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## 9 ENVIRONMENTAL EFFECT S: OZONE E FFECTS ON VEGETATION AND EC OSYSTEMS

9.1 Introduction

This chapter synthesizes and evaluates the relevant science to help form the scientific 

### 1 foundation for the review of a vegetation

- and ecologically

- based secondary NAAQS for 

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

### 3 definition of welfare effects includes, but is not limited to, effects on soils, water, 

### 4 wildlife, vegetation, visibility, weather, and climate, as well as effects on materials, 

### 5

eco nomic values, and personal comfort and well

- being. The effects of O 3 as a greenhouse 

### 6 gas and its direct effects on climate are discussed in Chapter 10 of this document. 

### 7
The intent of the ISA, according to the CAA, is to “ accurately reflect the latest scien tific 

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

### 9 and 42 U.S.C.7409 (1999) . This chapter of the ISA includes scientific research from 

### 10 biogeochemistry, soil science, plant physiology, and ecology conducted at multipl e scales 

### 11 (e.g., organ, organism, population, community, ecosystem). Key information and 

### 12 judgments formerly found in the AQCDs regarding O 3 effects on vegetation and 

### 13 ecosystems are found in this chapter. This chapter of the O 3 ISA serves to update and 

### 14 revis e Chapter 9 and AX9 of the 2006 O 3 AQCD ( U.S. EPA, 2006b )

. 

### 15

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

### 16 2006 O 3 AQCD. That document concluded that the effects of ambient O 3 on vegetation 

### 17 and eco systems appear to be widespread across the U.S., and experimental studies 

### 18 demonstrated plausible mechanisms for these effects. Ozone effect studies published 

### 19 from 2005 to July 2011 are reviewed in this document in the context of the previous O 3 

### 20 AQCDs. From 2005 to 2011, some areas have had very little new research published and 

### 21 the reader is referred back to sections of the 2006 O 3 AQCD for a more comprehensive 

### 22 discussion of those subjects. This chapter is focused on studies of vegetation and 

### 23 ecosystems tha t occur in the U.S. and that report endpoints or processes most relevant to 

### 24 the review of the secondary standard. Many studies have been published about vegetation 

### 25 and ecosystems outside of the U.S. and North America, largely in Europe and Asia. This 

### 26 docum ent includes discussion of studies of vegetation and ecosystems outside of North 

### 27 America only if those studies contribute to the general understanding of O 3 effects across 

### 28 species and ecosystems. For example, studies outside North America are discussed tha t 

### 29 consider physiological and biochemical processes that contribute to the understanding of 

### 30 effects of O 3 across species. Also, ecosystem studies outside of North America that 

### 31Draft

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- 2 September 2011 contribute to the understanding of O 3 effects on general ecosystem processes are 

### 1 discussed in the chapter. 

### 2
Sections of this chapter first discuss exposure methods, followed by effects on vegetation 

### 3 and ecosystems at various spatial scales and ends with policy

- relevant discussions of 

### 4 exposure indices and exposure

- response. Figure 9

- 1 i s a simplified illustrative diagram of 

### 5 the major pathway through which O 3 enters plants and the major endpoints O 3 may 

### 6 affect. First, Section 9.2 presents a brief overview of various methodologies that have 

### 7 been, and continue to be, central to quantifying O 3 effects on vegetation (AX9.1 of the 

### 8 2006 O 3 AQCD for more detailed discussion) ( U.S. EPA, 2006b ) . Sections 9.3 through 

### 9 9.4 begin with a discussion of effects at the cellular and subcellular level followed by 

### 10 consideration of the O 3 ef fects on plant and ecosystem processes (Figure 9

- 1). In Section 

### 11 9.3, research is reviewed from the molecular to the biochemical and physiological levels 

### 12 in impacted plants, offering insight into the mode of action of O 3 . Section 9.4 provides a 

### 13 review of th e effects of O 3 exposure on major endpoints at the whole plant scale 

### 14 including growth, reproduction, visible foliar injury and leaf gas exchange in woody and 

### 15 herbaceous plants in the U.S., as well as a brief discussion of O 3 effects on agricultural 

### 16 crop yi eld and quality. Section 9.4 also integrates the effects of O 3 on individual plants in 

### 17 a discussion of available research for assessing the effect of O 3 on ecosystems, along 

### 18 with available studies that could inform assessments of various ecosystem services (See 

### 19 section 9.4.1.2). The development of indices of O 3 exposure and dose modeling is 

### 20 discussed in Section 9.5. Finally, exposure

- response relationship s for a number of tree 

### 21 species, native vegetation, and crop species and cultivars are reviewed, tabulated, and 

### 22 compared in Section 9.6 to form the basis for an assessment of the potential risk to 

### 23 vegetation from current ambient levels of O 3 . 

### 24Draft

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### Figure 9

- 1 A n illustrative diagram of the major pathway through which O 3 

### enters plants and the major endpoints that O 3 

### may affect in plants and ecosystems.



## 9.2 Experimental Exposure Methodologies

9.2.1 Introduction

A variety of methods for studying plant response to O 3 exposure s have been developed 

### 1 over the last several decades. Methodological advancements since 2006 have not 

### 2 fundamentally altered our understanding of O 3 effects on plants or ecosystems. The 

### 3 majority of methodologies currently used have been discussed in detail i n the 1996 O 3 

### 4 AQCD and 2006 O 3 AQCD. This section will serve as a short overview of the 

### 5 methodologies and the reader is referred to the previous O 3 AQCDs for more in

- depth 

### 6 discussion. 

### 7 O 3 

### exposure

O 3 

### uptake & physiology (Fig 9

- 2)

• Antioxidant metabolism up

- regulated

• Decreased photosynthesis

• Decreased stomatal conductance or sluggish stomatal response



### Effects on leaves

• Visible leaf injury

• Altered leaf production

• Altered leaf chemical composition



### Plant growth (Fig 9.8)

• Decreased biomass accumulation

• Altered reproduction

• Altered carbon allocation

• Altered crop quality



### Belowground processes (Fig 9.8)

• Altered litter production and decomposition

• Altered soil carbon and nutrient cycling

• Altered soil fauna and microbial communitiesDifferential O3sensitivity



### Affected ecosystem services

• Decreased productivity

• Decreased C sequestration

• Altered water cycling (Fig 9

- 7)

• Altered community composition (i.e., plant, insect & microbe)Draft

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### 9.2.2 “ Indoor, ” Controlled Environment, and Greenhouse Chambers

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

### 1 glass or plastic

- covered chambers, often located within greenhouses, into which a flow of 

### 2 O 3 - enriched air or oxygen could be passed to provide the exposure. The types, shapes, 

### 3 styl es, materials of construction, and locations of these chambers have been numerous. 

### 4 Hogsett et al. ( 1987a ) have summarized the construction and performance of more 

### 5 elaborate and better instrumented chambers since the 1960s, including those installed in 

### 6 greenhouses (with or without some control of temperature and light intensity). 

### 7
One greenhouse chamber approach that continues to yield useful information on the 

### 8 relati onships of O 3 uptake to both physiological and growth effects employs continuous 

### 9 stirred tank reactors (CSTRs) first described by Heck et al. ( 1978 ) . Although originally 

### 10 developed to permit mass

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

### 11 widened to include short

- term physiological and growth studies of O 3 × CO 2 interactions 

### 12 ( Loats and Rebbeck, 1999 ; Reinert et al., 1997 ; Rao et al., 1995 ; Reinert and Ho, 1995 ; 

### 13 Heagle et al., 1994a ) , and validation of visible folia r injury on a variety of plant species 

### 14 ( Kline et al., 2009 ; Orendovici et al., 2003 ) . In many cases, supplementary lighting and 

### 15 temperature control of the surrounding structure have been used to control or modify the 

### 16 environmental conditions ( Heagle et al., 1994a )

. 

### 17
Many investigations have uti lized commercially available controlled environment 

### 18 chambers and walk

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

### 19 controlled air

- volume. Such chambers continue to find use in genetic screening and in 

### 20 physiological and biochemical s tudies aimed primarily at improving our understanding of 

### 21 modes of action. For example, some of the studies of the O 3 responses of common 

### 22 plantain ( Plantago major ) populations have been conducted in controlled environment 

### 23 chambers ( Whitfield et al., 1996 ; Reiling and Davison, 1994 )

. 

### 24
More recently, some researchers have been interested in attempting to investigate direct 

### 25 O 3 effects on reproductive processes, separate from the effects on vegetative processes 

### 26 ( Black et al., 2010 ) . For this purpose, controlled exposure systems have been e mployed 

### 27 to expose the reproductive structures of annual plants to gaseous pollutants independently 

### 28 of the vegetative component ( Black et al., 2010 ; Stewart et al., 1996 )

. 

### 29

9.2.3 Field Chambers

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

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

### 31 ( 1973 ) . The OTC method continues to be a widely used technique in the U.S. and Europe 

### 32Draft

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- 5 September 2011 for exposing plants to varying levels of O 3 . Most of the new information confirms earlier 

### 1 conclusions and provides addit ional support for OTC use in assessing plant species and in 

### 2 developing exposure

- response relationships. Chambers are generally ~3 m in diameter 

### 3 with 2.5

- m

- high walls. Hogsett et al. ( 1987b ) described in detail many of the various 

### 4 modifications to the original OTC designs that appeared subsequently, e.g., the use of 

### 5 larger chambers for exposing small trees ( Kats et al., 1985 ) or grapevines ( Mandl et al., 

### 6 1989 ) , the addition of a conical baffle at the top to improve ventilation ( Kats et al., 1976 )
, 

### 7 a frustum at the top to reduce ambient air incursions, and a plastic rain

- cap to exclude 

### 8 precipitation ( Hogsett et al., 1985 ) . All versions of OTCs included the discharge of air via 

### 9 ports in annular ducting or interiorly perforated double

- layered walls at the base of the 

### 10 chambers to provide turbulent mixing and the upward mas s flow of air. 

### 11
Chambered systems, including OTCs, have several advantages. For instance, they can 

### 12 provide a range of treatment levels including charcoal

- filtered (CF), clean

- air control, and 

### 13 several above ambient concentrations for O 3 experiments. Dependin g on experimental 

### 14 intent, a replicated, clean

- air control treatment is an essential component in many 

### 15 experimental designs. The OTC can provide a consistent, definable exposure because of 

### 16 the constant wind speed and delivery systems. Statistically robust c oncentration

- response 

### 17 (C

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

### 18 various alternative air quality scenarios on vegetation response. Nonetheless, there are 

### 19 several characteristics of the OTC design and operation th at can lead to exposures that 

### 20 might differ from those experienced by plants in the field. First, the OTC plants are 

### 21 subjected to constant air flow turbulence, which, by lowering the boundary layer 

### 22 resistance to diffusion, may result in increased uptake. Th is may lead to an 

### 23 overestimation of effects relative to areas with less turbulence ( Krupa et al., 1995 ; Legge 

### 24 et al., 1995 ) . H owever, other research has found that OTC ’ s may slightly change vapor 

### 25 pressure deficit (VPD) in a way that may decrease the uptake of O 3 into leaves ( Piikki et 

### 26 al., 2008b ) . As with all methods that expose vegetation to modified O 3 concentrations in 

### 27 chambers, OTCs create internal environments that differ from ambient air. This so

- called 

### 28

“ chamber effect ” refers to the modification of microclimatic variables, including reduced 

### 29 and uneven light intensity, uneven rainfall, constant wind speed, reduced dew formation, 

### 30 and increased air temperatures ( Fuhrer, 1994 ; Manning and Krupa, 1992 ) . However, in at 

### 31 least one case where canopy resistance was quantified in OTCs and in the field, it was 

### 32 determined that gaseous pollutant exposure to crops in OTCs was similar to that which 

### 33 would have occurred at the same concentration in the field ( Unsworth et al., 1984a
, b )

. 

