , 12

- h W126. Data are from the AQS and CASTNET monitors f or the years 2008 and 2009. (A) W126,

3 mo nth versus 6 mo nth , 2008 (Pearson correlation = 0.99); (B) W126, 3 mo nth

versus 6 mo nth , 2009 (Pearson correlation = 0.99).



## 9.7.4. Ozon e Upta ke/Do s e Mo d eling for Ve g eta tio n

1 Another approach for improving risk assessment o f vegetation response to ambient O 3 is based



### 2 on estimating the O 3 concentration from the atmosphere that enters the leaf (i.e., flux or deposition).

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36 Interest has been increasing in recent years, particularly in Europe, in using mathematically tractable

flu x models for O 3 assessments at the regional, national, and European scale (Matyssek et al., 2008,

191262 ) (Paoletti and Manning, 2007, 180174 ) (Emberson et al., 2000, 040350 ) (Emberson et al.,

2000, 042537 ) (ICP M&M, 2004, 677471 ) . Some researchers have claimed that using fl ux models

can be used to better predict vegetation responses to O 3 than exposure

- based approaches (Matyssek

et al., 2008, 191262 ) . However, other research has suggested that flux models do not predict

vegetation responses to O 3 better than exposure

- based models, such as AOT40 (Gonzalez

- Fernandez

et al., 2010, 381357 ) . While some efforts have been made in the U.S. to calculate O 3 flux into leaves

and canopies (Grantz et al., 1997, 026664 ) (Grantz et al., 1995, 026659 ) (Grulke et al., 2004,

042646 ) (Turnipseed et al., 2009, 588752 ) (Uddling et al., 2009, 596219 ) (Bergweiler et al., 2008,

191656 ) (Hogg et al., 2007, 199349 ) , little information has been published relating these fluxes to

effects on vegetation. The lack of flux data in the U.S. and the lack of understanding of

detoxification processes have made this technique less viable for vulnerability and risk assessments

in the U.S.

Flux calculations are data intensive and must be carefully implemented. Reducing

uncertainties in flux estimates for areas with diverse surfac e or terrain conditions to within ±50%

requires “very careful application of dry deposition models, some model development, and support

by experimental observations” (Wesely and Hicks, 2000, 025018 ) . As an example, the annual

average deposition velocity of O 3 among three nearby sites in similar vegetation was found to vary

by ±10%, presumably due to terrain (Brook et al., 1997, 041857 ) . Moreover, the authors stated that

the actual variation was even greater, because stomatal uptake was unrealistically assumed to be the

same among all sites, and flux is strongly influence d by stomatal conductance (Brook et al., 1997,

041857 ) . This uptake

- based approach to quantify the vegetation impact of O 3 requires inclusion of

those factors that control the diurnal and seasonal O 3 flux to vegetation (e.g., climate patterns,

species and/or vegetation

- type factors and site

- specific factors). The models have to distinguish

between stomatal and non

- stomatal components of O 3 deposition to adequately estimate actual

concentration reaching the target tissue of a plant to elicit a response (Uddling et al., 2009, 596219 )

. Determining this O 3 uptake via canopy and stomatal conductance by necessity relies on models to

predict flux and ultimately the “effective” flux (Grunhage et al., 2004, 056621 ) (Massman et al.,

2000, 01161 6 ) (Massman , 2004, 055350 ) . “Effective flux” has been defined as the balance between

O 3 flux and detoxification processes (Dammgen et al., 1993, 055312 ) (Grunhage and Haenel, 1997,

040392 ) (Musselman and Massman, 1999, 040706 ) (Heath et al., 2009, 196783 ) . The time -integrated

“effective flux” is termed “effective dose.” The uptake mechanisms and the resistances in this

process, including stomatal conductance and biochemical defense mechanisms, are d iscussed below.

The flux

- based index is the goal for the “Level II” critical level for assessment of O 3 risk to

vegetation and ecosystems across Europe (Ashmore MEmberson et al., 2004, 056624 ).

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## 9.7.4.1. Ca nopy Structure

1 A factor important in both O 3 exposure and uptake is how canopy structure affects O 3



### 2 concentration in and under forest canopies. There have been several investigations of O 3



### 3 concentrations under tree canopies (Enders, 1992, 040355 ) (Fontan et al., 1992,



### 4 040370 ) (Fredericksen et al., 1995, 038898 ) (Joss and Graber, 1996, 040408 ) (Kolb et al., 1997,

052597 ) (Lorenzini and Na li, 1995, 040691 ) (Neufeld et al., 1992, 038971 ) (Sam uelson and Kelly,



### 6 1997, 040832 ) . In general, they indicated a reduction in O 3 of ~20 to 40% in the area below the



### 7 canopy but above the shrub/herb layers. An essential component in the determination of the AOT40



### 8 as a critical level was the height at which the O 3 concentration was measured. The measurement



### 9 heights are related to the O 3 concentration measured at the top of the canopy, i.e., upper surface

boundary of the (quasi

- ) laminar layer (Grunhage and Jager, 2003, 052972 ) . This loc ation is



### 11 presumably more closely related to stomatal uptake. Weighting the O 3 concentration at this location



### 12 takes into account stomatal opening and, if weighted with the Jarvis

- Steward factors for radiation,



### 13 temperature, and soil moisture, the “toxicologi cally” effective AOT40 is obtained (Grunhage and



### 14 Jager, 2003, 052972 ) . A qu estion exists however as to whether this “canopy” O 3 concentration is

clearly connected to stomatal O 3 uptake. During site conditions that limit stomatal conductance (e.g.,



### 16 low soil moisture, high VPD), high concentrations of O 3 can occur at the top of the canopy with



### 17 minimal risk.



## 9.7.4.2. Site and Climate Factors

18 Soil moisture is a critical factor in controlling O 3 uptake through its effect on plant water status



### 19 and stomatal conductance. In an attempt to relate uptake, soil moisture, and ambient air quality to

ide ntify areas of potential risk, available O 3 monitoring data for 1983 to 1990 were used along with



### 21 literature

- based seedling exposure

- response data from regions within the southern Appalachian



### 22 Mountains that might have experienced O 3 exposures sufficient to inhibit growth (Lefohn et al.,



### 23 1997, 082871 ) . In a small number of areas within the region, O 3 exposures and soil moisture



### 24 availability were sufficient to possibly cause growth reductions in some O 3 sensitive species (e.g.,

black cherry). The conclusions were limited, however, because of the uncertainty in interpolating O 3



### 26 exposures in many of the areas and because the hydrologic index used might not reflect actual water



### 27 stress.



## 9.7.4.3. Pla nt De fe ns e Me c ha nis m

– De toxification

28 The non

- stomatal component of plant defenses are the most difficult to quantify, but some



### 29 studies are available (Barnes et al., 2002, 040313 ) (Chen et al., 1998, 040317 ) (Massman and Grantz,

1995, 040698 ) (Plochl et al., 2000, 04080 2 ) (Heath et al., 2009, 196783 ) . Massman et al. (2000,



### 31 011616 ) developed a conceptual model of a dose

- based index to determine how plant injury response



### 32 to O 3 relates to the traditional exposure

- based parameters. The index used time -varyi ng -weighted



### 33 fluxes to account for the fact that flux was not necessarily correlated with plant injury or damage.

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### 1 The model applied only to plant foliar injury and suggested that application of flux

- based models for



### 2 determining plant damage (yield or biomas s) would require a better understanding and quantification



### 3 of the relationship between injury and damage.



# 9.8. Ozone Exposure

- Plant Res pons e Relations hips

9.8.1. Introd uc tion

4 The adequate characterization of the effects of O 3 on plants for the purpose of setting air



### 5 q uality standards is contingent not only on the choice of the index used (i.e. SUM06, W126) to



### 6 summarize O 3 concentrations (Section 9.7), but also on quantifying the response of the plant



### 7 variables of interest at specific values of the selected index. The m any factors that determine the



### 8 response of plants to O 3 exposure have been discussed in previous sections. They include species,



### 9 genotype and other genetic characteristics (Section 9.4), biochemical and physiological status



### 10 (Section 9.4), previous and current exposure to other stressors (Section 9.5), and characteristics of



### 11 the exposure itself (Section 9.7). Establishing a secondary air quality standard requires the capability



### 12 to generalize those observations, in order to obtain predictions that are reliabl e enough under a broad



### 13 variety of scenarios, taking into account th e se factors. This section reviews results that have related



### 14 specific quantitative observations of O 3 exposure with quantitative observations of plant responses,



### 15 and the predictions of responses that have been derived from those observations through empirical



### 16 models.



### 17 For four decades, exposure to O 3 at ambient concentrations found in many areas of the U.S.



### 18 has been known to cause detrimental effects in plants (U.S. EPA, 2006, 088089 ) (U.S. EPA, 1996,



### 19 080827 ) (U.S. EPA, 1984, 029711 ) (U.S. EPA, 1978, 040586 ) . Results published after the 2006 O 3



### 20 AQCD continue to support this finding, and the following sections deal with the quantitative



### 21 characterizations of the relationship, and what new insights may have appeared since 2006.



### 22 Detrimental effects on plants include visible injury, decreases in the rate of photosynthesis, reduced



### 23 growth, and reduced yie ld of marketable plant parts. Most published exposure

- response data have



### 24 been reported O 3 effects on the yield of crops and the growth of tree seedlings, and those two



### 25 variables have been the focus of the characterization of ecological impacts of O 3 for th e purpose of



### 26 setting secondary air quality standards. In order to support quantitative modeling of exposure



### 27 response relationships, data should preferably include more than three levels of exposure, and some



### 28 control of potential confounding or interacting factors should be present in order to model the



### 29 relationship with sufficient accuracy. Letting potential confounders, such as other stressors, vary



### 30 freely when generating O 3 exposure

- response data might improve the ‘realism’ of the data, but it also



### 31 greatl y increases the amount of data necessary to extract a clear quantitative description of the



### 32 relationship. Conversely however, experimental settings should not be so exhaustively restrictive as



### 33 to make generalization outside of them problematic. During the last four decades, many of the



### 34 studies of the effects of O 3 on growth and yield of plants have not included enough levels of O 3 to

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39 parameterize more than the simplest linear model. The majority of these studies have only contrasted

two levels, ambient and elevated, or sometimes three by adding carbon filtration in OTC studies,

with little or no consideration of quantitatively relating specific values of exposure to specific values

of growth or yield. This is not to say that studies that did not include more than two or three levels of