### 34 Because of the standardized methodology and protocols used in National Crop Loss 

### 35 Assessment Network (NCLAN) and other programs, the database can be assumed to be 

### 36 internally consistent. 

### 37Draft

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- 6 September 2011 While it is clear that OTCs can alter some aspects of the microenvironment and plant 

### 1 growth, it is important to establish whether or not these differences affect the relative 

### 2 response of a plant to O 3 . As noted in the 1996 O 3 AQCD, e vidence from a number of 

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

### 4 essentially the same regardless of exposure system used and chamber effects did not 

### 5 significantly affect response. For example, a study of chamber effects examined the 

### 6 responses of tolerant and sensitive white clover clones ( Trifolium repens ) to ambient O 3 

### 7 in greenhouse, open top, and ambient plots ( Heagle et al., 1996 ) . The response found in 

### 8 OTCs was the same as in ambient plots. 

### 9
Another type of field chamber called a “ terracosm ” has been developed and used in 

### 10 recent studies ( Lee et al., 2009a ) . Concern over the need to establish realistic plant

- litter



### 11 soil relationships as a prerequisite to studies of the effects of O 3 and CO 2 enrichment on 

### 12 ponderosa pine ( Pinus ponderosa ) seedlings led Tingey et al. ( 1996 ) to develop closed, 

### 13 partially environmentally controlled, sun

- lit chambers ( “ terracosms ” ) incorporating 1

- m



### 14 deep lysimeters containing forest soil in which the appropriate horizon structure was 

### 15 retained. 

### 16

Other researchers h ave recently published studies using another type of out

- door chamber 

### 17 called recirculating Outdoor Plant Environment Chambers (OPECs) ( Flowers et al., 

### 18 2007 ) . These closed chambers are approximately 2.44 m×1.52 m with a growth volume 

### 19 of approximately 3.7 m 3 in each chamber. These chambers admit 90% of full sunlight and 

### 20 control temperature, humidity and vapor pressure ( Fiscus et al., 1999 )

. 

### 21

9.2.4 Plume and FACE

- Type Systems

Plume systems are chamberless exposure facilities in which the atmosphere surrounding 

### 22 plants in the field is modified by the injection of pollutant gas into the air above or 

### 23 around them from multiple orifices spaced to permit diffusion and turbulence, so as to 

### 24 establish relatively homogeneous conditions as the individual plumes disperse and mix 

### 25 with the ambient air. They can only be used to increase the O 3 levels in the ambient air. 

### 26
The most common plume system used in the U.S. is a modification of the free

- air carbon



### 27 dioxide/ozone enrichment (FACE) system ( Hendrey et al., 1999 ; Hendrey and Kimball, 

### 28 1994 ) . Although originally designed to provide chamberless field facilities for studying 

### 29 the CO 2 effects of climate change, FACE systems have been adapted to include the 

### 30 dispensing of O 3 ( Karnosky et al., 1999 ) . This method has been employed in Illinois 

### 31 (SoyFACE) to study soybeans ( Morgan et al., 2004 ; Rogers et al., 2004 ) and in 

### 32 Wisconsin (Aspen FACE) to study trembling aspen ( Populus tremuloides ), birch ( Betu la 

### 33 papyrifera ) and maple ( Acer saccharum ) ( Karnosky et al., 1999 ) . Volk et al. ( 2003 ) also 

### 34Draft

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- 7 September 2011 described a similar system for exposing grasslands that uses 7

- m diameter plots. FACE 

### 1 systems discharge the pollutant gas (O 3 and/or CO 2 ) through orifices spaced along an 

### 2 annular ring (or torus) or at different heights on a ring of vertical pipes. Computer



### 3 controlled feedback from the monitoring of gas concentration regulates the feed rate of 

### 4 enriched air to the dispersion p ipes. Feedback of wind speed and direction information 

### 5 ensures that the discharges only occur upwind of the treatment plots, and that discharge is 

### 6 restricted or closed down during periods of low wind speed or calm conditions. The 

### 7 diameter of the arrays and their height (25

- 30 m) in some FACE systems requires large 

### 8 throughputs of enriched air per plot, particularly in forest tree systems. The cost of the 

### 9 throughputs tends to limit the number of enrichment treatments, although Hendrey et al. 

### 10 ( 1999 ) argued that the cost on an enriched volume basis is comparable to that of chamber 

### 11 systems. 

### 12

Altho ugh plume systems make virtually none of the modifications to the physical 

### 13 environment that are inevitable with chambers, their successful use depends on selecting 

### 14 the appropriate numbers, sizes, and orientations of the discharge orifices to avoid “ hot



### 15
spo ts ” resulting from the direct impingement of jets of pollutant

- enriched air on plant 

### 16 foliage ( Werner and Fabian, 2002 ) . Because mixing is unassisted and completely 

### 17 dependent on wind turbulence and diffusion, local gradients are inevitable especially in 

### 18 large

- scale systems. FACE systems have provisions for shutting down under low wind 

### 19 speed or calm conditions and for an experimental area that is usually defined within a 

### 20 generous border in order to strive for homogeneity of the exposure concentrations within 

### 21 the treatment area. They are also dependent upon continuous computer

- controlled 

### 22 feedback of the O 3 concentrations in the mixed treated air and of the meteorological 

### 23 conditions. Plume and FACE systems also are unable to reduce O 3 levels below ambient 

### 24 in areas where O 3 concentrations are phytotoxic. 

### 25

9.2.5 Ambient Gradients

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

### 26 responses over multiple levels of exposure. However, few such gradients can be found 

### 27 that meet the rigorous statistical requirements for comparable site characteristics such as 

### 28

s oil type, temperature, rainfall, radiation, and aspect ( Manning and Krupa, 1992 ) ; 

### 29 although with small plants, soil variability can be avoided by the use of plants in large 

### 30 pots. The use of soil monoliths transported to various locations along natural O 3 gradients 

### 31 is another possible app roach to overcome differences in soils; however, this approach is 

### 32 also limited to small plants. 

### 33Draft

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- 8 September 2011 Studies in the 1970s used the natural gradients occurring in southern California to assess 

### 1 yield losses of alfalfa and tomato ( Oshima et al., 1977 ; Oshima et al., 1976 ) . A transect 

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

### 3 confounded by differences in the concurrent gradients of SO 2 and NO 2 pollution 

### 4 ( Ashmore et al., 1988 ) . Studies of forest tree species in national parks in the eastern U.S. 

### 5 ( Winner et al., 1989 ) revealed increasing gradients of O 3 and visible foliar injury with 

### 6 increased elevation. 

### 7
Several studies have used the San Bernardino Mountains Gradient Study in southern 

### 8 Cal ifornia to study the effects of O 3 and N deposition on forests dominated by ponderosa 

### 9 and Jeffrey pine ( Jones and Paine, 2006 ; Arbaugh et al., 2003 ; Grulke, 1999 ; Miller and 

### 10 Elderman, 1977 ) . However, it is difficult to separate the effects of N and O 3 in some 

### 11 instances in these studies ( Arbaugh et al., 2003 ) . An O 3 gradient in Wisconsin has been 

### 12 used to study foliar injury in a series of trembling a spen clones ( Populus tremuloides ) 

### 13 differing in O 3 sensitivity ( Maňkovská et al., 2005 ; Karnosky et al., 1999 )

. 

### 14
More recently, studies have been published that have used natural gradients to study a 

### 15 variety of endpoints and species. For exa mple, Gregg et al. ( 2003 ) studied cottonwood 

### 16 saplings grown in an urban to rural gradient of O 3 in the New York City area. The 

### 17 secondary nature of the reactions of O 3 formation and NO X titration reactions within the 

### 18 city center resulted in significantly higher cumulative O 3 exposures in the rural sites. The 

### 19 results of this gradient study were similar to those of a parallel OTC study. Also, the U.S. 

### 20 forest service Forest Inventory and An alysis (FIA) program uses large

- scale O 3 exposure 

### 21 patterns across the continental U.S. to study occurrences of foliar injury due to O 3 

### 22
exposure ( Smith et al., 2003 ) (Sect ion 9.4.2). Finally, McLaughlin et al. ( 2007a ; 2007b ) 

### 23 used spatial and temporal O 3 gradients to study forest growth and water use in the 

### 24 southern Appalach ians. These studies found varying O 3 exposures between years and 

### 25 between sites. 

### 26

9.2.6 Comparative Studies

All experimental approaches used to expose plants to O 3 have strengths and weaknesses. 

### 27 One potential weakness of laboratory, greenhouse, or field chamber st udies is the 

### 28 potential effect of the chamber on micrometeorology. In contrast, plume, FACE and 

### 29 gradient systems are limited by the very small number of possible exposure levels 

### 30 (almost always no more than two), small replication and an inability to reduce O 3 levels 

### 31 below ambient. In general, experiments that aim at characterizing the effect of a single 

### 32 variable, e.g., exposure to O 3 , must not only manipulate the levels of that variable, but 

### 33 also control potentially interacting variables and confounders, or else account for them. 

### 34Draft

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- 9 September 2011 However, while increasing control of environmental variables makes it easier to discern 

### 1 the effect of the variable of interest, it must be balanced with the ability to extend 

### 2 conclusions to natural, non

- experimental settings. More na turalistic exposure systems, on 

### 3 the other hand, let interacting factors vary freely, resulting in greater unexplainable 

### 4 variability. The various exposure methodologies used with O 3 vary in the balance each 

### 5 strikes between control of environmental inputs, c loseness to the natural environment, 

### 6 noisiness, and ability to make general inferences. 

### 7
Studies have examined the comparability of results obtained though the various exposure 

### 8 methodologies. As noted in the 1996 O 3 AQCD, evidence from the comparative stud ies 

### 9 of OTCs and from closed chamber and O 3 - exclusion exposure systems on the growth of 

### 10 alfalfa (Medicago sativa ) by Olszyk et al. ( 1986 ) suggested that, since significant 

### 11 differences were found for fewer than 10% of the growth parameters measured, the 

### 12 responses were, in general, essentially the same regardless of exposure system used, and 

### 13 chamber effects did not significantly affect respon se. In 1988, Heagle et al. ( 1988 ) 

### 14 concluded: “ Although chambe r effects on yield are common, there are no results showing 

### 15 that this will result in a changed yield response to O 3 . ” A study of the effects of an 

### 16 enclosure examined the responses of tolerant and sensitive white clover clones ( Trifolium 

### 17

repens ) to ambient O 3 in a greenhouse, open

- top chamber, and ambient (no chamber) 

### 18 plots ( Heagle et al., 1996 ) . For individua l harvests, greenhouse O 3 exposure reduced the 

### 19 forage weight of the sensitive clone 7 to 23% more than in OTCs. However, the response 

### 20 in OTCs was the same as in ambient plots. Several studies have shown very similar 

### 21 response of yield to O 3 for plants grown in pots or in the ground, suggesting that even 

### 22 such a significant change in environment does not alter the proportional response to O 3 , 

### 23 providing that the plants are well watered ( Heagle et al., 1983 ; Heagle, 1979 )

. 

### 24

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

### 25 experiments, and gradient studies. For example, a series of studies undertaken at Aspen 

### 26 FACE ( Isebrands et al., 2001 ; Isebrands et al., 2000 ) showed that O 3 symptom 

### 27 expression was generally similar in OTCs, FACE, and ambient O 3 gradient sites, and 

### 28 supported the previously observed variation among trembling aspen clones using OTCs 

### 29 ( Maňkovská et al., 2005 ; Karnosky et al., 1999 )

. In the SoyFACE experiment in Illinois, 

### 30 soybean (Pioneer 93B15 cultivar) yield loss data from a two

- year study was published 

### 31 ( Morgan et al., 2006 ) . This cultivar is a recent selection and, like most modern cultivars, 

### 32 has been selected under an already high current O 3 exposure. It was fou nd to have 

### 33 average sensitivity to O 3 compared to 22 other cultivars tested at SoyFACE. In this 

### 34 experiment, ambient hourly O 3 concentrations were increased by approximately 20% and 

### 35 measured yields were decreased by 15% in 2002 as a result of the increased O 3 exposure 

### 36 ( Morgan et al., 2006 ) . To compare these results to chamber studies, Morgan et al. ( 2006 ) 

### 37 calculated the expected yield loss from a linear relationship constructed from chamber 

### 38Draft

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- 10 September 2011 data using seven

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

### 1 expected yield loss from the 2002 O 3 exposure using that linear relationship. In another 

### 2 study, Gregg et al. ( 2006
, 2003 ) found similar O 3 effects on cottonwood sapling biomass 

### 3 growth along an ambie nt O 3 gradient in the New York City area and a parallel OTC 

### 4 study. 

### 5
Finally, EPA conducted comparisons of exposure

- response model predictions based on 

### 6 OTC studies, and more recent FACE observations. These comparisons include yield of 

### 7 annual crops, and bioma ss growth of trees. They are presented in section 9.6.3 of this 

### 8 document. 

### 9



## 9.3 Mechanisms Governing Vegetation Response to Ozone

9.3.1 Introduction

This section focuses on the effects of O 3 stress on plants and their responses to that stress 

### 10 on the molecular, biochem ical and physiological levels. First, the pathway of O 3 uptake 

### 11 into the leaf and the initial chemical reactions occurring in the substomatal cavity and 

### 12 apoplast will be described (Section 9.3.2); additionally, direct effects of O 3 on the 

### 13 stomatal apparatus will be discussed. Once O 3 has entered the substomatal cavity and 

### 14 apoplast, it is thought that the cell must be able to sense the presence of O 3 or its 

### 15 breakdown products in order to initiate the rapid changes in signaling pathways and gene 

### 16 expression tha t have been measured in O 3 - treated plants. While it remains unclear exactly 

### 17 how O 3 and/or its breakdown products are sensed in the apoplast, much progress has been 

### 18 made in examining several different mechanisms that may contribute both to sensing the 

### 19
prese nce of O 3 and its breakdown products, and also initiating a signal transduction 

### 20 cascade, which will be described in Section 9.3.3.1. The next section focuses on changes 

### 21 in gene and protein expression measured in plants exposed to O 3 , with particular 

### 22 emphas is on results from transcriptome (all RNA molecules produced in a cell) and 

### 23 proteome (all proteins produced in a cell) analyses (Section 9.3.3.2). Subsequently, the 

### 24 role of phytohormones such as salicylic acid (SA), ethylene (ET), jasmonic acid (JA), and 

### 25

a bscisic acid (ABA) and their interactions in both signal transduction processes and in 

### 26 determining plant response to O 3 is discussed in Section 9.3.3.3. After O 3 uptake and 

### 27 sensing, some plants can respond to the oxidative stress with detoxification to min imize 

### 28 damage. These mechanisms of detoxification, with particular emphasis on antioxidant 

### 29 enzymes and metabolites, are reviewed in Section 9.3.4. The next section focuses on 

### 30 changes in primary and secondary metabolism in plants exposed to O 3 , looking at 

### 31 ph otosynthesis, respiration and several secondary metabolites, some of which may also 

### 32Draft

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- 11 September 2011 act as antioxidants and protect the plant from oxidative stress (Section 9.3.5). For many 

### 1 of these topics, information from the 2006 O 3 AQCD has been summarized, as this 

### 2
in formation is still valid and supported by more recent findings. For other topics, such as 

### 3 genomics and proteomics, which have arisen due to the availability of new technologies, 

### 4 the information is based solely on new publications with no reference to the 2 006 O 3 

### 5 AQCD. 

### 6
As Section 9.3 focuses on mechanisms underlying effects of O 3 on plants and their 

### 7 response to it, the conditions that are used to study these mechanisms do not always 

### 8 reflect conditions that a plant may be exposed to in an agricultural setting or natural 

### 9 ecosystem. The goal of many of these studies is to generate an O 3 effect in a relatively 

### 10 short period of time and not always to simulate ambient O 3 exposures. Therefore, plants 

### 11 are often exposed to unrealistically high O 3 concentrations for sev eral hours or days 

### 12 (acute exposure), and only in some cases to ambient or slightly elevated O 3 

### 13 concentrations for longer time periods (chronic exposure). Additionally, the plant species 

### 14 utilized in these studies are often not agriculturally important or co mmonly found as part 

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

### 16 as they are easy to work with, and mutants or transgenic plants are easy to develop or 

### 17 have already been developed. Furthermore, the Arabidopsis ge nome has been sequenced, 

### 18 and much is known about the molecular basis of many biochemical and cellular 

### 19
processes. 

### 20
Many of the studies described in this section focus on changes in the expression of genes 

### 21 in O 3 - treated plants. Changes in gene expression (i.e., either up

- or down

- regulation of 

### 22 gene expression) do not always translate into changes in protein quantity and/or activity, 

### 23 as there are many levels of post

- transcriptional and post

- translational modifications 

### 24 which impact protein quantity and activ ity. Many studies do not evaluate whether the 

### 25 observed changes in gene expression lead to changes at the protein level and, therefore, it 

### 26 is not always clear how relevant the changes in gene expression are in determining plant 

### 27 response to O 3 . However, with the advent of proteomics, some very recent studies have 

### 28 evaluated changes in protein expression for large numbers of proteins in O 3 treated 

### 29 plants, and the findings from these studies support the previous results regarding changes 

### 30 in gene expression studi es as a result of O 3 exposure. The next step in the process is to 

### 31 determine the implications of the measured changes occurring at the cellular level to 

### 32 whole plants and ecosystems, which is an important topic of study which has not been 

### 33 widely addressed. 

### 34

T he most significant new body of research since the 2006 O 3 AQCD is on the 

### 35 understanding of molecular mechanisms underlying how plants are affected by O 3 ; a 

### 36 significant number of recent studies reviewed here focus on changes in gene expression 

### 37Draft

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- 12 September 2011 in plants exp osed to elevated O 3 . Conclusions from the 2006 O 3 AQCD have been 

### 1 supported by these new studies, and the advent of new technologies has allowed for a 

### 2 more comprehensive understanding of the mechanisms governing how plants are affected 

### 3 by O 3 . 

### 4

In summary, t hese new studies have increased knowledge of the molecular, biochemical 

### 5 and cellular mechanisms occurring in plants in response to O 3 by often using artificial 

### 6 exposure conditions and model organisms. This information adds to the understanding of 

### 7 the basic biology of how plants are affected by oxidative stress in the absence of any 

### 8 other potential stressors. The results of these studies provide important insights, even 

### 9 though they may not always directly translate into effects observed in other plants under 

### 10 more realistic exposure conditions. 

### 11

9.3.2 Ozone Uptake into the Leaf

Appendix AX9.2.3 of the 2006 O 3 AQCD clearly described the process by which O 3 

### 12 enters plant leaves through open stomata ( U.S. EPA, 2006b ) . This information continues 

### 13 to be valid and is only summarized here. 

### 14
Stomata provide the principal pathway for O 3 to enter and affect plants ( Massman and 

### 15 Grantz, 1995 ; Fuentes et al., 1992 ; Reich, 1987 ; Leuning et al., 1979 ) . Ozone moves into 

### 16 the leaf interior by diffusing through open stomata, and environmental conditions which 

### 17 promote high rates of gas exchange will favor the uptake of the pollutant by the leaf. 

### 18 Factors that may limit uptake include boundary layer resistanc e and the size of the 

### 19 stomatal aperture (Figure 9

- 2) ( U.S. EPA, 2006b ) . Once inside the substomatal cavity, O 3 

### 20 is thought to rapidly react with the aqueous apoplast to form breakdown products known 

### 21 as reactive oxygen species (ROS), such as hydrogen peroxide (H 2 O 2 ), superoxide (O 2 ), 

### 22 hydroxyl radicals (HO · ) and peroxy radicals (HO 2 · ) (Figure 9

- 3). Hydrogen peroxide is 

### 23 not only a toxic breakdown product of O 3 , but has been shown to function as a signaling 

### 24 molecule, which is activated in res ponse to both biotic and abiotic stressors. The role of 

### 25
H 2 O 2 in signaling was described in detail in the 2006 O 3 AQCD. Additional organic 

### 26 molecules present in the apoplast or cell wall, such as those containing double bonds or 

### 27 sulfhydryls that are sensitiv e to oxidation, could also be converted to oxygenated 

### 28 molecules after interacting with O 3 (Figure 9

- 4). These reactions are not only pH 

### 29 dependent, but are also influenced by the presence of other molecules in the apoplast 

### 30 ( U.S. EPA, 2006b ) . The 2006 O 3 AQCD provided a comprehensive summary of what is 

### 31 known about the possible interactions of O 3 with other biomolecules ( U.S. EPA, 2006b )

. 

### 32 It is in the apoplast that initial detoxification reactions by antioxidant metabolite s and 

### 33 enzymes take place, and these initial reactions are critical to reduce concentrations of the 

### 34Draft

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- 13 September 2011 oxidative breakdown products of O 3 ; these reactions are described in more detail in 

### 1 Section 9.3.4 of this document. 

### 2

9.3.2.1 Changes in Stomatal Function

The effects of O 3 exposure on stomatal conductance have been reviewed in detail in 

### 3 previous O 3 AQCDs. Although the nature of these effects depends upon many different 

### 4 factors, including the plant species, concentration and duration of the O 3 exposure, and 

### 5 prevailing meteorological conditions, stomatal conductance is often negatively affected 

### 6 by plant exposure to O 3 ( Wittig et al., 2007 ) . Decreases in conductance have been shown 

### 7 to result from declines in photosynthetic carboxylation capacity, leading to a buildup of 

### 8 CO 2 in the substomatal cavity and subsequent stomatal closure ( Wittig et al., 2007 )

. 

### 9 However, results from the use of Arabidopsis mutants and new technologies, which allo w 

### 10 for analysis of guard cell function in whole plants rather than in isolated guard cells or 

### 11 epidermal peels, suggest that O 3 may also have a direct impact on stomatal guard cells, 

### 12 leading to alterations in stomatal conductance. The use of a new simultaneo us O 3 

### 13 exposure/gas exchange device has demonstrated that exposure of Arabidopsis ecotypes 

### 14 Col

- 0 and Ler to 150 ppb O 3 resulted in a 60

- 70% decline in stomatal conductance within 

### 15
9

- 12 minutes of beginning the exposure. Twenty to thirty minutes later, stomat al 

### 16 conductance had returned to its initial value, even with continuing exposure to O 3 , 

### 17 indicating a rapid direct effect of O 3 on stomatal function ( Kollist et al., 2007 ) . This 

### 18 transient dec rease in stomatal conductance was not observed in the abscisic acid 

### 19 insensitive (ABI2) Arabidopsis mutant. As the ABI2 protein is thought to regulate the 

### 20 signal transduction process involved in stomatal response downstream of ROS 

### 21 production, the authors su ggest that the transient decrease in stomatal conductance in the 

### 22 Col

- 0 and Ler ecotypes results from the biological action of ROS in transducing signals, 

### 23 rather than direct physical damage to guard cells by ROS ( Kollist et al., 2007 ) . This rapid 

### 24 transient decrease in stomatal conductance was also not observed when exposing the 

### 25 Arabidopsis mutant slac1 (slow anion channel

- associated 1) to 200 ppb O 3 ( Vahisalu et 

### 26 al., 2008 ) . The SLAC1 protein was shown to be essential for guard cell slow anion 

### 27 channel functioning and for stomatal closure in response to O 3 . Based on additional 

### 28 studies using a variety of Arabidopsis mutants impaired in various aspects of stomat al 

### 29 function, Vahisalu et al. ( 2010 ) suggest that the presence of ROS in the guard cell 

### 30 apoplast (formed either by O 3 breakdown or through ROS production from NADPH 

### 31 oxidase activity) leads to the activation of a signaling pathway in the guard cells, which 

### 32 includes SLAC1, and results in stomatal closure. 

### 33
A review by McAinsh et al. ( 2002 ) discusses the role of calcium as a part of the signal 

### 34 transduction pathway involved in regulating stomatal responses to pollutant stress. A 

### 35Draft

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- 14 September 2011 number of studies in this review provide some evidence that exposure to O 3 increases the 

### 1 cytosolic free calcium concentration ([Ca 2+ ]cyt) in guard cells, which may result in an 

### 2 inhibition of the plasma membrane inward

- rectifying K + channels in guard cells, which 

### 3 allow for the K + uptake needed for stomatal opening ( McAinsh et al., 2002 ; Torsethaugen 

### 4 et al., 1999 ) . This would comprom ise the ability of the stomata to respond to various 

### 5 stimuli, including light, CO2 concentration and drought. Pei et al. ( 2000 ) reported that the 

### 6 presence of H 2 O 2 activated Ca 2+

- permeable channels, which mediate increases in 

### 7 [Ca 2+ ]cyt in guard cell plasma membranes of Arabidopsis. They also determined that 

### 8 abscisic acid (ABA) induced H 2 O 2 productio n in guard cells, leading to ABA

- induced 

### 9 stomatal closure via activation of the membrane Ca 2+ channels. Therefore, it is possible 

### 10 that H 2 O 2 , a byproduct of O 3 breakdown in the apoplast, could disrupt the Ca 2+

- ABA 

### 11 signaling pathway that is involved in regul ating stomatal responses ( McAinsh et al., 

### 12 2002 ) . The studies described here provide some evidence to suggest that O 3 and its 

### 13 breakdown products can directly affect stomatal functioning by impacting the signal 

### 14 transduction pathways which regulate guard cells. Stomatal sluggishness has been 

### 15 described as a delay in stomatal response to changing environmental conditions in 

### 16 sensitive species exposed to higher concentrations and/or longer

- term O 3 exposures 

### 17 ( Paoletti and Grulke, 2 010
, 2005 ; McAinsh et al., 2002 ) . It is possible that the signaling 

### 18 pathways described above could be involved in mediating this stomatal sluggishness in 

### 19 some plant species under certain O 3 exposure conditions ( Paoletti and Grulke, 2005 ; 

### 20 McAinsh et al., 2002 )

. 