O 3 exposure, or studies that were conducted in uncontrolled environments, do not provide exposure

response information that is highly relevant to reviewing air quality standards. In fact, they can be

essential in verifying the agreement between predictions obtained through the empirical models

derived from experiments such as NCLAN, and observations. The consensus of model predictions

and observations from a variety of studies conducted in other locations, at other times, and us ing

different exposure methods, greatly increases confidence in the reliability of both. Furthermore, if

they are considered in the aggregate, studies with few levels of exposure or high unaccounted

variability can provide additional independent estimates of decrements in plant growth and yield, at

least within a few broad categories of exposure.

Extensive exposure

- response information on a wide variety of plant species has been produced

by t wo long

- term projects that were designed with the explicit aim of obtaining quantitative

characterizations of the response of such an assortment of crop plants and tree seedlings to O 3 under

North American conditions: the NCLAN project for crops, and the EPA National Health and

Environmental Effects Research Laboratory

, Western Ecology Division tree seedling project

( NHEERL/WED ) . The NCLAN project was initiated by the EPA in 1980 primarily to improve

estimates of yield loss under field conditions and to estimate the magnitude of crop losses caused by

O 3 throughout the U. S. (Heck et al., 1 982, 039525 ) (Heck et al., 1991, 042621 ) . The cultural

conditions used in the NCLAN studies approximated typical agrono mic practices, and the primary

objectives were: (1) to define relationships between yields of major agricultural crops and O 3 exposure as required to provide data necessary for economic assessments and development of O 3
NAAQS; (2) to assess the national ec onomic consequences resulting from O 3 exposure of major

agricultural crops; and (3) to advance understanding of cause

- and

- effect relationships that determine

crop re sponses to pollutant exposures.

NCLAN experiments yielded 54 exposure

- response curves for 12 crop species, some of which

were represented by multiple cultivars at several of 6 locations throughout the U.S. The

NHEERL/WED project was initiated by EPA in 1988 with the same objectives for tree species, and

yielded 49 exposure

- responses curves for m ultiple genotypes of 11 tree species grown for up to three

years in Oregon, Michigan, and the Great Smoky Mountain National Park. Both projects used OTCs

to expose plants to three to five levels of O 3 . Eight of the 54 crop datasets were from plants grown

u nder a combination of O 3 exposure and experimental drought conditions. Figure 9

- 13 through 9

- 16

summarize some of the NCLAN and NHEERL/WED results.

It should be noted that data from FACE experiments might also be used for modeling

exposure

- response. They only use two levels of O 3 (ambient concentration at the site and a multiple

of it), but given that the value of both levels of exposure change s every year, and that they are

typically run for many consecutive years, aggregating data over time produces twice as many levels

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39 of O 3 as there are years. As described in Section 9.3.4 , FACE experiments seek to impose fewer

constraints on the growth environment then OTCs . As a consequence, FACE studies have to contend

with larger variability, especially year

- to

- year variability , but the difference in experimental

conditions between the two methodologies makes comparisons between their results especially

useful

.

G rowth and yield of at least one crop (soybean) has been investigated in yearly experiments

since 2001 at a FACE facility in Illinois (Morgan et al., 2006, 079186 ) (University of Illinois, 2010,

670286 ) , however almost all analyses of SoyFACE published so far have been based on subsets of

one or two years, and have only contrasted ambient versus elevated O 3 as categorical variables. They

have not modeled the respo nse of growth and yield to O 3 exposure continuously over the range of

exposure values that have occurred over time. The only exception is a study by Betzelberger et al.

(2010, 644183 ) , who used a linear regression model on data pooled over 2 years. Likewise, trees of

th

ree species (trembling aspen, paper birch, and sugar maple) were grown between 1998 and 2009 in

a FACE experiment located in Rhinelander, Wisconsin (Dickson et al., 2000, 628220 ) (Pregitzer et

al., 2008, 191677 ) . Th e Aspen FACE experiment has provided extensive data on responses of trees

beyond the seedling stage under long

- term exposure, and also on ecosystem

- level responses (Section

9.6), but the only attem pt to use those data in a continuous model of the response of tree growth to

O 3 exposure (Percy et al., 2007, 093287 ) suffered severe methodological problems, some of which

are discussed in Section 9.8.3. Finally, one experiment was able to exploit a naturally occurring

gradient of O 3 concentrations to fit a linear regression model to t he growth of cottonwood (Gregg et

al., 200 3, 046996 ; Gregg et al., 2006, 186961 ) . Factors such as ge notype, soil type and soil moisture

were under experimental control, and the authors were able to partition out the effects of potential

confounders such as temperature, atmospheric N deposition, and ambient CO 2

. A serious difficulty in assessing results o f exposure

- response research is the multiplicity of O 3 metrics that have been used in reporting. As described in Section 9.7, metrics that entail either

weighting or thresholding of hourly values cannot be converted into one another, or into unweighted

met rics such as hourly average. When using weighted or thresholded metrics, which include W126,

AOTx or SUMx metrics ; O 3 exposure at every exposure

- response data point must be computed

separately for each metric, starting with the hourly data. Comparisons of exposure

- response models

can only be made between studies that used the same metric, and the value of exposure at which a

given plant response is expected on one scale of exposure cannot be exactly converted to another

scale. Determining the exposure value at which an effect would be observed in a different metric can

only be accomplished by first computing the experimental exposures in this metric from the hourly

data, then estimating (fitting) model coefficients again. This problem is irremediable, althou gh useful

comparisons might be made using categorical exposures such as ‘current ambient exposure’ or ‘2050

projected exposure’, which can serve as a common reference for quantitative values expressed in

various metrics. Studies that contained growth or yi eld exposure

- response data at few levels of

exposure, and/or using metrics other than W126 are summarized in Tables 9 -16 and 9 -17.

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## 9.8.2. 1996 and 2006 Ozo ne AQCDs Es tima te s Of Crop Yield Lo s s And Tre e S ee dlin g Bio mas s Lo s s

1 The 1996 and 2006 O 3 AQCDs relied ext ensively on analyses of NCLAN and NHEERL/WED



### 2 by Lee et al. (1987, 042135 ; 1988, 042136 ; 1989, 042137 ; 1994, 043268 ) , Hogsett et al. (1997,



### 3 040402 ) , Lee and Hogsett (1999, 040451 ) , Heck et al. (1984, 039380 ) , Rawlings and Cure (1985,



### 4 039419 ) , Lesser et al. (1990, 043015 ) , and Gumpertz and Rawlings (1992, 043259 ) . Those analyses



### 5 concluded that a three

- parameter Weibull model

–

**β** 

###  W 126 ****

**− **  ** **  ** **  **η ** 

﷒ Equation 9 -2 

# e **α =**  6 

# Y

6 is the most appropriate model for the re sponse of absolute yield and growth to O 3 exposure, because



### 7 of the interpretability of its parameters, its flexibility ( given the small number of parameters ) , and its



### 8 tractability for estimation. In addition, removing the intercept α results in a model of relative yield



### 9 (yield relative to [ yield at exposure=0 ] ) without any further reparameterization. Formulating the



### 10 model in terms of relative yield or relative yield loss (yield loss= [1 − relative yield ] ) is essential in



### 11 comparing exposure

- response across sp ecies, genotypes, or experiments for which absolute values of



### 12 the response may vary greatly. In the 1996 and 2006 O 3 AQCDs, the two

- parameter model of relative



### 13 yield was used in deriving common models for multiple species, multiple genotypes within species

, 

### 14 and multiple locations.



### 15 Given the disparate species, genotypes, and locations that were included in the NCLAN and



### 16 NHEERL/WED projects, and in the absence of plausible distributional assumptions with respect to



### 17 those variables, a three step process using robust methods was used to obtain parameter estimates



### 18 that could be generalized. The models that were derived for each species or group of species were



### 19 referred to as median composite functions. In the first step, the three parameters of the Weibull



### 20 model were estimated (fitted) for absolute yield or biomass data from each NCLAN and



### 21 NHEERL/WED experiment (54 crop datasets and 49 tree seedling datasets), using nonlinear



### 22 regression. When data were only available for three levels of exposure because of experim ental



### 23 problems, the shape parameter β was constrained to 1, reducing the model to an exponential decay



### 24 model. In the second step, α was dropped, and predicted values of relative yield or biomass were



### 25 then computed for 12

- hr W126 exposures between 0 and 60 ppm

- h. At each of these W126 exposure



### 26 values, the 25th, 50th, and 75th percentiles of the response were identified among the predicted



### 27 curves of relative response. For example, for the 34 NCLAN studies of 12 crop species grown under



### 28 non

- droughted condition s for a complete cropping cycle (Figure 9

- 13), the 3 quartiles of the



### 29 response were identified at every integer value of W126 between 0 and 60. The third step fitted a



### 30 two

- parameter Weibull model to those percentiles, yielding the median composite function for the



### 31 relative yield or biomass response to O 3 exposure for each grouping of interest (e.g., all crops, all



### 32 trees, all datasets for one species), as well as composite functions for the other quartiles . In the 1996



### 33 and 2006 O 3 AQCDs, this modeling of cro p yield loss and tree seedling biomass loss was conducted

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### 1 using the SUM06 metric for exposure. This section updates those results by using the 12

- hr W126 as



### 2 proposed in 2007 (72 FR 37818 (2007, 684055 ) ) and 2010 (75 FR 2938 (2010, 684211 ) , p. 3003 ).



### 3 Figures 9

- 13 through 9

- 16 present quantiles of predicted relative yield or biomass loss at seven



### 4 values of the 12

- h W126 for some representative gro upings of NCLAN and NHEERL/WED results.