### 21Draft

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- 15 September 2011 

### Figure 9

- 2 The microarchitecture of a dicot leaf. While details among species vary, the general overview remains the same. Light that drives photosynthesis generally falls upon the upper (adaxial) leaf surface. Carbon dioxide and ozone enter through the stomata on the lower (abaxial) leaf surface, 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.Draft

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- 16 September 2011 Source: Adapted from Mudd ( 1996 )

. 

### Figure 9

- 4 The Crigee mechanism of ozone attack of a double bond. (a ) The typical Crigee mechanism is shown in which several reactions paths from the initial product is shown. (b) Typical reaction of ascorbic acid with ozone.Draft

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- 17 September 2011 

### 9.3.3 Cellular to Systemic Responses

9.3.3.1 Ozone Sensing and Signal Transduction

New technologies allowing for large

- scale analysis of oxidative stress

- induced changes in 

### 1 gene expression have facilitated the study of signal transduction processes associated 

### 2 with the perception and integration of responses to the stress. Many of these studies have 

### 3 been conducted us ing Arabidopsis or tobacco plants, for which a variety of mutants are 

### 4 available and/or which can be easily genetically modified to generate either loss

- of



### 5 function or over

- expressing genotypes. Several comprehensive review articles provide an 

### 6 overview of w hat is known of O 3 - induced signal transduction processes and how they 

### 7 may help to explain differential sensitivity of plants to the pollutant ( Ludwikow and 

### 8 Sadowski, 2008 ; Baier et al., 2005 ; Kangasjarvi et al., 2005 ) . Additionally, analysis of 

### 9 several studies of transcriptome changes has also al lowed for the compilation of these 

### 10 data to determine an initial time

- course for O 3 - induced activation of various signaling 

### 11 compounds ( Kangasjarvi et al., 2005 )

. 

### 12
A number of different mechanisms for plant sensing of O 3 have been proposed; however, 

### 13 there is still much that is not known about this process. Some of the earliest events that 

### 14 occur in plants exposed to O 3 have been described in the guard cells of stomata. Reactive 

### 15 oxygen species were observed in the chloroplasts of guard cells in the O 3 tolerant Col

- 0 

### 16 Arabidopsis thaliana ecotype plants within 5 minutes of plant exposure to 350 ppb O 3 

### 17 ( Joo et al., 2005 ) . Reactive o xygen species from the breakdown of O 3 in the apoplast are 

### 18 believed to activate GTPases (G

- proteins), which, in turn, activate several intracellular 

### 19 sources of ROS, including ROS derived from the chloroplasts. G

- proteins are also 

### 20 believed to play a role in activating membrane

- bound NADPH oxidases to produce ROS 

### 21 and, as a result, propagate the oxidative burst to neighboring cells ( Joo et al., 2005 )

. 

### 22 Therefore, G

- proteins are recognized as important molecules involved in plant responses 

### 23 to O 3 and may play a role in perceiving ROS from the breakdown of O 3 in the apoplast 

### 24 ( Kangasjarvi et al., 2005 ; Booker et al., 2004b )

. 

### 25
A change in the redox state of the plant and the oxidation of sensitive molecules in its elf 

### 26 may represent a means of perception and signaling of oxidative stress in plants. 

### 27 Disulfide

- thiol conversions in proteins and the redox state of the glutathione pool may be 

### 28 important components of redox sensing and signal transduction ( Foyer and Noctor, 

### 29 2005a
, b )

. 

### 30

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

### 31 response to oxidative stress. The influx of Ca 2+ from the apoplast into the cell occurs 

### 32 early during plant exposure to O 3 , and it is thought to play a role in regulating the activity 

### 33Draft

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- 18 September 2011 of protein kinases, which are discussed below ( Baier et al., 2005 ; Hamel et al., 200 5 )

. 

### 1 Calcium channel blockers inhibited O 3 - induced activation of protein kinases in tobacco 

### 2 suspension cells exposed to 500 ppb O 3 for 10 minutes, indicating that the opening of 

### 3 Ca 2+ channels is an important upstream signaling event or that the as yet unknown 

### 4 upstream process has a requirement for Ca 2+ ( Samuel et al., 2000 )

. 

### 5

Further transmission of information re garding the presence of ROS to the nucleus t 

### 6 involves mitogen

- activated protein kinases (MAPK), which phosphorylate proteins and 

### 7 activate various cellular responses ( Hamel et al., 2005 ) . Mitogen

- activated protein 

### 8 kinases are induced in several different plant species in response to O 3 exposure, 

### 9 including tobacco ( Samuel et al., 2005 )
, Arabidopsis ( Ludwikow et al., 2004 ) , the shrub 

### 10 Phillyrea latifolia ( Paolacci et al., 2007 ) and poplar ( Hamel et al., 2005 ) . Disruption of 

### 11 these signal transduction pathways by over

- expressing or suppressing MAP kinase 

### 12 activity in different Arabidopsis and tobacco lines resulted in increased plant sensitivity 

### 13 to O 3 ( Miles et al., 2005 ; Samuel and Ellis , 2002 ) . Additionally, greater O 3 tolerance of 

### 14 several Arabidopsis ecotypes was correlated with greater up

- regulation of MAP kinase 

### 15 signaling pathways upon O 3 exposure than in more sensitive A rabidopsis ecotypes ( Li et 

### 16 al., 2006b ; Mahalingam et al., 2006 ; Overmyer et al., 2005 ) , indicating that determination 

### 17 of plant sensitivity and plant response to O 3 may, in part, be determined not only by 

### 18 whether th ese pathways are turned on, but also by the magnitude of the signals moving 

### 19 through these communication channels. 

### 20
In conclusion, experimental evidence suggests that there are likely several different 

### 21 mechanisms by which the plant senses the presence of O 3 or its breakdown products. 

### 22 These mechanisms may vary by species or developmental stage of the plant, or may co



### 23 exist and be activated by different exposure conditions. Calcium and protein kinases are 

### 24 likely involved in relaying information about the presen ce of the stressor to the nucleus 

### 25 and other cellular compartments as a first step in determining whether and how the plant 

### 26 will respond to the stress. 

### 27

9.3.3.2 Gene and Protein Expression Changes in Response to Ozone

The advent of DNA microarray technology has all owed for the study of gene expression 

### 28 in cells on a large scale. Rather than assessing changes in gene expression of individual 

### 29 genes, DNA microarrays facilitate the evaluation of entire transcriptomes, providing a 

### 30 comprehensive picture of simultaneous alt erations in gene expression. In addition, these 

### 31 studies have provided more insight into the complex interactions between molecules, how 

### 32 those interactions lead to the communication of information in the cell (or between 

### 33Draft

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- 19 September 2011 neighboring cells) , and which role these interactions play in determining tolerance or 

### 1 sensitivity and how a plant may respond to stresses such as O 3 ( Ludwik ow and 

### 2 Sadowski, 2008 ) . Transcriptome analysis of O 3 - treated plants has been performed in 

### 3 several species, including Arabidopsis thaliana ( Li et al., 2006b ; Tosti et al., 2006 ; 

### 4 Heidenreich et al., 2005 ; Mahalingam et al., 2005 ; Tamaoki et al., 2003 ) , pepper 

### 5 ( C apsicum annuum) ( Lee and Yun, 2006 ) , clover ( Medicago truncatula ) ( Puckette et al., 

### 6 2008 )
, Phillyrea latifolia ( Paolacci et al., 2007 ) , poplar ( Street et al., 2011 ) , and European 

### 7 beech ( Fagus sylvatica ) ( Olbrich et al., 2010 ; Olbrich et al., 2009 ; Olbrich et al., 2005 )

. 

### 8 In some cases, researchers compared transcriptomes of two or more cultivars, ecotypes or 

### 9 mutants that differed in their sen sitivity to O 3 ( Puckette et al., 2008 ; Rizzo et al., 2007 ; 

### 10 Lee and Yun, 2006 ; Li et al., 2006b ; Tamaoki et al., 2003 )

. Species, O 3 exposure 

### 11 conditions (concentration, duration of ex posure) and sampling times varied significantly 

### 12 in these studies. However, functional classification of the genes that were either up

- or 

### 13 down

- regulated by plant exposure to O 3 exhibited common trends. Genes involved in 

### 14 plant defense, signaling and those a ssociated with the synthesis of plant hormones and 

### 15 secondary metabolism were generally up

- regulated, while those related to photosynthesis 

### 16 and general metabolism were typically down

- regulated in O 3 - treated plants ( Puckette et 

### 17 al., 2008 ; Lee and Yun, 2006 ; Li et al., 2006b ; Tosti et al., 2006 ; Olbrich et al., 2005 ; 

### 18 Tamaoki et al., 2003 )

. 

### 19
Analysis of the transcriptome has been used to evaluate differences in gene expression 

### 20 between O 3 sensitive and tolerant plants. In pepper, 67% of the 180 genes studi ed that 

### 21 were affected by O 3 were differentially regulated in the sensitive and tolerant cultivars. 

### 22 At both 0 hours and 48 hours after a 3

- day exposure at 150 ppb, O 3 responsive genes 

### 23 were either up

- or down

- regulated more markedly in the sensitive than in the tolerant 

### 24 cultivar ( Lee and Yun, 2006 ) . Transcriptome analysis also revealed differences in timing 

### 25 and magnitude of changes in gene expression between sensitive and tolerant clovers. 

### 26 Acute exposure (300 ppb O 3 for 6 hours) led to the production of an oxidative burst in 

### 27 both clovers ( Puckette et al., 2008 ) . How ever, the sensitive Jemalong cultivar exhibited a 

### 28 sustained ROS burst and a concomitant down

- regulation of defense response genes at 

### 29
12 hours after the onset of exposure, while the tolerant JE 154 accession showed much 

### 30 more rapid and large

- scale transcript ome changes than the Jemalong cultivar ( Puckette et 

### 31 al., 2008 )

. 

### 32
Arabidopsis ecotypes WS and Col

- 0 were exposed to 1.2 × ambient O 3 concentrations for 

### 33 8

- 12 days at the SoyFACE site ( Li et al., 2006b ) . The sensitive WS ecotype showed a far 

### 34 greater number of changes in gene expression in response to this low

- level O 3 exposure 

### 35 than the tolerant Col

- 0 ecotype. In a different study, exposure of the WS e cotype to 

### 36 300 ppb O 3 for 6 hours showed a rapid induction of genes leading to cell death, such as 

### 37Draft

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- 20 September 2011 proteases, and down

- regulation or inactivation of cell signaling genes, demonstrating an 

### 1 ineffective defense response in this O 3 sensitive ecotype ( Mahalingam et al., 2006 )

. 

### 2
The temporal response of plants to O 3 exposure was evaluated in the Arabidopsis Col

- 0 

### 3 ecotype during a 6

- h exposure at 350 ppb O 3 and for 6 hours after the exposure was 

### 4 completed. Results of this study, shown in Figure 9

- 5, indicate that genes associated with 

### 5 signal transduction and regulation of transcription were in the class of early up

- regulated 

### 6 genes, while genes associated with redox homeostasis and defense/stress response were 

### 7 in the class of late up

- regulated genes ( Mahalingam et al., 2005 )

. 

### 8

A few studies have been conducted to evaluate transcriptome changes in response to 

### 9 longer term chronic O 3 exposures in woody plant species. Longer term exposures 

### 10 resulted in the up

- regulation of genes associated with secondary metabolites, including 

### 11 isopren oids, polyamines and phenylpropanoids in 2

- year

- old seedlings of the 

### 12 Mediterranean shrub Phillyrea latifolia exposed to 110 ppb O 3 for 90 days ( Paolacci et 

### 13 al., 2007 ) . In 3

- year

- old European beech saplings exposed to O 3 for 20 months, with 

### 14 monthly average twice ambient O 3 concentrations ranging from 11 to 80 ppb, O 3 - induced 

### 15 changes in gene transcription were similar to those observed for herbaceous species 

### 16 ( Olbrich et al., 2009 ) . Genes encoding proteins associated with plant stress response, 

### 17 including ethylene biosynthesis, pathogenesis

- related proteins and enzymes detoxifying 

### 18 ROS, were up

- regulated. Some genes associated with primary metabolism, cell structure, 

### 19 cell division and cell growth were reduced ( Olbrich et al., 2009 ) . In a similar study using 

### 20 adult European beech trees, it was determined that the magnitude of the transcriptional 

### 21 changes described above was far greater in the saplings than in the adult trees exposed to 

### 22 the sa me O 3 concentrations for the same time period ( Olbrich et al., 2010 )

. 

### 23
The results from transcriptome studies described above have been substantiated by results 

### 24 from proteome analysis in rice, poplar, European beech, wheat, and soybean. Exposure of 

### 25 soybean to 120 ppb O 3 for 12

- h/day for 3 days in growth chambers resulted in decreases 

### 26 in the quantity of proteins associated with photosynthesis, while proteins involved with 

### 27 antioxidant defense and carbon metabolism increased ( Ahsan et al., 2010 ) . Young pop lar 

### 28 plants exposed to 120 ppb O 3 in a growth chamber for 35 days also showed significant 

### 29 changes in proteins involved in carbon metabolism ( Bohler et al., 2007 ) . Declines in 

### 30 enzymes associated with carbon fixation, the Calvin cycle and photosystem II were 

### 31 measured, while ascorbate peroxidase and enzymes associated with glucose catabolism 

### 32 increased in abundance. In another study to determine the impacts of O 3 on both 

### 33 developing and fully expanded poplar leaves, young poplars were exposed to 120 ppb O 3 

### 34 for 13

- h per day for u p to 28 days ( Bohler et al., 2010 ) . Impacts on protein quantity only 

### 35 occurred after the plants had been exposed to O 3 for 14 days, and at this point in time, 

### 36 several Calvin cycle enzymes were reduced in quantity, while the effects on the light 

### 37Draft

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- 21 September 2011 reactions appeared later, at 21 days after beginning treat ment. Some of the antioxidant 

### 1 enzymes increased in abundance with O 3 treatment, while others (ascorbate peroxidase) 

### 2 did not. In relationship to leaf expansion, it was shown that O 3 did not affect protein 

### 3 quantity until leaves had reached full expansion, af ter about 7 days ( Bohler et al., 2010 )

. 

### 4

Two

- w eek

- old rice seedlings exposed to varying levels of O 3 (4, 40, 80, 120 ppb) in a 

### 5 growth chamber for 9 days showed reductions in quantities of proteins associated with 

### 6 photosynthesis and energy metabolism, and increases in some antioxidant and defense 

### 7 relat ed proteins ( Feng et al., 2008a ) . A subsequent study of O 3 - treated rice seedlings 

### 8 (exposed to 200 ppb O 3 for 24

- h) focusing on the integration of transcriptomics and 

### 9 proteomics, supported and further enhanced these results ( Cho et al., 2008 ) . The authors 

### 10 found that of the 22,000 genes analyzed from the rice genome, 1,535 were differentially 

### 11 regulated by O 3 . Those differentially regulated genes were functionally categorized as 

### 12 transcription factors, MAPK cascades, those encoding for enzymes involved in the 

### 13 synthesis o f jasmonic acid (JA),ethylene (ET), shikimate, tryptophan and lignin, and 

### 14 those involved in glycolysis, the citric acid cycle, oxidative respiration and 

### 15 photosynthesis. The authors determined that the proteome and metabolome (all small 

### 16 molecule metabolites in a cell) analysis supported the results of the transcriptome 

### 17 changes described above ( Cho et al., 2008 ) . This type of study, which ties together results 

### 18 from changes in gene expression, protein quantity and activity, and metabolite levels, 

### 19 provides the most complet e picture of the molecular and biochemical changes occurring 

### 20 in plants exposed to a stressor such as O 3 . 

### 21

Sarkar et al. ( 2010 ) compared proteomes of two cultivars of wheat grown in OTCs at 

### 22 several O 3 concentrations, including filtered air, ambient O 3 (mean concentration 

### 23
47 ppb), ambient + 10 ppb and ambient + 20 ppb for 5

- h/day for 50 days. Declines in the 

### 24 rate of photosynthesis and stomatal conductance were related to decreases in p roteins 

### 25 involved in carbon fixation and electron transport and increased proteolysis of 

### 26 photosynthetic proteins such as the large subunit of ribulose

- 1,6

- bisphosphate 

### 27 carboxylase/oxygenase (Rubisco). Enzymes that take part in energy metabolism, such as 

### 28
ATP synthesis, were also down

- regulated, while defense/stress related proteins were up



### 29 regulated in O 3 treated plants. In comparing the two wheat cultivars, Sarkar et al. ( 2010 ) 

### 30 found that while the qualitative changes in protein expression between the two cultivars 

### 31 were similar, the magnitude of these changes differed between the sensitive and tolerant 

### 32 wheat cultivars. Greater foliar injury and a smaller decline in stomatal conductanc e was 

### 33 observed in the sensitive cultivar as compared to the more tolerant cultivar, along with 

### 34 greater losses in photosynthetic enzymes and higher quantities of antioxidant enzymes. 

### 35 Results from a three year exposure of European beech saplings to elevated O 3 (AOT 40 

### 36 value was 52.6 ul l

- 1

- h for 2006 when trees were sampled) supported the results from the 

### 37 short

- term exposure studies described above ( Kerner et al., 2011 ) . The O 3 treatment of 

### 38Draft

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- 22 September 2011 the saplings resulted in reductions in enzymes associated with the Calvin cycle, which 

### 1 could lead to reduced carbon fixation. Enzymes associated with carbon 

### 2 metabolism/catabolism were in creased, and quantities of starch and sucrose were reduced 

### 3 in response to the O 3 treatment in these trees, indicating a potential impact of O 3 on 

### 4 overall carbon metabolism in long

- term exposure conditions ( Kerner et al., 2011 )

. 

### 5Draft

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- 23 September 2011 Source: Used with permission from Springer ( Mahalingam et al., 2005 )

. (A) Temporal profile of the oxidative stress response to ozone. The biphasic ozone

- induced oxidative burst is represented in black, with the ROS control measurements shown as a broken line. A verage transcript profiles are shown for early up

- regulated genes (yellow, peaks at 0.5

- 1 hours), and the 3 hours (blue), 4.5 hours (red) and 9

- 12 hours (green) late up

- regulated genes and for the down

- regulated genes coding for photosynthesis proteins (br own). (B) Diagrammatic representation of redox regulation of the oxidative stress response.


### Figure 9

- 5 Composite diagram of major themes in the temporal evolution of the genetic response to ozone stress. All of these studies describe common 

### 1
trends for cha nges in gene and protein expression which occur in a variety of plant 

### 2 species exposed to O 3 . While genes associated with carbon assimilation and general 

### 3 metabolism are typically down

- regulated, genes associated with signaling, catabolism, 

### 4 and defense are u p

- regulated. The magnitude of these changes in gene and protein 

### 5 expression appears to be related to plant species, age and their sensitivity or tolerance to 

### 6 O 3 . 

### 7averaged (normalized)transcript profiles forgene groupsDraft

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- 24 September 2011 

### 9.3.3.3 Role of Phytohormones in Plant Response to Ozone

Many studies of O 3 effects on plants have ana lyzed the importance of plant hormones 

### 1 such as SA, ET and JA in determining plant response to O 3 ; some of the roles of these 

### 2 hormones were described in the 2006 O 3 AQCD. Transcriptome analysis and the use of a 

### 3 variety of mutants have allowed for further el ucidation of the complex interactions 

### 4 between SA, ET, JA and the role of abscisic acid (ABA) in mediating plant response to 

### 5 O 3 ( Ludwikow and Sadowski, 2008 ) . In addition to their roles in signaling pathways, 

### 6 phytohormones also appear to regulate, and be regulated by, the MAPK signaling 

### 7 cascades described previously. Most evidence suggests that while ET and SA are needed 

### 8

to develop O 3 - induced leaf lesions, JA acts antagonistically to SA and ET to limit the 

### 9 lesions (Figure 9

- 6) ( Kangasjarvi et al., 2005 )

. 

### 10
The rapid production of ET in O 3 treated plants has been described in many plant species 

### 11 and has been further characterized through the use of a variety of mutants that eithe r 

### 12

over

- produce or are insensitive to ET. Production of stress ET in O 3 - treated plants, which 

### 13 is thought to be part of a wounding response, was found to be correlated to the degree of 

### 14 injury development in leaves ( U.S. EPA, 2006b ) . More r ecent studies have supported 

### 15 these conclusions and have also focused on the interactions occurring between several 

### 16 oxidative

- stress induced phytohormones. Yoshida et al. ( 2009 ) determined that ET likely 

### 17 amplifies the oxidative signal generated by ROS, thereby promoting lesion formation. By 

### 18 analyzing the O 3 - induced transcriptome of several Arabidopsis mutants of the Col

- 0 

### 19 ecotype, Tamaoki et al. ( 2003 ) determined th at at 12 hours after initiating the O 3 

### 20 exposure (200 ppb for 12 hours), the ET and JA signaling pathways were the main 

### 21 pathways used to activate plant defense responses, with a lesser role for SA. The authors 

### 22 also demonstrated that low levels of ET product ion could stimulate the expression of 

### 23 defense genes, rather than promoting cell death which occurs when ET production is 

### 24 high. Tosti et al. ( 2006 ) supported these findings by showing that plant exposure to O 3 

### 25 not only results in activation of the biosynthetic path ways of ET, JA and SA, but also 

### 26 increases the expression of genes related to the signal transduction pathways of these 

### 27 phytohormones in O 3 - treated Arabidopsis plants (300 ppb O 3 for 6 hours). Conversely, in 

### 28 the O 3 sensitive Ws ecotype, its sensitivity may, in part, be due to intrinsically high ET 

### 29 levels leading to SA accumulation, and the high ET and SA may act to repress JA



### 30 associated genes, which would serve to inhibit the spread of lesions ( Mahalingam et al., 

### 31 2006 ) . Ogawa et al. ( 2005 ) found that increases in SA in O 3 - treated plants leads to the 

### 32 formation of leaf lesions in tobacco plants exposed to 200 ppb O 3 for 6 hours. 

### 33 Furthermore, in transgenic tobacco plants with reduced levels of ET production in 

### 34

res ponse to O 3 exposure, several genes encoding for enzymes in the biosynthetic pathway 

### 35 of SA were suppressed, suggesting that SA levels are, in part, controlled by ET in the 

### 36 presence of O 3 . 

### 37Draft

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- 25 September 2011 Exposure of the Arabidopsis mutant rcd1 to acute doses of O 3 (250 pp b O 3 for 8

- h/day 

### 1 for 3 days) resulted in programmed cell death (PCD) and the formation of leaf lesions 

### 2 ( Overmyer et al., 2000 ) . They determined that the observed induction of ET synthesis 

### 3 promotes cell death, and that ET perception and signaling are required for th e 

### 4 accumulation of superoxide, which leads to cell death and propagation of lesions. . 

### 5 Jasmonic acid, conversely, contains the spread of leaf lesions ( Overmyer et al., 2000 )

. 