### 5 Tables 9 -8 through 9

- 10 give the 90

- day 12

- h W126 O 3 exposure values at which 10 and 20% yield



### 6 or biomass losses are predicted in 50 and 75% of crop or tree species using the composite functions.

100Percent Yield Loss (%) 90

80

70

60

50

40

30

20

10

0 10 th Pctile 25 th Pctile 50 th Pctile 75 th Pctile 90 th Pctile 34 crop datasets

0 10 20 30 40 50 60

12 hr W126 (ppm

- hr)

Source of Weibull parameters: Lee and Hogsett (1996, 670278 ).



### Quantiles ﷒ Figure 9

- 13. Quantiles of predicted relative yield loss for 34 NCLAN crop experiments.
of the predicted relative yield loss at 7 values of 12 -h our W126 for 34 Weibull curves estimated using nonlinear regression on data from 34 studies of 12 crop species grown under well

- watered conditions for the full duration of 1 cropping cycle.

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90

80

70

60

50

40

30

20

10

0 25 th Pctile 50 th Pctile 75 th Pctile 11 Soybean datasets 100

90

80

70

60

50

40

30

20

10

0 25 th Pctile 50 th Pctile 75 th Pctile 7 Wheat datasetsPercent Yield Loss (%)Percent Yield Loss (%)

100

90

80

70

60

50

40

30

20

10

0 0 10 20 30 40 50 60 0 10 20 30 40 50 60

100

5 Cotton datasets 90 2 Corn datasets

80

70

60

75 th Pctile

50

40

30

20 50th Pctile

10

0

0 10 20 30 40 50 60 0 10 20 30 40 50 60

12 hr W126 (ppm

- hr 12 hr W126 (ppm

- hr)

Source of Weibull parameters: Lee and Hogsett (1996, 670278 ). 25 th Pctile 50 th Pctile



### ﷒ Figure 9

- 14. Quantiles of predicted relative yield loss for 4 crop species in NCLAN experiments. Qua ntiles of the predicted relative yield loss at 7 values of 12

- h W126 for Weibull curves estimated using nonlinear regression for 4 species grown under well

watered conditions for the full duration of 1 cropping cycle. The number of studies available for each species is indicated on each plot.

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0 10 20 30 40 50 60 70 80 90 100Percent Biomass Loss (%) 49 tree seedlings datasets

0 10 20 30 40 50 60

90 day 12 hr W126 (ppm

- hr)

Source of Weibull parameters: Lee and Hogsett (1996, 670278 ).



### Figure 9

- 15. Quantiles of predicted relative biomass loss for 49 tree spe cies in NHEERL/WED Quantiles of the predicted relative above

- ground biomass loss at 7 ﷒ experiments.
values of 12

- h W126 for 49 Weibull curves estimated using nonlinear regression on data from 49 studies of 11 tree species grown under well

- watered conditions for 1 or 2 years. Curves were standardized to 90

- day W126.

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0 100

90 11 Ponderosa pine datasets

80

25 th Pctile 50 th Pctile 75 th Pctile 14 Aspen datasets

0 10 20 30 40 50 60Percent Biomass Loss (%)Percent Biomass Loss (%)

70 70

60 60

50

40

25 th Pctile 50 th Pctile 75 th Pctile

0 10 20 30 40 50 60 30 30

20 20

10 10

0 0

100 100

50 th Pctile 7 Douglas f ir datasets

0 10 20 30 40 50 60 50 th Pctile 3 Tulip poplar datasets

0 10 20 30 40 50 60

90 day 12 hr W126 (ppm

- hr) 90

80

70

60

50

40

30

20

10 90

80

70

60

50

40

30

20

10

0

90 day 12 hr W126 (ppm

- hr)

Source of Weibull parameters: Lee and Hogsett (1996, 67027 8 ).



### Figure 9

- 16. Quantiles o f predicted relative biomass loss for 4 tree species in NHEERL/WED Quantiles of the predicted relative above

- ground biomass loss at 7 ﷒ experiments. exposure values of 12

- h W126 for Weibull curves estimated using nonlinear

reg ression on data for 4 tree species grown under well

- watered conditions for 1 or 2

year . Curves were standardized to 90

- day W126. The number of studies available for each species is indicated on each plot.

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### Table 9 -8 . O zone exposures at which 10 and 20% yi eld loss is predicted for 50 and 75% of crop species, based on composite functions for the 50th and 75th percentiles of 34 Weibull curves for relative yield loss data from 34 non

- droughted NCLAN studies of 12 crop species;

curves were standardized to 90

- da y W126

90

- day 12

- h W126 for 10% yield loss (ppm

- h) 90

- day 12

- h W126 for 20% yield loss (ppm

- h)

Model for the 50th P er c en tile of 34 curves

Relative yield=exp(

- (W126/104.82)**1.424) 22 37

Model for the 75th P er c en tile of 34 curves

Relative yield=ex p(

- (W126/78.12)**1.415) 16 27

Source of parameters for the 34 curves: Lee and Hogsett (1996, 670278 )



### Table 9 -9 . O zone exposures at which 10 and 20 % yield loss is predicted for 50 and 75% of crop species under drought conditions and adequate moisture, based on composite functions for the 50th and 75th percentiles of 16 Weibull curves for relative yield loss data from 8 NCLAN studies that paired droughted and watered conditions for the same genotype;

curves were standardized to 90

- day W126

90 day 12

- h W126 for 10% yield loss (ppm

- h) 90 day 12

- h W126 for 20% yield loss (ppm

- h)

Model for the 50th Percentile of 2 × 8 curves

Watered Relative yield=exp (

- (W126/132.86)**1.170) 19 37

Droughted Relative yield=exp(

- (W126/179.84)**1.713) 48 75

Model for the 75th Percentile of 2 × 8 curves

Watered Relative yield=exp(

- (W126/90.43)**1.310) 16 29

Droughted Relative yield=exp(

- (W126/105.16)**1.833) 31 46

So urce of parameters for the 16 curves: Lee and Hogsett (1996, 670278 )

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### Table 9

- 10 . O zone exposures at which 10 and 20% biomass loss is predicted for 50 and 75 % of tree species , based on composite functions for the 50th and 75th percentiles of 49 Weibull
curves for relative above

- ground biomass loss data from 49 studies of 11 tree species grown under well

- watered conditions for 1 or 2 year ; curves were standardized to 90

- day

W126

90 day 12 h W126 for 10% yield loss 90 day 12 h W126 for 20% yield loss (ppm

- h) (ppm

- h)

Model for the 50th P er c en tile of 49 curves

Relative yield=exp(

- (W126/131.57)**1.242) 21 39

Model for the 75th P er c en tile of 49 curve s

Relative yield=exp(

- (W126/65.49)**1.500) 15 24

Source of parameters for the 49 curves: Lee and Hogsett (1996, 670278 )



## 9.8.3. Valida tio n o f 1996 a nd 2006 Ozo n e AQCD Mo d els an d Meth od olo gy Us ing th e 90 day 12

- h W126 a nd Cu rre n t FACE Data.