### 6 Transcriptome analysis of several Arabidopsis mutants, which are insensitive to SA, ET 

### 7 and JA, exposed to 12

- h of 200 ppb O 3 showed that approximately 78 of the up

- regulated 

### 8 genes measured in this study were controlled by ET and JA signaling pathways, while SA 

### 9 signaling pathways were suggested to antagonize ET and JA pathways ( Tamaoki e t al., 

### 10 2003 ) . In a subsequent transcriptome study on the Col

- 0 ecotype exposed to 150 ppb O 3 

### 11 for 48

- h, JA and ET synthesis was down

- regulated, while SA was up

- regulated in O 3 - 

### 12 treated plants. In cotton plants exposed to a range of O 3 concentrations (0

- 12 0 ppb) and 

### 13 methyl jasmonate (MeJA), Grantz et al. ( 2010a ) determined that exogenous applications 

### 14 of MeJA did not protect plants from chronic O 3 exposure. 

### 15
Abscisic acid has been investigated for its role in regulating stomatal aperture and also 

### 16 for its contribution to signaling pathways in th e plant. The role of ABA and the 

### 17 interaction between ABA and H 2 O 2 in O 3 - induced stomatal closure was described in the 

### 18 2006 O 3 AQCD. More recently, it was determined that synthesis of ABA was induced in 

### 19 O 3 - treated Arabidopsis plants (250

- 350 ppb O 3 for 6 hours), with a more pronounced 

### 20 induction in the O 3 sensitive rcd3 mutant as compared to the wildtype Col

- 0 ( Overmyer 

### 21 et al., 2008 ) . The rcd3 mutant also exhibited a lack of O 3 - induced stomatal closure, and 

### 22 the RCD3 protein has been shown to be required for slow anion channels ( Overmyer et 

### 23 al., 2008 ) (see Section 9.3.4.1). Ludwikow et al. ( 2009 ) used Arabidops is ABI1td 

### 24 mutants, in which a key negative regulator of ABA action (abscisic acid insensitive1 

### 25 protein phosphatase 2C) has been knocked out, to examine O 3 responsive genes in this 

### 26 mutant compared to the Arabidopsis Col

- 0. Results of this study indicate a r ole for ABI1 

### 27 in negatively regulating the synthesis of both ABA and ET in O 3 - treated plants (350 ppb 

### 28 O 3 for 9 hours). Additionally, ABI1 may stimulate JA

- related gene expression, providing 

### 29 evidence for an antagonistic interaction between ABA and JA signali ng pathways 

### 30 ( Ludwikow et al., 2009 )

. 

### 31
Nitric oxide (NO) has also been shown to play a role in regu lating gene expression in 

### 32 plants in response to O 3 exposure. However, little is known to date about NO and its role 

### 33 in the complex interactions of molecules in response to O 3 . Exposure of tobacco to O 3 

### 34 (150 ppb for 5 hours) stimulated NO and NO

- dependent E T production, while NO 

### 35 production itself did not depend on the presence of ET ( Ederli et al., 2006 ) . Analysis of 

### 36 O 3 - treated Arabidopsis indicated the possibility of a dual role for NO in the initiation of 

### 37 cell death and later lesion containment ( Ahlfors et al., 2009 )

. 

### 38Draft

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- 26 September 2011 While much work remains to be done to better elucidate how plants sense O 3 , what 

### 1 determines their sensitivity to the pollutant and how they might respond to it, it is clear 

### 2 that the mechanism for O 3 sensing and signal transduction is very complex. M any of the 

### 3 phytohormones and other signaling molecules thought to be involved in these processes 

### 4 are interactive and depend upon a variety of other factors, which could be either internal 

### 5 or external to the plant. This results in a highly dynamic and compl ex system, capable of 

### 6 resulting in a spectrum of plant sensitivity to oxidative stress and generating a variety of 

### 7 plant responses to that stress. 

### 8

Source: Used with permission from Blackwell Publishing Ltd. ( Kangasjarvi et al., 2005 )

. Ozone

- derived radicals induce endogenous ROS production (1) which resul ts 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, S A and jasmonic acid (JA) are indicated with red bars.


### Figure 9

- 6 The oxidative cell death cycle. Detoxification

9.3.4.1 Overview of Ozone

- Induced Defense Mechanisms

Plants are exposed to an oxidizing environment on a continual basis, and many reactions 

### 9 that are p art of the basic metabolic processes, such as photosynthesis and respiration, 

### 10 generate ROS. As a result, there is an extensive and complex mechanism in place to 

### 11 detoxify these oxidizing radicals, including both enzymes and metabolites, which are 

### 12 located in several locations in the cell and also in the apoplast of the cell. As O 3 enters 

### 13 the leaf through open stomata, the first point of contact of O 3 with the plant is likely in 

### 14 the apoplast, where it breaks down to form oxidizing radicals such as H 2 O 2 , O 2 - , H O· and 

### 15Draft

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- 27 September 2011 HO 2 . Another source of oxidizing radicals is an oxidative burst, generated by a 

### 1 membrane

- bound NADPH oxidase enzyme, which is recognized as an integral 

### 2 component of the plant ’ s defense system against pathogens ( Schraudner et al., 1998 )

. 

### 3 Antioxidant metabolites and enzymes located in the apoplast are thought to form a first 

### 4 line of defense by detoxifying O 3 and/or the ROS that are formed as breakdown products 

### 5 of O 3 (Section 9.3.2.). However, even with the presence of several antioxidants, 

### 6 including ascorbate, the redox buffering capacity of the apoplast is far less than that of 

### 7 the cytoplasm, as it lacks the regeneration systems necessary to retain a reduced pool of 

### 8 antioxidants ( Foyer and Noctor, 2005b )

. 

### 9
Redox homeostasis is regulated by the presence of a pool of antioxidants, which are 

### 10 typically found in a reduced state and detoxify ROS produced by oxidases or electron 

### 11 transport components. As ROS increase due to environmental stress such as O 3 , it is 

### 12 unclear whether the antioxidant pool can maintain its reduce d state ( Foyer and Noctor, 

### 13 2005b ) . As such, not only the quantity and types of antioxidant enzymes and metabolites 

### 14 present, but also the cellular ability to regenerate those antioxidants are importa nt 

### 15 considerations in mechanisms of plant tolerance to oxidative stress ( Dizengremel e t al., 

### 16 2008 ) . Molecules such as glutathione (GSH), thioredoxins and NADPH play very 

### 17 important roles in this regeneration process; additionally, it has been hypothesized that 

### 18 alterations in carbon metabolism would be necessary to supply the needed reduci ng 

### 19 power for antioxidant regeneration ( Dizengremel et al., 2008 )

. 

### 20

9.3.4.2 Role of Antioxi dants in Plant Defense Responses

Ascorbate has been the focus of many different studies as an antioxidant metabolite that 

### 21 protects plants from exposure to O 3 . It is found in several cellular locations, including the 

### 22 chloroplast, the cytosol and the apoplas t ( Nocto r and Foyer, 1998 ) . Ascorbate is 

### 23 synthesized in the cell and transported to the apoplast. Apoplastic ascorbate can be 

### 24 oxidized to dehydroascorbate (DHA) with exposure to O 3 and is then transported back to 

### 25 the cytoplasm. Here, DHA is reduced to ascorbate by the enzyme dehydroascorbate 

### 26 reductase (DHAR) and reduced GSH, which is part of the ascorbate

- glutathione cycle 

### 27 ( Noctor and Foyer, 1998 ) . Many studies have focused on evaluating whether ascorbate is 

### 28 the primary determining factor in differential sensitivity of plants to O 3 . An evaluation of 

### 29 several species of wildflowers in Great Smoky Mountains National Park showed a 

### 30 correlation between higher quantities of reduced apoplastic ascorbate and lower levels of 

### 31 foliar injury from O 3 exposure in the field in tall m ilkweed plants ( Asclepsias exaltata L.) 

### 32 ( Burkey et al., 2006 ; Souza et al., 2006 ) . Cheng et al. ( 2007 ) exposed two soybean 

### 33 cultivars to elevated O 3 (77 ppb) and filtered air for 7

- h/day for 6 days. The differences in 

### 34 sensitivity between the two cultivars could not be explained by differential O 3 uptake or 

### 35Draft

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- 28 September 2011 by the fraction of reduced ascorbate present in the apoplast. However, total antioxidant 

### 1 capacity of the apoplast was twofold higher in the tolerant Essex cultivar as compared to 

### 2 the sensitive Forrest cultivar, indicating that there may be other compounds in the leaf 

### 3 apoplast that scavenge ROS. D ’ Haese et al. ( 2005 ) exposed the NC

- S (sensitive) and NC



### 4 R (resistant) clones of white clover ( Trifolium repens ) to 60 ppb O 3 for 7

- h/day for 

### 5 5 days in environmental chambers. Surprisingly, the NC

- S clone had a higher constitutive 

### 6

conc entration of apoplastic ascorbate with a higher redox status than the NC

- R clone. 

### 7 However, the redox status of symplastic GSH was higher in NC

- R, even though the 

### 8 concentration of GSH was not higher than in NC

- S. In addition, total symplastic 

### 9 antioxidative capacity was not a determining factor in differential sensitivity between 

### 10 these two clones. Severino et al. ( 2007 ) also examined t he role of antioxidants in the 

### 11 differential sensitivity of the two white clover clones by growing them in the field for a 

### 12 growing season and then exposing them to elevated O 3 (100 ppb for 8

- h/day for 10 days) 

### 13 in OTC at the end of the field season. The NC

- R clone had greater quantities of total 

### 14 ascorbate and total antioxidants than the NC

- S clone at the end of the experiment. In snap 

### 15 bean, plants of the O 3 tolerant Provider cultivar had greater total ascorbate and more 

### 16 ascorbate in the apoplast than the sens itive S156 cultivar after exposure to 71 ppb O 3 for 

### 17
10 days in OTC ( Burkey et al., 2003 ) . While most of the apoplastic ascorbate was in the 

### 18 oxidized form, the ratio of reduced ascorbate to total ascorbate was higher in Provider 

### 19 than S156, indicating that Provider is better able to maintain this ratio to maximize plant 

### 20 protection from oxidative stress. Exposure of two wheat varieties to ambient (7

- h average 

### 21 44 ppb O 3 ) and elevated (7

- h average 56 ppb O 3 ) for 60 days in open

- air field conditions 

### 22 showed higher concentrations of reduced ascorbate in the apoplast in the tolerant Y16 

### 23 variety than the more sensitive Y2 variety, however no varietal dif ferences were seen in 

### 24 the decrease in reduced ascorbate quantity in response to O 3 exposure ( Feng et al., 2010 )

. 

### 25 There is much evidence that supports an important role for ascorbate, particularly 

### 26 apoplastic ascorbate, in protecting plants from oxidative stressors such as O 3 ; however, it 

### 27 is also clear that there is much variation in the importance of ascorbate for different plant 

### 28 species and differing exposure conditions. Additionally, the work of several authors 

### 29 suggests that there may be other compounds in the apoplast which have the capacity to 

### 30 act as antioxidants. 

### 31
While the quantities of antioxidant metabolites such as ascorbate are an important 

### 32 indicator of plant tolerance to O 3 , the ability of the plant to recycle oxidized ascorbate 

### 33 efficiently also plays a large role in determining the plant ’ s ability to effectively protect 

### 34 itself from sustained exposure to oxidative stress. Tobacco plants over

- expressing DHAR 

### 35 were better protected from exposure to either chronic (100 ppb O 3 4

- h/day for 30 days) or 

### 36

a cute (200 ppb O 3 for 2 hours) conditions than control plants and those with reduced 

### 37 expression of DHAR ( Chen and Gallie, 2005 ) . The DHAR over

- expressing plants 

### 38 exhibited an increase in guard cell ascorbic acid, leading to a decrease in stomatal 

### 39Draft

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- 29 September 2011 responsiveness to O 3 and an increase in stomatal conductance and O 3 uptake. Despite 

### 1 this, the presence of higher levels of ascorbic acid led to a lower oxidative load and a 

### 2 higher level of photosynthetic activity in the DHAR over

- expressing plants ( Chen and 

### 3 Gallie, 2005 ) . A subsequent study with tobacco plants over

- expressing DHA R confirmed 

### 4 some of these results. Levels of ascorbic acid were higher in the transgenic tobacco 

### 5 plants, and they exhibited greater tolerance to O 3 exposure (200 ppb O 3 ) as demonstrated 

### 6 by higher photosynthetic rates in the transgenic plants as compared to the control plants 

### 7 ( Eltayeb et al., 2006 ) . Over

- expression of monodehydroascorbate reductase (MDAR) in 

### 8 tobacco plants also showed enhanced stress tolerance in response to O 3 ex posure 

### 9 (200 ppb O 3 ), with higher rates of photosynthesis and higher levels of reduced ascorbic 

### 10 acid as compared to controls ( Eltayeb et al., 2007 ) . Results of these studies demonstrate 

### 11 the importance of ascorbic acid as a detoxification mechanism in some plant species, and 

### 12 al so emphasize that the recycling of oxidized ascorbate to maintain a reduced pool of 

### 13 ascorbate is a factor in determining plant tolerance to oxidative stress. 

### 14
The roles of other antioxidant metabolites and enzymes, including GSH, catalase (CAT), 

### 15 and superox ide dismutase (SOD), were comprehensively reviewed in the 2006 O 3 

### 16 AQCD. Additional studies have supported the findings reported in that document. 

### 17 Superoxide dismutase (SOD) and peroxidase (POD) activities were measured in both the 

### 18 tolerant Bel B and sensit ive Bel W3 tobacco cultivars exposed to ambient O 3 

### 19 concentrations for 2 weeks 3 times throughout a growing season ( Borowiak et al., 2009 )

. 

### 20 In this study, SOD and POD activity, including that of several different isofor ms, 

### 21 increased in both the sensitive and tolerant tobacco cultivars with exposure to O 3 , 

### 22 however the isoenzyme composition for POD differed between the sensitive and tolerant 

### 23 tobacco cultivars ( Borowiak et al., 2009 ) Tulip poplar ( Liriodendron tulipifera ) trees 

### 24 exposed to increasing O 3 concentrations (from 100 to 300 ppb O 3 during a 2

- week 

### 25 period) showed increases in activities of SOD, ascorbate peroxidase (APX), glutathione 

### 26 reductase (GR), MDAR, DHAR, CAT and POD in t he 2

- week period, although 

### 27 individual enzyme activities increased at different times during the 2

- week period ( Ryang 

### 28 et al., 2009 )

. 

### 29
Longer, chronic O 3 exposures in trees revealed increases in SOD and APX activity in 

### 30 Quercus mongolica after 45 days of plant exposure to 80 ppb O 3 , which were followed 

### 31 by declines in the activities and quantities of these enzymes after 75 days of exposure 

### 32 ( Yan et al., 2010 ) . Similarly, activities of SOD, APX, DHAR, MDAR, and GR increased 

### 33
in Gingko biloba trees during th e first 50 days of exposure to 80 ppb O 3 , followed by 

### 34 decreases in activity below control values after 50 days of exposure ( He et al., 2006 )

. 

### 35 Soybean plants exposed to 70 or 100 ppb O 3 for 4

- h/day over the course of a growing 

### 36 season showed elevated POD activity and a decrease in CAT activity at 40 and 60 days 

### 37 after germination ( Singh et al., 2010a )

. 

### 38Draft

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- 30 September 2011 Antioxidant enzymes and metabolites have been shown to play an important role in 

### 1 determining plant tolerance to O 3 and mediating plant responses to O 3 . However, there is 

### 2 also some evidence to suggest that the direct reaction of ascorbate with O 3 could lead to 

### 3 the formation of secondary toxicants, such as peroxy compounds, which may act upon 

### 4 signal transduction pathways and modulate plant response to O 3 ( Sandermann, 2008 )

. 

### 5 Therefore, the role of ascorbate and other antioxidants and their intera ction with other 

### 6 plant responses to O 3 , such as the activation of signal transduction pathways, is likely far 

### 7 more complex than is currently understood. 

### 8

9.3.5 Effects on Primary and Secondary Metabolism

9.3.5.1 Light and Dark Reactions of Photosynthesis

Declines in the rate of photosynthesis and stomatal conductance in O 3 - treated plants have 

### 9 been documented for many different plant species ( Booker et al., 2009 ; U.S. EPA, 2006b ) 

### 10 ( Wittig et al., 2007 ) . The 2006 O 3 AQCD outlined what is known about the ef fects of O 3 

### 11 on carbon assimilation, and the more recent scientific literature confirms these findings. 

### 12 While several measures of the light reactions of photosynthesis are sensitive to exposure 

### 13 to O 3 (see below), photosynthetic carbon assimilation is genera lly considered to be more 

### 14 affected by pollutant exposure, resulting in an overall decline in photosynthesis ( Guidi 

### 15 and Degl'lnnocenti, 2008 ; Heath, 2008 ; Fiscus et al., 2005 ) . Loss of carbon assimilation 

### 16 capacity has been shown to result primarily from declines in the quantity of Rubisco 

### 17 ( Singh et al., 2009 ; Calatayud et al., 2007a ) . Experimental evidence suggests that both 

### 18 decreases in Rubisco synthesis and enhanced degradation of the protein contribute to the 

### 19 measured reduction in its quantity ( U.S. EPA, 2006b ) . Reduced carbon assimi lation has 

### 20 been linked to reductions in biomass and yield ( Wang et al., 2009b ; He et al., 2007 ; 

### 21 Novak et al., 2007 ; Gregg et al., 2006 ; Keutgen et al., 2005 ) . Recent studies evaluating 

### 22 O 3 induced changes in the transcriptome and proteome of sever al different species 

### 23 confirm these findings. Levels of mRNA for the small subunit of Rubisco (rbcS) declined 

### 24 in European beech saplings exposed to 300 ppb O 3 for 8

- h/day for up to 26 days ( Olbrich 

### 25 et al., 2005 ) . Similar declines in rbcS mRNA were also measured in the beech saplings in 

### 26 a free air exposure system over a course of two growing seasons ( Olbrich et al., 2009 )

. 

### 27 Proteomics studies have also confirmed the effects of O 3 on proteins involved in carbon 

### 28 assimilation. Reductions in quantities of the small and large subunit (rbcL) o f Rubisco 

### 29 and Rubisco activase were measured in soybean plants exposed to 120 ppb O 3 for 3 days 

### 30 in growth chambers ( Ahsan et al., 2010 ) . Exposure of young poplar trees to 120 ppb O 3 

### 31 for 35 days in exposure chambers resulted in reductions of Rubisco, Rubisco activase, 

### 32 and up to 24 isoforms of Calvin cycle enzymes, m ost of which play a role in regenerating 

### 33Draft

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- 31 September 2011 the CO 2 acceptor molecule, ribulose

- 1.5

- bisphosphate ( Bohler et al., 2007 ) . Reductions 

### 1 in protein quantity of both the small and large subunit of Rubisco were seen in wheat 

### 2 plants exposed to ambient (average concentration 47.3 ppb O 3 ) and elevated O 3 (ambient 

### 3 + 10 or 20 ppb O 3 ) in open

- top chambers for 5

- h/day for 50 days ( Sarkar et al., 2010 )

. 

### 4 Lettuce plants exposed to 100 ppb O 3 in growth chambers for 8

- h/da y for 3 weeks also 

### 5 showed reductions in transcript and protein levels of the small and large subunits of 

### 6 Rubisco and Rubisco activase ( Goumenaki et al., 2010 ) . The reductions in carbon 

### 7 assimilation have been associated with declines in both the mRNA of the small and large 

### 8 subunits of Rubisco, and with reduc tions in Rubisco activase mRNA and protein. 

### 9 Additionally, the reduction in Rubisco quantity has also been associated with the O 3 - 

### 10 induced oxidative modification of the enzyme, which is evidenced by the increases in 

### 11 carbonyl groups on the protein after plant exposure to O 3 . 

### 12
In addition to impacts on carbon assimilation, the deleterious effects of O 3 on the 

### 13 photosynthetic light reactions have received more attention in recent years. Chlorophyll 

### 14 fluorescence provides a useful measure of changes to the photosyn thetic process from 

### 15 exposure to oxidative stress. Decreases in the Fv/Fm ratio (a measure of the maximum 

### 16 efficiency of Photosystem II) in dark adapted leaves indicate a decline in the efficiency of 

### 17 the PSII photosystems and a concomitant increase in non

- ph otochemical quenching 

### 18 ( Guidi and Degl'lnnocenti, 2008 ; Scebba et al., 2006 ) . Changes in these parameters have 

### 19 been correlated to differential sens itivity of plants to the pollutant. In a study to evaluate 

### 20 the response of 4 maple species to O 3 (exposed to an 8

- h avg of 51 ppb for ambient and 

### 21
79 ppb for elevated treatment in OTC), the 2 species which were most sensitive based on 

### 22 visible injury and dec lines in CO 2 assimilation also showed the greatest decreases in 

### 23 Fv/Fm in symptomatic leaves. In asymptomatic leaves, CO 2 assimilation decreased 

### 24 significantly but there was no significant decline in Fv/Fm ( Calatayud et al., 2007a ) . Degl 

### 25

‘ Innocenti et al. ( 2007 ) measured significant decreases in Fv/Fm in young and 

### 26 symptomatic leaves of a resistant tomato genotype (line 93.1033/1) in response to O 3 

### 27 exposure (150 ppb O 3 for 3 hours in a growth chamber), but only minor decreases in 

### 28 asymptomatic leaves with no associated changes in net photosynthetic rate. In the O 3 

### 29 sensitive tomato cultivar Cuor Di Bue, the Fv/Fm ratio did not change, while the 

### 30 photosynthetic rate declined significantly in asymptomatic leaves ( Degl'Innocenti et al., 

### 31 2007 ) . In two soybean cultivars, Fv/Fm also declined significantly with pl ant exposure to 

### 32 O 3 ( Singh et al., 2009 ) . It appears that in asymptomatic leaves, p hotoinhibition, as 

### 33 indicated by a decrease in Fv/Fm, is not the main reason for a decline in photosynthesis. 

### 34
An evaluation of photosynthetic parameters of two white clover ( Trifolium repens cv. 

### 35 Regal) clones that differ in their O 3 sensitivity revealed tha t O 3 (40

- 110 ppb O 3 for 7

- 

### 36 h/day for 5 days) increased the coefficient of non

- photochemical quenching (q NP ) in both 

### 37 the resistant (NC

- R) and sensitive (NC

- S) clones, however q NP was significantly lower 

### 38Draft

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- 32 September 2011 for the sensitive clone ( Crous et al., 2006 ) . Sensitive Acer c lones had a lower coefficient 

### 1 of non

- photochemical quenching, while exposure to O 3 increased q NP in both sensitive 

### 2 and tolerant clones ( Calatayud et al., 2007a ) . While exposure to O 3 also increased q NP in 

### 3 tomato, there were no differences in the coefficient of photochemical quenching between 

### 4 cultivars thought to be differentially sensitive to O 3 . ( Degl'Innocenti et al., 2007 ) . Higher 

### 5
q NP as a result of exposure to O 3 indicates a reduction in the pr oportion of absorbed light 

### 6 energy being used to drive photochemistry. A lower coefficient of non

- photochemical 

### 7 quenching in O 3 sensitive plants could indicate increased vulnerability to ROS generated 

### 8 during exposure to oxidative stress ( Crous et al., 2006 )

. 

### 9

Most of the research on O 3 effects on photosynthesis has focused on C3 (Calvin cycle) 

### 10 plants because C4 (Hatch

- Slack) plants have lower stomatal conductance and are, 

### 11 therefore, thought to be less sensitive to O 3 stress. However, a few studies have been 

### 12 conduct ed to evaluate the effects of O 3 on C4 photosynthesis. In older maize leaves, 

### 13 Leitao et al. ( 2007b ; 2007c ) found that the activity, quantity and transcript levels of both 

### 14 Rubisco and phosphoenolpyruvate carboxylase (PEPc) decreased as a function of rising 

### 15 O 3 concentration. In younger maize leaves, the qua ntity, activity, and transcript levels of 

### 16 the carboxylases were either increased or unaffected in plants exposed to 40 ppb O 3 for 

### 17
7

- h/day for 28

- 33 days, but decreased at 80 ppb ( Leitao et al., 2007a ; Leitao et al., 

### 18 20 07b )

. 

### 19

9.3.5.2 Respiration and Dark Respiration

While much research emphasis regarding O 3 effects on plants has focused on the 

### 20 negative impacts on carbon assimilation, ot her studies have measured impacts on 

### 21 catabolic pathways such as shoot respiration and photorespiration. Generally, shoot 

### 22 respiration has been found to increase in plants exposed to O 3 . Bean plants exposed to 

### 23 ambient (average 12

- h mean 43 ppb) and twice amb ient (average 12

- h mean 80 ppb) O 3 

### 24 showed increases in respiration. When mathematically partitioned, the maintenance 

### 25 coefficient of respiration was significantly increased in O 3 treated plants, while the 

### 26 growth coefficient of respiration was not affected ( Amthor, 1988 ) . Loblolly pines were 

### 27 exposed to ambient (12

- h daily mean was 45 ppb) and twice ambient (12 hours daily 

### 28 mean was 86 ppb) O 3 for 12

- h/day for approximately seven months per yea r for 3 and 

### 29
4 years. While photosynthetic activity declined with the age of the needles and increasing 

### 30 O 3 concentration, enzymes associated with respiration showed higher levels of activity 

### 31 with increasing O 3 concentration ( Dizengremel et al., 1994 ) . In their review on the role of 

### 32 metabolic changes in plant redox status after O 3 exposure, Dizengremel et al. ( 2009 ) 

### 33 summarized multiple studies in which several different tree species were exposed to O 3 

### 34 concentrations ranging from ambient to 200 ppb O 3 for at least several weeks. I n all 

### 35Draft

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- 33 September 2011 cases, the activity of enzymes, including phosphofructokinase, pyruvate kinase and 

### 1 fumarase, which are part of several catabolic pathways, were increased in O 3 treated 

### 2 plants. 

### 3
Photorespiration is a light

- stimulated process which consumes O 2 and relea ses CO 2 . 