1 Since the completion of the NCLAN and NHEERL/WED projects, almost no studies have



### 2 been published that could provide a basis for estimates of exposure

- response that can be compared to



### 3 those of the 1996 and 2006 O 3 AQCDs. Most experiments, regardless of exposure methodology,



### 4 include only two levels of exposure. In addition, very few studies have included measurements of



### 5 exposure using the W126 metric, or the hourly O 3 con centration data that would allow computing



### 6 exposure using the W126. Two FACE projects, however, were conducted over multiple years, and by



### 7 adding to the number of exposure levels over time, may support independent model estimation and



### 8 prediction using the same model and the same robust process as summarized in Section 9.8.2.



### 9 Hourly O 3 data were available from both FACE projects.



### 10 The SoyFACE project is situated near Champaign, IL, and comprises 32 octagonal rings ( 20m



### 11 diameter ) , 4 of which in a given year are exposed to ambient conditions, and 4 of which are exposed



### 12 to elevated O 3 as a fixed proportion of the instantaneous ambient concentration (Betzelberger et al.,



### 13 2010, 644183 ; Morgan et al., 2004, 072764 ; Morgan et al., 2006, 079186 ) (University of Illinois,



### 14 2010, 670286 ) . Since 2002, yield data have been collected for up to 8 genotypes of soybean grown



### 15 in subplots within each ring. The Aspen FACE project is situated in Rhinelander, WI, and comprises



### 16 12 rings ( 30m

- diameter ) , 3 of which are exposed to ambient conditions, and 3 of which are exposed



### 17 to O 3 as a fixed proportion of the instantaneous ambient concentration (Dickson et al., 2000, 628220 ;



### 18 Karnosky et al., 2005, 095556 ; Pregitzer et al., 2008, 191677 ) . In the summer of 1997, half the area



### 19 of each ring was planted with small (five to seven leaf sized ) clonally propagated plants of five



### 20 genotypes of t rembling a spen, which were left to grow in those environments until 2009. Biomass



### 21 data are currently available for the years 1997

- 2005 (King et al., 2005, 191701 )

. Ozone exposure in



### 22 these two FACE projects can be viewed as a categorical variable with two levels: ambient, and



### 23 elevated. However, this overlooks the facts that yearly ambient and elevated exposure both vary with



### 24 every year, and that the proportionality between them also changes. This change has two sources:

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### 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37 first, the dispensing of O 3 into the elevated exposure rings varies from the proportionality set point to

some extent, and for SoyFACE, the set point changed between years. Second, the proportionality

does n ot propagate predictably from the hourly data to the yearly value when using thresholded or

concentration

- weighted cumulative metrics (such as AOT40, SUM06 or W126). Hourly average

elevated exposures that are, for example, 1.5 times ambient do not result i n AOT40, SUM06 or

W126 values that are some constant multiple of the ambient values of those indices. The greater the

fraction of elevated hourly values that are above the threshold or heavily weighted, compared to the

fraction of hourly ambient values tha t are, the greater the difference between ambient and elevated

yearly exposure, as measured using weighted cumulative indices. When elevated exposure is a

multiple of ambient hourly intervals, the number of hours for which elevated exposure meets the

thres hold for inclusion can vary widely, even though the hourly mean for the year retains the

proportionality. As a consequence, the number of exposure levels in multi

- year experiments is twice

the number of years. In the case of SoyFACE for the period between 2002 and 2008, ambient

exposure in the highest year was approximately equal to elevated exposure in the lowest year, with

14 levels of O 3 exposure evenly distributed from lowest to highest. The particular conditions of the

Aspen FACE experiment resulted in 12 exposure levels between 1998 and 2003, but they were not as

evenly distributed between minimum and maximum over the 6- year period.

There are necessary differences in the modeling of exposure

- response in annual plants such as

soybean, and in perennial plants such as aspen trees, when exposure takes place over multiple years.

In annual plants, responses recorded at the end of the life cycle, i.e., yearly, are analyzed in

relationship to that year's exposure. Yield of soybeans is affected by exposure durin g the year the

crop was growing, and a new crop is planted every year. Thus an exposure

- response relationship can

be modeled from yearly responses matched to yearly exposures, with those exposure

- response data

points having been generated in separate years. For perennial organisms, which are not harvested

yearly and continue to grow from year to year, such pairing of exposure and response cannot be done

without accounting for time. Not only does the size of the organism at the beginning of each year of

expo sure increase, but size is also dependent on the exposure from previous years. Therefore the

relationship of response and exposure must be analyzed either one year at a time, or by standardizing

the response as a yearly increment relative to size at the be ginning of each year. Furthermore, the

relevant measurement of exposure is cumulative

, or cumulative yearly average exposure

, starting in

the year exposure was initiated, up to the end of the year of interest. When analyzing the growth of

trees over severa l years, it would be evidently incorrect to pair the exposure level in every discrete

year with absolute size of the trees that year, and posit a direct relationship between them. In the

Aspen FACE experiment, for example, one could not establish an exposu re

- response relationship by

matching 12 yearly exposures and 12 yearly tree sizes, as if size did not also depend on time. This is

the basis of the 2007 study of Aspen FACE data by Percy et al. (2007, 093287 ) , and that study was

therefore not informative.

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### 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

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22

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24

25

26

27

28

29

30

31

32

33

34

35

36

37

38 

## 9.8.3.1. Co mpa ris on of NCLAN

- Ba s e d P re dic tion a nd SoyFACE Da ta .

For this ISA , EPA conducted a comparison between yield as predicted by the composite

function three- step process (Section 9.8.2) using NCLAN data for soybean yield as observed in

SoyFACE. The median composite function for relative yield was derived for the 11 NCLAN soybean

Weibull fu nctions for non

- droughted studies, and several comparisons between prediction and

SoyFACE observations were conducted as follows.

For the years 2007 and 2008, SoyFACE yield data were available for 7 and 6 genotypes,

respectively. The EPA used those data to compare the change in relative yield observed in a given

year between ambient O 3 and elevated O 3 in SoyFACE, versus the change in relative yield predicted

by the NCLAN

- based median composite function between those same two values of O 3 exposure.

The two para meter median composite function for relative yield of soybean w as used to predict yield

response at the two observed values of exposure in each year, and the change between yield under

ambient and elevated was compared to the change observed in SoyFACE for the relevant year (Table

9- 11). This approach results in a direct comparison of predicted versus observed change in yield.

Because the value of relative response between any two values of O 3 exposure is inde pe ndent of the

intercept α, this comparison does not require prediction of the absolute values of the responses.

Since comparisons of absolute values might be of interest, the predictive functions were also

scaled to the observed data, using two distinct met hods. In the first method, the intercept α was

calculated algebraically by entering the observed W126 value at ambient exposure and the

corresponding value of the response into the three parameter model with the shape and scale

parameters (β and η) set to their value for the NCLAN predictive model. This method provides a

comparison between the response observed under elevated exposure

, and the response that would

have been predicted with only the knowledge of what the response was under ambient exposure

(Ta ble 9

- 12; Method 1). In the second method, the intercept for the NCLAN predictive model was

estimated by regression using both ambient and elevated data. This method gives a comparison of

prediction and observation that takes all the observed information i nto account to provide the best

possible estimate of the intercept, and thus the best possible scaling (Table 9- 12, Method 2 and

Figure 9- 17) . It should be noted that the similarity to each other of the predictions obtained by these

two scaling methods is a reflection of the accuracy of the predictions: the distance between the

predictions from the two methods increases as the distance between prediction and observation

increases.

For the comparison of NCLAN and SoyFACE, this validation was possible for 2007 and 2008,

where data for 7 and 6 soybean genotypes, respectively, were available. The median composite

function for relative yield was derived for the 11 NCLAN soybean Weibull functions for

nondroughted studies, and the values of median yield under ambient exposure at SoyFACE in 2007

and 2008 were used to obtain an estimate of the intercept α for the NCLAN median function in each

of the two years

Table 9 -11 presents the results of ambient/elevated relative yield comparisons between the

NCLAN

- derived predi ctions and SoyFACE observations. Table 9 -12 presents the results of

March 20 11 9

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### 1 comparisons between NCLAN

- derived predictions and SoyFACE observations of yield, using two



### 2 methods for scaling the predictive function. Figure 9

- 17 presents yield observed in two years in



### 3 SoyFACE, and predicted by the median composite function derived from NCLAN data using the



### 4 second scaling method as described, with the intercept estimated using 14 observations in 2007, and



### 5 12 in 2008.



### 6 Finally, data were also available for one additional genotype from 2003 to 2007. By



### 7 aggregating data for each genotype over the years (5 years for one genotype, 2 years for the 6



### 8 others), a composite function for the 25th, 50th, and 75th percentiles was then developed for



### 9 SoyFACE, and compared to the corresp onding NCLAN

- based function. NCLAN functions were



### 10 obtained using 12

- hr W126 standardized to 90 days. SoyFACE 12

- hr W126 was cumulated over the



### 11 highest 90 days of the growing season.



### 12

Table 9 -11 . Comparison between relative yield observed in the SoyFACE exp eriment, and relative yield predicted at the same values of ozone by the median composite function for NCLAN (two

parameter relative yield model)

Year 90

- day 12

- h W126 (ppm

- h) Yield,

Elevated Relative to Ambient

Ambient Elevated Observed in SoyFACE Predi cted by NCLAN

2007 4.39 46.23 0.76 0.75

2008 3.23 28.79 0.88 0.85



### Table 9 -12 . Comparison between yield observed in the SoyFACE experiment and yield predicted at the
same values of ozone by the median composite function for NCLAN (three

- parameter
absolut e yield model), using two scaling methods to calculate the intercept

Year 90

- day 12

- h W126 (ppm

- h)

Ambient Elevated Yield observed in SoyFACE ambient (g/m 2 ) Yield observed in SoyFACE elevated (g/m 2 ) Yield predicted by NCLAN in elevated,

Method 1 ( g/m 2 ) Y ield predicted by NCLAN in ambient,

Method 2 (g/ m 2 ) Yield predicted by NCLAN in elevated,

Method 2 (g/m 2 )

2007 4.39 46.23 305.2 230.6 227.6 309.2 230.6

2008 3.23 28.79 344.8 304.4 293.5 350.3 298.2

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350

300 2007, 7 genotypes 400

350
2008, 6 genotypes

300

250 250Yield (g/m2)Yield (g/m2)

200

150 200

150

100 100

50 50

0 0

0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70

90 day 12 hr W126 (ppm

- hr) 90 day 12 hr W126 (ppm

- hr)

Source of data: Betzelberger et al. (2010, 644183 ) ;Morgan et al. (2006, 079186 ) ; Lee and Hogsett (1996, 670278 ).

Note: Black dots are median of 7 or 6 soybean genotypes in SoyFACE (2007, 2008); bars are IQR for genotypes; dashed line is median

composite model for 11 studies in NCLAN.



### Figure 9

- 17 . Comparison of yield observed in SoyFACE experiment in a given year with yield predicted by the median composite function based on NCLAN

.

1 Finally, a composite function for the 25th, 50th, and 75th percentiles was developed from



### 2 SoyFACE annual yield data by pooling one genotype from 2003 to 2007, and six genotypes in 2007



### 3 and 2008. NCLAN functions were obtained using 12

- h W126 standardized to 90 days. SoyFACE



### 4 12

- h W126 was cumulated over the highest 90 days of the growing season. The correlation between



### 5 W1 26 cumulated for the entire season in SoyFACE and W126 for the highest 90 days was greater



### 6 than 0.99 in all years. The same process was used for SoyFACE: first, the three parameter Weibull



### 7 model described in Section 9.8.2 was estimated using nonlinear regression on exposure

- yield data



### 8 for each genotype separately, over the years for which data were available, totaling seven curves.



### 9 The 25th, 50th, and 75th percentiles of the predicted values for the two parameter relative yield



### 10 curves were then identified a t every integer of W126 between 0 and 60, and a two

- parameter Weibull



### 11 model estimated by regression for the three quartiles. The comparison between these composite



### 12 functions for the quartiles of relative yield loss in SoyFACE and the corresponding composit e



### 13 functions for NCLAN is presented in Figure 9

- 18.

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FACE median
FACE 25 th Pctile NCLAN 75 th Pctile

NCLAN median

NCLAN 25 th Pctile

0 10 20 30 40 50 60 70 80 90 100Percent Yield Loss (%)

0 10 20 30 40 50 60 70

90 day 12 hr W126 (ppm

- hr)

Source of data: Betzelberger et al. (2010, 644183 ) ;Morgan et al. (2006, 079186 ) ; Lee and Hogsett (1996, 670278 ).



### Figure 9

- 18. Comparison of composite functions for the quartiles of 7 curves for 7 genotypes of soybean grown in the SoyFACE experiment, and for the quartiles of 11 curves for 5 genotypes of soybean grown in the NCLAN project.

1 As seen in Tables 9 -11 and 9 -12 , and in Figure 9

- 17, the agreement between predictions based



### 2 on NCLAN da ta and SoyFACE observations was notably close in single

- year comparisons. Together



### 3 with the very high agreement between median composite models for NCLAN and SoyFACE, it



### 4 provides very strong mutual confirmation of those two projects' results with respect t o the response



### 5 of yield of soybeans to O 3 exposure. It is readily apparent from these results that the methodology



### 6 described in Section 9.8.2 for obtaining predictions of yield or yield loss from NCLAN data is



### 7 strongly validated by SoyFACE results. As described in Section 9.3, the exposure technologies used



### 8 in the two projects were in sharp contrast, specifically with respect to the balance each achieved



### 9 between control of potential interacting factors or confounders, and fidelity to real world conditions.



### 10 The comparisons that EPA conducted therefore demonstrate that the methodology used in developing



### 11 the composite functions is resistant to the influence of nuisance variables, and that predictions are



### 12 reliable. They may also suggest that the aspects in which the two exposure technologies differ have



### 13 less influence on exposure

- response than initially supposed.



## 9.8.3.2. Co mpa ris on of NHEERL/WED

- Ba s e d Pre dic tion of Tre e Bioma s s Re s p ons e a nd As pe n FACE Da ta

14 EPA also conducted two comparisons between prediction of above -gr ound biomass loss based



### 15 on NHEERL/WED results and observations from Aspen FACE. The median composite function was



### 16 developed from NHEERL/WED data for 11 studies that used wild

- type seedlings of a spen as well as

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### 1 four clonally propagated genotypes. All plants were grown in OTCs for one growing season before



### 2 being destructively harvested. Aspen FACE data were from clonally propagated trees of five



### 3 genotypes grown from 1998 to 2003, with above

- ground biomass calculated using allometric



### 4 equations derived from data for trees harvested destructively in 2000 and 2002 (King et al., 2005,



### 5 191701 )

. 

### 6 The two parameter median composite function for relative biomass was used to predict



### 7 biomass response under the observed elevated exposure, relative to its value under observed ambient



### 8 exposure

. EPA first tested the accuracy of the prediction of biomass at elevated exposure relative to



### 9 biomass at ambient exposure, for each separate year of Aspen FACE. Comparisons between



### 10 observed and predicted biomass values were then conducted for each year by scaling the predictive



### 11 function to yearly Aspen FACE data using the two scaling methods described in Section 9.8.3.1.



### 12 Yearly 90 day 12 -hou r W126 values for Aspen FACE were computed as the cumulative average



### 13 from the year of planting up to the year of interest. A comparison of composite functions between



### 14 NHEERL/WED and Aspen FACE, similar to the one performed for NCLAN and SoyFACE, was not



### 15 possible: as discussed in the introduction to Section 9.8, the pairing of 12 exposure values from



### 16 separate years and 12 values of biomass cannot be the basis for a model of exposure

- response,



### 17 because the trees continued growing for the six

- year period of exposure. Table 9 -13 presents the



### 18 results of ambient/elevated relative biomas s comparisons between the NHEERL/WED

- derived



### 19 predictions and Aspen FACE observations. Table 9 -14 presents the results of comparisons between



### 20 NHEERL/WED

- derived predictions and Aspen FACE observations of biomass, using two methods



### 21 for scaling the predictive function. Figure 9

- 19 presents biomass observed in six years at Aspen



### 22 FACE, and predicted by the median composite function derived from NHEERL/WED data using the



### 23 second scaling method as described, with the intercept estimated using 2 observations in each year.



### Table 9 -13 . Comparison between above

- ground biomass observed under elevated ozone in Aspen FACE experiment in 6 year , relative to above

- ground biomass observed under ambient

ozone and relative above

- ground biomass above

- ground biomass at the same va lues of ozone predicted by the median composite function for NHEERL/WED (two

- parameter relative biomass model)

90

- day 12

- h W126 (ppm

- h) Above

- Ground Biomass, Year Cumulative Average Elevated Relative To Ambient

Ambient Elevated Observed in Aspen FACE Pre dicted by NHEERL/WED

1998 3.19 30.08 0.75 0.74

1999 2.61 33.85 0.7 0 0. 70

2000 2.43 30.16 0.71 0. 74

2001 2.55 31.00 0.7 1 0.7 3

2002 2.51 30.27 0. 69 0.7 4

2003 2.86 29.12 0. 71 0.75

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### Table 9 -14 . Comparison between above

- ground biomass observed in Aspen FACE experiment in

6 year and biomass predicted by the median composite function based on NHEERL/WED (three

- parameter absolute biomass model), using 2 scaling methods to calculate the intercept

Year 90 day 12

- h W126 (ppm

- h) Cumulative Average Biomass Observe d in Aspen
FACE (g/m 2 ) Biomass Predicted by NHEERL/WED,

Method 1 (g/m 2 ) Biomass Predicted by NHEERL/WED,

Method 2 (g/m 2 )

Ambient Elevated Ambient Elevated Elevated Ambient Elevated

1998 3.19 30.08 274.7 204.9 202.3 27 6.0 203.2

1999 2.61 33.85 955.3 6 73.3 665.9 958.7 668.3

2000 2.43 30.16 1400.3 998.6 1036.0 1382.4 1022.8

2001 2.55 31.00 1620.7 1154.9 1183.7 160 7.0 1173.7

2002 2.51 30.27 2125.9 1468.41 1566.7 207 9.0 1532.1

2003 2.86 29.12 2695.2 1907.8 2022.5 2640. 1 1981.2

1998 1999 2000 2001 2002 2003

0 500 1000 1500 2000 2500 3000Biomass (g/m2)

0 10 20 30 40 50 60 70

90 day 12 hr W126 (yearly cumulative average, ppm

- hr)

Source of data: K ing et al. (2005, 191701 ) , Lee and Hogsett (1996, 670278 ).

Note: Black dots are a spen biomass /m 2 for 3 FACE rings filled with an assemblage of 5 clonal genotypes of a spen at Aspen FACE; bars

are SE for 3 rings; dashed line is median composite model for 4 clonal genotypes and wild

- type seedlings in 11 NHEERL/WED 1

- year
OTC studies.



### Figure 9

- 19. Comparison between above

- ground biomas s observed in Aspen FACE experiment

in 6 year and biomass predicted by the median composite function based on
NHEERL/WED

.

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### 5

10

15

20

25

30

35

1 As in the comparisons between NCLAN and SoyFACE, the agreement between predictions



### 2 based on NHEERL/WED data and A s pen FACE observatio ns was exceptionally close. The results of



### 3 the two projects strongly reinforce each other with respect to the response of a spen biomass to O 3



### 4 exposure. The methodology used for obtaining the median composite function is shown to be

capable of deriving a pr edictive model despite potential confounders, and despite the added



### 6 measurement error that is expected from calculating biomass using allometric equations. In addition,



### 7 the function based on one year of growth was shown to be applicable to subsequent years

. 

### 8 The results of experiments that used different exposure methodologies, different genotypes,



### 9 locations, and durations converged to the same values of response to O 3 exposure for each of two

very dissimilar plant species, and predictions based on the ear lier experiments were validated by the



### 11 data from current ones. However, in these comparisons, the process used in establishing predictive



### 12 functions involved aggregating data over variables such as time, locations, and genotypes, and the



### 13 use of a robust sta tistic (quartiles) for that aggregation. The validating data, from SoyFACE and



### 14 Aspen FACE, were in turn aggregated over the same variables. The accuracy of predictions is not

expected to be conserved for individual values of those variables over which aggregation occurred.



### 16 For example, the predicted values for soybean, based on data for five genotypes, are not expected to



### 17 be valid for each genotype separately. As shown in the validation, however, aggregation that



### 18 occurred over different value s of the same v ariable did not affect accuracy: composite functions



### 19 based on one set of genotypes were predictive for another set, as long as medians were used for both

sets. A study of cottonwood ( Populus deltoides ) conducted using a naturally occurring gradient of O 3



### 21 e xposure (Gregg et al., 2003, 046996 ; Gregg et al., 2006, 186961 ) may provide an illustration of the



### 22 response of an individual species whose response is far from the median response for an aggregation



### 23 of species.



## 9.8.3.3. Expos ure

- Res pons e in a Gra dient Study

24 Gregg et al. (2003, 046996 ) grew saplings of one clonally propagated genotype of cottonwood

( Populus deltoides ) in seven locations within New York City and in the surrounding region between



### 26 July and September in 1992, 1993 and 1994, and harvested them 72 days after planting. Owing to



### 27 regional gradients of atmospheric O 3 concentration, the experiment yielded eight levels of exposure



### 28 (Fig ure 9

- 20 ), and the authors were able to rule out environmental variables other than O 3 to account



### 29 for the large differences in biomass observed after one season of growth . The deficit in growth

increased substantially faster with increasing O 3 exposure than has been observed in a spen, another



### 31 species of the same genus ( Populus tremuloides , Section 9.8.3.2). Using a three parameter Weibull



### 32 model (Figure 9

- 20), the biomass of cottonwood at a W126 exposure of 15 ppm

- h, relative to



### 33 biomass at 5 ppm

- h, is estimated to be 0.18 (18% of growth at 5 ppm

- h). The relative biomass of



### 34 trembling aspen within the same 5

- 15 ppm

- h range of exposure is estimated to be 0.92, using the

median composite model for aspen whose very close agreement with Aspen FACE data was shown



### 36 in Section 9.8.3.2. Using a median composite function for all deciduous trees in the NHEERL/WED



### 37 project (6 species in 21 studies) also gives predictions that are very dista nt from the cottonwood

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### 1 response observed in this experiment. For all deciduous tree species in NHEERL/WED, biomass at a



### 2 W126 exposure of 15 ppm

- h, relative to biomass at 5 ppm

- h, was estimated to be 0.87.

100

90

80

70

0 10 20 30 40 50 60 70Biomass (g) 60

50

40

30

20

10

0

72 day 12 hr W126 (ppm

- hr)

Source: Modified with permission from Nature Publishing Group, Gregg et al. (2003, 046996 ).



### Figure 9

- 20. Above

- ground biomass for one genotype of cottonwood grown in seven locations Line represents the three

- parameter Weibull model. ﷒ for one season in 3 y ea rs

.

3 These cottonwood data confirms that, as should be expected, some individual tree species are



### 4 substantially more sensitive than the median of NHEERL/WED (Fig ure 9- 15). As shown in



### 5 Section 9.8.2, the median models available for trembling asp en and soybean have verifiable



### 6 predictive ability for those particular species. This suggests that the corresponding NCLAN - and



### 7 NHEERL/WED

- based models for multiple crop and tree species can provide reliable estimates of



### 8 losses for similar assortments of s pecies. However, their predictive ability would likely be poor for



### 9 individual species not tested.



### 10 An alternative hypothesis for the difference between the response of cottonwood in this



### 11 experiment and deciduous tree species in NHEERL/WED, or the differenc e between the response of



### 12 cottonwood and aspen in NHEERL/WED and Aspen FACE, could be the presence of confounding



### 13 factors in the environments where the experiment was conducted. However, variability in



### 14 temperature, moisture, soil fertility, and atmospheric deposition of N were all ruled out by Gregg



### 15 et al. (2003, 046996 ) as contributing to the observed response to O 3 . In addition, this hypothesis



### 16 would imply that the unrecognized confounder(s) were e ither absent from both OTC and FACE



### 17 studies, or had the same value in both. This is not impossible, but the hypothesis that cottonwood is



### 18 very sensitive to O 3 exposure is more parsimonious, and sufficient.

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## 9.8.3.4. Meta -a nalys es of growth and yield s tudies

1 Since th e 2006 O 3 AQCD, five studies have used meta

- analytic methods to integrate results



### 2 from experimental studies of crops or tree species relevant to the U.S. It is possible to obtain



### 3 exposure

- response data for growth and yield from those meta -a nalyses, but bec ause all of them



### 4 provided summary measurements of O 3 exposure as hourly averages of various lengths of exposures,



### 5 comparisons with exposure

- response results where exposure is expressed as W126 are problematic.



### 6 Table 9 -15 summarizes the characteristics of t he five meta -a nalyses. They all included studies



### 7 conducted in the U.S. and other locations worldwide, and all of them expressed responses as



### 8 comparative change between levels of exposure to O 3 , with carbon filtered air (CF) among those



### 9 levels. Using hourly average concentration to summarize exposure, CF rarely equates absence of O 3 , 

### 10 although it almost always near zero when exposure is summarized as W126, SUM06, or AOT40.



### Table 9 -15. Meta -a nalyses of growth or yield studies published since 2005

Years of Number of articles Number of Duration of Study publication Crop, species or genera Response included O3 levels exposure surveyed

Ainsworth (2008, 12 1980

- 2007 rice Yield 2 unreported 191646 )

Feng et al. (2008, 53 1980

- 2007 wheat Yield 5 > 10 days 191453 )

Feng and Kobayashi Potato, barley, wheat, rice, All crops togethe r : 81 1980

- 2007 Yield 3 > 10 days (2009, 199223 ) bean, soybean

34 herb aceous dicots Grantz et al. (2006, Relative 16 1992

- 2004 21 herbaceous monocots 2 2- 24 weeks 191545 ) Growth Rate 5 tree species

All responses:263 Wittig et al. (2009, 4 gymnosperm tree genera Articles that included 1970

- 2006 Total biomass 4 > 7 days 191631 ) 11 angiosperm tree genera biomass:unreported



### 11 The only eff ect of O 3 exposure on yield of rice reported in Ainsworth (2008, 191646 ) was a



### 12 decrease of 14% with exposure increasing from CF to 62 ppb average concentration. Feng et al.



### 13 (2008, 191453 ) were able to separate exposure of wheat into four classes with average



### 14 concentrations of 42, 69, 97, and 153 ppb, in data where O 3 was the only treatment. Mean responses



### 15 relative to CF were yield decreases of 17, 25, 49, and 61% respectively. Feng et al. (2008, 191453 )



### 16 observed that wheat yield losses were smaller under conditions of drought, and that Spring wheat



### 17 and Winter wheat appeared similarly affected. However, mean exposure in studies of Winter wheat



### 18 was substantially higher than in studies of Spring wheat (86 v ersu s 64 ppb), which suggests that the



### 19 yield of Spring wheat was in fact more se verely affected, since yield was approximately the same,



### 20 even though Spring wheat was exposed to lower concentrations. Exposures of the six crops



### 21 considered in Feng and Kobayashi (2009, 199223 ) were classified into two ranges, each compared to



### 22 CF air. In the lower range of exposure (41

- 49 ppb), potato studies had the highest average exposure



### 23 (45 ppb), and wheat and rice the lowest (41 ppb). In the higher range (51

- 75 ppb), wheat studies had



### 24 the highest average exposure (65 ppb), and potato, barley and rice the lowest (63 ppb). In other



### 25 words, across the studies included, all crops were exposed to very similar levels of O 3 . At

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### 1 approximately 42 ppb, the yield of potato, barley, wheat, rice, bean, and soybean declined by 5.3,



### 2 8.9, 9.7, 17.5, 19, and 7.7% respectively, relative to CF air. At approximately 64 ppb O 3 , declines



### 3 were 11.9, 12.5, 21.1, 37.5, 41.4, and 21.6%. Grant z et al. (2006, 191545 ) reported Relative Growth



### 4 Rate (RGR) rather than growth, and did not report O 3 exposure values in a way that would allow



### 5 calculation of mean exposure for each of the three categories of plants for which RGR changes are



### 6 reported. All studies used only two levels of exposure, with CF air as the lower one, and most used



### 7 elevated exposure in the range of 40 to 70 ppb. Decline in RGR was 8.2% for the 34 herbaceous



### 8 dicots, 4.5% for the 21 herbaceous monocots, and 17.9 for the 5 tree species. Finally, Wittig et al.



### 9 (2009, 191631 ) divided the studies analyzed into three classes of comparisons: CF versus ambient,



### 10 CF versus elevated, and ambient ver sus elevated, but reported comparisons between three average



### 11 levels of exposure besides CF: 40 ppb, 64 ppb, and 97 ppb. Corresponding decreases in total biomass



### 12 relative to CF were 7, 17, and 17%.



### 13 These meta -a nalyses provide very strong confirmation of EP A's conclusions from previous O 3



### 14 AQCDs: compared to lower levels of ambient O 3 , current levels in many locations are having a



### 15 substantial detrimental effect on the growth and yield of a wide variety of crops and natural



### 16 vegetation. They also confirm strong ly that decreases in growth and yield continue at exposure levels



### 17 higher than current ambient levels. However, direct comparisons with the predictions of exposure



### 18 response models that use concentration

- weighted cumulative metrics are difficult.



## 9.8.3.5. Ad ditiona l e xpos ure

- res pons e data

19 The studies summarized in Tables 9 -16 and 9 -17 contain growth or yield exposure

- response



### 20 data at too few levels of exposure for exposure

- response models, and/or used metrics other than



### 21 W126. These tables u pdate Tables AX9

- 16 throug h AX 9- 19 of the 2006 O 3 AQCD.

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### Table 9 -16. Summary of studies of effects of ozone exposure on growth and yield of agricultural crops

Species Facility

Location Exposure

Duration O 3 Exposure (Additional Treatment) Response Measured percent change from

CF ( per cent change from ambient) Reference

Alfalfa ( Medicago

sativa )

OTC; 0.27m 3 pots

Federico, Italy 2 yr , 2005,

2006 AOT40: CF 0 ppm

- h 13.9 ppm

- h (2005), 10.1 ppm

- h (2006)

(NaCl: 0.29, 0.65, 0.83,

1.06 deciSiemens/meter) Total shoot yield n.s. (N/A) Maggio et a l. (2009, 191645 )

Bean ( Phaseolus vulgaris I. cv Borlotto)

OTC; ground

- p lanted

Curno, Italy 3 mo n ths

, 2006 Season al AOT40: CF (0.5 ppm

- h); ambient (4.6 ppm

- h)

(N/A) # Seeds per plant;

100

- seed weight - 33 (N/A)

n.s. (N/A) Gerosa et a l. (2009, 191403 )

Big Blue Stem ( Andropogon gerardii )

OTC

Alabama 4 mo n ths

, 2003 12

- h avg: CF (14 ppb), Ambient (29 ppb), Elevated (71 ppb)

(N/A) Final harvest biomass;

RVF n.s. ( n.s. )

- 7 (

- 7) Lewis et al. (2006, 191542 )

Brassica napus cv. Westar

Growth chambers

Finland 17

- 26 days 8- h avg:
CF (0 ppb) , 100 ppb

(Bt/non

- Bt; herbivory) Shoot biomass

- 30.70 (N/A) Himanen et al. (2009, 191338 )

Corn ( Zea mays cv. Chambord)

OTC

France 33 days AOT40 pp m-h : 1.1; 1.3; 4.9; 7.2; 9.3; 12.8

(N/A) Total above

- ground

biomass N/A (Highest treatment

caused

- 26% change) Leitao et al. (2007, 191456 )

Cotton cv. Pima

OTC; 9

- L pots

France 8 wk 12

- h avg: 12.8 ± 0.6; 79.9 ± 6.3;

122.7 ± 9.7

(N/A) Above

- ground biomass

- 76 (n.s.) Grantz and Shrestha (2006

, 191702 )

Eastern Gamagrass ( Tripsacum dactyloides )

OTC

Alabama 4 mo n ths

, 2003 12

- h avg: CF (14ppb), Ambient (29 ppb), Elevated (71 ppb)

(N/A) Final harvest b iomass;

RVF +68 (+42);

- 17 (

- 12) Lewis et al. (2006, 191542 )

Grapevine ( Vitis

vinivera )

OTC

Austria 3 yr, May

- Oct AOT40 ppm

- h: CF (0), Ambient (7

- 20), Elevated. 1 (20

- 30), Elevated. 2 (38

- 48) Total fruit yield / Sugar yield - 20 to

- 80 in different yr (- 20 to

- 90 in different yr) Soja et al. (2004,

094397 )

Mustard ( Brassica campestris )

Chambers;

7.5

- cm pots 10 days CF & 67.8 ppb for 7 h

(N/A) Seeds/plant n.s. (N/A) Black et al. (2007, 191558 )

Oilseed Rape ( Brassica napus )

OTC

Yangtze Delta, China 39 days Daily avg: 100 ppb, one with

diurnal variation and one with

constant co ncentrat ion

(N/A) Biomass and pods per plant Diurnal variability

reduced both biomass and pod number more than constant fumigation (N/A) Wang et al. (2008, 191276 )

Peanut ( Arachis hypogaea )

OTC 3 yr 12

- h avg: CF (22 ppb), Ambient (46 ppb), Elevated (75ppb) Yield (seed weight, g/m)

- 33 (

- 8) Burkey et al. (2007, 191371 )

Raleigh, NC (CO 2 : 375 ppm; 548 ppm; 730 ppm)

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Location Exposure

Duration O 3 Exposure (Additional Treatment) Response Measured percent change from
CF ( per cent change from ambient) Reference

Poa prat ensis

OTC

Braunschweig, Germany 2000

- 2002:

4- 5 wk in the
S pr ing 8- h avg: CF+25 (21.7), NF+50 (73.1)

(Competition) Total biomass (g DW/pot) N/A (n.s.) Bender et al. (2006, 191437 )

Potato ( Solanum

tuberosum )

OTC; CHIP

6 northern European

locations 1988,1999. Emergence to

harvest AOT40: CF ( 0 ); Ambient ( 0.27

- 5.19 ); NF ( 0.002

­ 2.93 ) NF+ (3.10

- 24.78

(N/A) Tuber yiel d averaged
across 5 field

- s ites;

Tuber starch content regressed against [O 3 ] report sig. ± slope with increasing [O 3 ] N/A (- 27 %

- +27%, most comparisons n.s. ) Linear regression slope =

- 0.0098) Vandermeiren

et al. (2005,

179992 )

Rice ( Oryza sativa )

OTC

Raleigh, NC 1997

- 1998,
June

September 12

- h mean ppb: CF (27.5),

Elevat ed (74.8)

(CO 2) Total biomass;
S eed y ield - 25 (N/A)

- 13 to 20 (N/A) Reid, et al. (2008, 191561 )

Rice ( Oryza sativa ) 20

Asian cultiva rs OTC

Gunma Prefecture, Japan 2008 growing

season Daily avg (ppb): CF (2), 0.8×ambient (23);

1 ×ambient (28); 1.5×ambient (42); 2×ambient (57)

(Cultivar comparisons) Yield From n.s. to

- 30 across

all cultivars Sawada and Kohno (2009,

199426 )

Seminatural grass

FACE 5 yr Seasonal AOT40: Ambient (0.1

- 7.2 ppm -h ); Elevated. (1.8

- 24.1 ppm -h) Relati ve annual yield N/A (2×faster decrease in yield/yr) Volk et al. (2006,

191434 )

Le Mouret, Switzerland (N/A)

Soybean

OTC; CRA

Bari, Italy 2003 -2 005 growing

seasons Seasonal AOT40 ppm

- h: CF (0), Ambient (3.4), High (9.0)

(Drought) Yield

- 46 (

- 9) Jaoude et a l. (2008, 191223 )

Soybean ( Glycine max cv. 93B15)

SoyFACE

Urbana, IL 2002

, 2003 growing

seasons 8- h avg: Ambient (62 & 50 ppb), Elevated (75 & 63 ppb)

(N/A) Yield N/A (- 15 in 2002;

- 25 in 2003) Morgan et a l. (2006, 079186 )

Soybean ( Glycine max cv. Essex)

Chambers; 21 L

Raleigh, NC 2×3 mo n th s 12

- h avg: CF (28), Elevated (79), Elevated flux (112)

(CO 2 : 365 & 700) Seed mass per plant

- 30 (N/A) Booker and

Fiscus (2005,

191652 )

Soybean ( Glycine max cv. Essex)

OTCs; 21

- L pots

Raleigh, NC 2×3 mo n ths 12

- h avg: CF (18); Elevated (72)

(CO 2 : 367 & 718) Seed mass per plant

- 34 (N/A) Booker et a l. (2004, 079138 )

Soybean ( Glycine max cv. Tracaja)

Chambers; pots 20 days 12

- h avg: CF & 30 ppb

(N/A) Biomass

- 18 (N/A) Bulbovas et al. (2007, 199411 )

Brazil

Soybean ( Glycine

max ) 10 cultivars

SoyFACE

Urbana, IL 2007 & 2008 8- h avg: Ambient (46.3 & 37.9), Elevated (82.5 & 61.3)

(Cultivar comparisons) Yield N/A (

- 17.20) Betzelberger

et al. (2010,

644183 )

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Location Exposure

Duration O 3 Exposure (Additional Treatment) Response Measured percent change from
CF ( per cent change from ambient) Reference

Spring Wheat ( Triticum

aestivum cv. Minaret; Satu; Drabant; Dragon)

OTCs

Belgium, Finland, &
Sweden 1990

- 2006 Seasonal AOT40s ranged from 0 to16 ppm -h

(N/A) Seed protein content;

1,000

- seed weight regressed across all experiments N/A (signific ant negat ive

correlation )

N/A ( sig negative correlation) Piikki et al. (2008, 199812 )

Strawberry ( Fragaria x

ananassa Duch. Cv Korona & Elsanta)

Growth chambers 2 mo n ths 8- h avg: CF (0 ppb) & Elevated (78 ppb)

(N/A) Fruit yield (weight/plant)

- 16 (N/A) Keutgen et al. (2005, 191295 )

Bonn, Germany

Sugarbeet ( Beta vulgaris cv. Patriot)

OTC 2003

, 2004;

5 mo n ths 8

- h avg: Ambient (36 ppb); Elevated (62 ppb)

(N/A) Sugar yield N/A (

- 9) De Temmerman

et al. (2007,

191361 )

Belgium

Sugarcane ( Saccharum spp )

CSTR 2007;

11

- 13 wk. 12

- h avg: CF (4 ppb);

Amb ient (58); Elevated (147) Total biomass (g/plant)

- 40 (

- 30) Grantz and Vu (2009, 195237 )

San Joaquin Valley, CA (N/A)

Sweet Potato

Growth chambers 4 wk 8

- h avg: CF (0 ppb), Ambient (<40 ppb) Elevated (255 ppb) T uber weight

- 14 (

- 11.5) Keutgen et al. (2008, 191690 )

Bonn, Germany (N/A)

Tomato ( Lycopersicon

esculentu m )

OTC

Valencia, Spain 133 days in

1998 8

- h mean ppb: CF 16.3, NF 30.1,

NF+ 83.2

(Various cultivars; early & late harvest) Yield n.s (n.s.) Calvo et al. (2005, 191570 )

Trifolium

Subterraneum

OTC; 2.5

- L pots 29 days 12

- h avg: CF (<7.9±6.3); Ambient (34.4±10.8); Elevated (56.4±22.3) Above

- ground biomass

- 45 (

- 35) Sanz et al. (2005,

196963 )

Madrid, Spain (N: 5, 15 & 30 kg/ha)

Watermelon ( Citrullus lanatus)

OTC 2000, 2001. 90 days AOT40: CF (0 ppm

- h) Ambient (5.7 ppm

- h), Ele vated (34.1 ppm

- h) total fruit yield (kg) n.s. (54) Calatayud et al. (2006, 191482 )

Valencia, Spain (N:0, 14.0 & 29.6 g/pot)

Yellow Nutsedge

OTC; 9

- L pots 8 wk 12

- h avg: 12.8 ± 0.6; 79.9 ± 6.3;

122.7 ± 9.7

(N/A) above

- ground biomass n.s. (n.s.) Grantz and Shrestha (2006,

191702 )

In studies where variables other than O 3 were included in the experimental design, response to O 3 is only provided for the control level of those variables

.

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### Table 9 -17. Summary of studies of effects of ozone exposure on growth of natural vegetation

Species Facility

Location Exposure

Duration O 3 Exposure (Additional Treatment) Response Measured Response Reference

Yellow nutsedge ( Cyperus

esculentus )

CSTR 53 days in 2008 12

- h mean ppb: CF (4); CF+ (60); CF2+ (115) Above

- ground biomass; tubers (g/plant) ns; CF(4.1) CF+(3.9) CF2+(2.7) Grantz et al. (2010,

102161 )

Parlier, CA

35 herbaceous species

OTC

Corvallis, OR 1999

- 2002, May

- August 4

- yr avg; yearly W126 ppm

- h: CF (0), CF+ (21), CF 2+ (49.5) Total community above

ground biomass (35 species) after 4 y ea rs CF (459 g /m 2 ), CF+ (457 g /m 2 ), CF2+ (398 g/m 2 ) Pfleeger et al. (2010,

644281 )

Highbush blackberry ( Rubus argutus )

OTC

Auburn, AL 2004, May

- Aug ust 12

- h mean ppb: CF (21.7), Ambient (32.3), Elevated (73.3) Vegetative regrowth after pruning CF (75.1 g/plant), Ambient (76.4 g/plant), Elevated (73.1 g/plant) Ditchkoff et al. (2009,

192230 )

Horseweed ( Conyza

canadensis )

CSTR

San Joaquin Valley, CA 2005, 2 runs, 28 days each (July

- Aug, Sept) W126 ppm

- hr: CF(0), CF+ (11), CF 2+ (30)

(Glyphosate resistance) Total biomass (g/plant ) Glyphosate sensitive: CF (0.354) CF+ (0.197) CF2+ (0.106)

Glyphosate resistant: CF(0.510) CF+ (0.313) CF2+ (0.143) Grantz et al. ( (2008,

191312 )

AOT60:

Red Oak ( Quercus rubrum )

Forest sites

Look Rock & Twin Creeks Forests, TN 2001

- 2003, April

- October 2001 (11.5), 2002 (24.0), 2003 (11.7)

(Observational study with multiple

environmental Annual circumference increment (change relative to 2001 in y ea r 2002;2003) - 42.8%; +1% McLaughlin et al. (2007,

090348 )

va riables)

AOT60:

Pine spec ies

Forest sites

Look Rock Forest, TN 2001

- 2003, April

- October 2001 (11.5), 2002 (24.0), 2003 (11.7)

(Observational study with multiple

environmental Annual circumference increment (change relative to 2001 in y ea r 2002;2003) - 62.5%;

- 2.9% McLaughlin et al. (2007,

090348 )

variables)

AOT60:

Hickory species

Forest sites

Look Rock Forest, TN 2001

- 2003, April

- October 200 1 (11.5), 2002 (24.0), 2003 (11.7)

(Observational study with multiple

environmental Annual circumference increment (change relative to 2001 in y ea r 2002;2003) - 14%; +30% McLaughlin et al. (2007,

090348 )

variables)

AOT60:

Chestnut Oak ( Quercus prinus )

Forest sites

Look Rock Forest, TN 2001

- 2003, April

- October 2001 (11.5), 2002 (24.0), 2003 (11.7)

(Observational study with multip le

environmental Annual circumference increment (change relative to 2001 in y ea r 2002;2003) +44%; +55% McLaughlin et al. (2007,

090348 )

variables)

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Location Exposure

Duration O 3 Exposure (Additional Treatment) Response Measured Response Reference

AOT60:

Black Cherry ( Prunus rigida )

Forest sites

Twin Creeks Forest, TN 2002

- 2003, April

- October 2002 (24.0), 2003 (11.7)

(Observational study with multiple

environmental Annual circumference increment (change

relative to 200 3 in y ea r 2002) - 75% McLaughlin et al. (2007,

090348 )

variables)

AOT60:

Shortleaf pine ( Pinus echinata )

Forest sites

Twin Creeks Forest, TN 2002

- 2003, April

- October 2002 (24.0), 2003 (11.7)

(Observational study with multiple

environmental Annual circumference increment (change relative to 2003 in y ea r 2002) - 16.8% McLaughlin et al. (2007,

090348 )

variables)

AOT60:

Hemlock ( Tsuga canadensis )

Forest sites

Twin Creeks Forest, TN 2002

- 2003, April

- October 2002 (24.0), 2003 (11.7)

(Observational study with multiple

environmen ta l Annual circumference increment (change relative to 2003 in y ea r 2002) - 21.9% McLaughlin et al. (2007,

090348 )

variables)

AOT60:

Red Maple ( Acer rubrum )

Forest sites

Twin Creeks Forest, TN 2002

- 2003, April

- October 2002 (24.0), 2003 (11.7)

(Observational study with multiple

environmental Annual circumference increment (change relative to 2003 in y ea r 2002) - 59.6% McLaughl in et al. (2007,

090348 )

variables)

Yellow Poplar ( Liriodendron tulipifera )

Forest sites

Look Rock, Oak Ridge, & Twin Creeks Forest, TN 2002 -2 003, April

- October AOT60: 2002 (24.0), 2003 (11.7)

(Observational study with multiple

environmental variables) Annual circumference increment (change relative to 2001 in y ea rs 2002; 2003) - 45.9%;

- 15.25% McLaughlin et al. (2007,

090348 )

AOT60:

Sugar Maple ( Acer saccharum )

Forest sites

Twin Creeks Forest, TN 2002

- 2003, April

- October 2002 (24.0), 2003 (11.7)

(Observational study with mu ltiple

environmental Annual circumference increment (change relative to 2003 in y ea r 2002) - 63.8% McLaughlin et al. (2007,

090348 )

variables)

Trembling aspen ( Populus

tremuloides ), 5 genotypes

Aspen FACE 1998

- 2004,

M ay

- October Cumulative avg

90

- day 12

- h W126. Ambient 3.1 ppm

- h

Elevated: 27.2

ppm -h main stem volume after 7

y ea rs Ambient: 6.22 dm 3 ; Elevated: 4.73 dm 3 Kubiske et al. (2006,

093284 )

Rhinelander, WI (Competition with birch, maple)

Hybrid Poplar ( Populus trichocarpa x Populus deltoides )

OTC 2003, 3 mon ths Daily mean (μg/g): CF(<9), Elevated (85

- 128) Total biomass CF to elevated:

- 12.9% Woo and Hinckley (2005,

191359 )

Seattle, WA

In studies where variables other than O 3 were included in the experimental design, response to O 3 is only provided for the control level of those variables.

March 20 11 9

- 136 DRAFT
– DO NOT CITE OR QUOTE

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