### 4 While it has been regarded as a wasteful process, more recent evidence suggests that it 

### 5 may play a role in photoprotection during photosynthesis ( Bagard et al., 2008 ) . The few 

### 6 studie s that have been conducted on O 3 effects on photorespiration suggest that rates of 

### 7 photorespiration decline concomitantly with rates of photosynthesis. Soybean plants were 

### 8 exposed to ambient (daily averages 43

- 58 ppb) and 1.5 ambient O 3 (daily averages 63

- 

### 9 83 ppb) O 3 in OTCs for 12

- h/day for 4 months. Rates of photosynthesis and 

### 10 photorespiration and photorespiratory enzyme activity declined only at the end of the 

### 11 growing season and did not appear to be very sensitive to O 3 exposure ( Booker et al., 

### 12 1997 ) . Young hybrid poplars exposed to 120 ppb O 3 for 13

- h/day for 35 days in 

### 13 phytotron chambers showed that effects on photorespiration and photosynthesis were 

### 14 dependent upon the developmental stage of the leaf. While young leaves were not 

### 15 impacted, reductions in photosynthesis and photorespiration were measured in fully 

### 16 expanded leaves ( Bagard et al., 2008 )

. 

### 17

9.3.5.3 Secondary Metabolism

Transcriptome analysis of Arabidopsis plants has revealed modulation of several genes 

### 18 involved in plant secondary metabolism ( Ludwikow a nd Sadowski, 2008 ) . Phenylalanine 

### 19 ammonia lyase (PAL) has been the focus of many studies involving plant exposure to O 3 

### 20 due to its importance in linking the phenylpropanoid pathway of plant secondary 

### 21 metabolism to primary metabolism in the form of the s hikimate pathway. Genes encoding 

### 22 several enzymes of the phenylpropanoid pathway and lignin biosynthesis were up



### 23 regulated in transcriptome analysis of Arabidopsis plants (Col

- 0) exposed to 350 ppb O 3 

### 24 for 6 hours, while 2 genes involved in flavonoid biosynt hesis were down

- regulated 

### 25 ( Ludwikow et al., 2004 ) . Exposure of Arabidopsis (Col

- 0) to lower O 3 concentrations 

### 26 (150 ppb for 8

- h/day for 2 days) resulted in the induction of 11 transcripts involved in 

### 27 flavonoid synthesis. In their exposure of 2

- year

- old Mediterranean shrub Phillyrea 

### 28 latifolia to 110 ppb O 3 for 90 days, Paolacci et al. ( 2007 ) identified four clones that were 

### 29
up

- regul ated and corresponded to genes involved in the synthesis of secondary 

### 30 metabolites, such as isoprenoids, polyamines and phenylpropanoids. Up

- regulation of 

### 31 genes involved in isoprene synthesis was also observed in Medicago trunculata exposed 

### 32 to 300 ppb O 3 fo r 6 hours, while genes encoding enzymes of the flavonoid synthesis 

### 33 pathway were either up

- or down

- regulated ( Puckette et al., 2008 ) . Exposure of red clover 

### 34 to 1.5 × ambient O 3 (average co ncentrations of 32.4 ppb) for up to 9 weeks in an open 

### 35Draft

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- 34 September 2011 field exposure system resulted in increases in leaf total phenolic content. However, the 

### 1 types of phenolics that were increased in response to O 3 exposure differed depending 

### 2 upon the developmental stag e of the plant. While almost all of the 31 different phenolic 

### 3 compounds measured increased in quantity initially during the exposure, after 3 weeks 

### 4 the quantity of isoflavones decreased while other phenolics increased ( Saviranta et al., 

### 5 2010 ) . Exposure of beech saplings to ambient and 2 × ambient O 3 concen trations over 2 

### 6 growing seasons resulted in the induction of several enzymes which contribute to lignin 

### 7 formation, while enzymes involved in flavonoid biosynthesis were down

- regulated 

### 8 ( Olbrich et al., 2009 ) . Exposure of tobacco Bel W3 to 160 ppb O 3 for 5 hours showed up



### 9 regulation of almost all genes encoding for enzymes which a re part of the prechorismate 

### 10 pathway ( Janzik et al., 2005 ) . Isoprenoids can serve as antioxidant compounds in plants 

### 11 exposed to oxidative stress ( Paolacci et al., 2007 )

. 

### 12
The prechorismate pathway is the pathway leading to the fo rmation of chorismate, a 

### 13 precursor to the formation of the aromatic amino acids tryptophan, tyrosine and 

### 14 phenylalanine. These amino acids are precursors for the formation of many secondary 

### 15 aromatic compounds, and, therefore, the prechorismate pathway repre sents a branch



### 16 point in the regulation of metabolites into either primary or secondary metabolism ( Janzik 

### 17 et al., 2005 ) . Exposure of the O 3 sensitive Bel W3 tobacco cultivar at 160 ppb for 5 hours 

### 18 showed an increase in transcript levels of most of the genes encoding enzymes of the 

### 19 prechorismate pathway. However, shikimate kinase (SK) did not show any change in 

### 20 transcript levels and only one o f three isoforms of DAHPS (3

- deoxy

- D

- arabino



### 21 heptulosonat

- 7

- phosphate synthase), the first enzyme in this pathway, was induced by O 3 

### 22
exposure ( Janzik et al., 2005 ) . Differential induction of DAHPS isoforms was also 

### 23 observed in European beech after 40 days of exposure to 150

- 190 ppb O 3 . At this time 

### 24 point in the beech experiment, transcript levels of shikimate pathway enzymes, including 

### 25 SK, were generally strongly induced after an only weak initial i nduction after the first 

### 26 40 days of exposure. Both soluble and cell

- wall bound phenolic metabolites showed only 

### 27 minimal increases in response to O 3 for the duration of the exposure period ( Alonso et 

### 28 al., 2007 ) . Total leaf phenolics decreased with leaf age in Populus nigra ex posed to 

### 29 80 ppb O 3 for 12

- h/day for 14 days. Ozone increased the concentration of total leaf 

### 30 phenolics in newly expanded leaves, with the most significant increases occurring in 

### 31 compounds such as quercitin glycoside, which has a high antioxidant capacity ( Fares et 

### 32 al., 2010b ) . While several phenylpropanoid pathway enzymes were induced in two poplar 

### 33 clones exposed to 60 ppb O 3 for 5

- h/day for 15 days, the degree of induction differed 

### 34 between the two clones. In the tolerant I

- 214 clone, PAL activity increased nine fold in 

### 35 O 3 - t reated plants as compared to controls, while there was no significant difference in 

### 36 PAL activity in the sensitive Eridano clone ( Di Baccio et al., 2008 )

. 

### 37Draft

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- 35 September 2011 Polyamines such as putrescine, spermidine and spermin e play a variety of roles in plants 

### 1 and have been implicated in plant defense responses to both abiotic and biotic stresses. 

### 2 They exist in both a free form and conjugated to hydroxycinnamic acids. Investigations 

### 3 on the role of polyamines have found that le vels of putrescine increase in response to 

### 4 oxidative stress. This increase stems largely from the increase in the activity of arginine 

### 5 decarboxylase (ADC), a key enzyme in the synthesis of putrescine ( Groppa and 

### 6 Benavides, 2008 ) . Langebartels et al. ( 1991 ) described differences in putrescine 

### 7 accumulation in O 3 - treated tobacco plants exposed to several O 3 concentrations, ranging 

### 8 from 0

- 400 ppb for 5

- 7 hours. A large and rapid increase in putrescine occurred in the 

### 9 tolerant Bel B cultivar and only a small increase in the sensitive Bel W3 cultivar, which 

### 10 occurred only after the formation of necrotic leaf lesions. Van Buuren et al. ( 2002 ) 

### 11 further examined the role of polyamines in these two tobacco cultivars during an acute 

### 12 (130 ppb O 3 for 7

- h in a growth chamber) exposure. They found that while free 

### 13
p utrescine accumulated in undamaged tissue of both cultivars, conjugated putrescine 

### 14 predominantly accumulated in tissues undergoing cell death after plant exposure to O 3 

### 15 ( van Buuren et al., 2002 ) . The authors suggest that while free putrescine may not play a 

### 16 role in conferring tolerance in the Bel B cult ivar, conjugated putrescine may play a role in 

### 17
O 3 - induced programmed cell death in Bel W3 plants. 

### 18
Isoprene is emitted by some plant species and represents the predominant biogenic source 

### 19 of hydrocarbon emissions in the atmosphere ( Guenther et a l., 2006 ) . In the atmosphere, 

### 20 the oxidation of isoprene by hydroxyl radicals can enhance O 3 formation in the presence 

### 21 of NO X , thereby impacting the O 3 concentration that plants are exposed to. While 

### 22 isoprene emission varies widely between species, it ha s been proposed to stabilize 

### 23 membranes and provide those plant species that produce it with a mechanism of 

### 24 thermotolerance ( Sharkey et al., 2008 ) . It has also been suggested that isoprene may act 

### 25 as an antioxidant compound to scavenge O 3 ( Loreto and Velikova, 2001 ) . Recent studies 

### 26 using a variety of plant species have shown conflicting results in trying to unders tand the 

### 27 effects of O 3 on isoprene emission. Exposure to acute doses of O 3 (300 ppb for 3

- h) in 

### 28 detached leaves of Phragmites australis resulted in stimulation of isoprene emissions 

### 29 ( Velikova et al., 2005 ) . Similar increases in isoprene emissions were measured in 

### 30 Populus nigra after exposure to 100 ppb O 3 for 5 days continuously ( Fares et al., 2008 )

. 

### 31 Isoprene emission in attached leaves of Populus alba , which were exposed to 150 ppb O 3 

### 32 for 11

- h/day for 30 days inside cuvette s, was inhibited, while isoprene emission and 

### 33 transcript levels of isoprene synthase mRNA were increased in the leaves exposed to 

### 34 ambient O 3 (40 ppb), which were located above the leaves enclosed in the exposure 

### 35

cuvettes ( Fares et al., 2006 ) . Exposure of 2 genotypes of hybrid poplar to 120 ppb O 3 for 

### 36
6

- h/day for 8 days resulted in a significant reduction in isoprene emission in the O 3 - 

### 37

s ensitive but not the tolerant genotype ( Ryan et al., 2009 ) . Similarly, O 3 treatment 

### 38 (80 ppb 12

- h/day for 14 days) of Populus nigra showed that isoprene emission was 

### 39Draft

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- 36 September 2011 reduced in the treated plants relative to the control plants ( Fares et al., 2010b ) . Based on 

### 1 results of this and other studies, Fares et al. ( 2010b ) concluded that the isoprenoid 

### 2 pathway may be induced in plants exposed to acute O 3 doses, while at lower doses 

### 3 isoprene emission may be inhibited. Vickers et al. ( 2009 ) developed transgenic t obacco 

### 4 plants with the isoprene synthase gene from Populus alba and exposed them to 120 ppb 

### 5 O 3 for 6

- h/day for 2 days. They determined that the wildtype plants showed significantly 

### 6 more O 3 damage, including the development of leaf lesions and a decline in 

### 7 photosynthetic rates, than the transgenic, isoprene

- emitting plants. Transgenic plants also 

### 8 accumulated less H 2 O 2 and had lower levels of lipid peroxidation following exposure to 

### 9 O 3 than the wildtype plants ( Vickers et al., 2009 ) . These results indicate that isoprene 

### 10 may have a protective role for plants exposed to oxidative stress. 

### 11

9.3.6 Summary

The results of recent studies on the effects of O 3 stress on plants support and strengthen 

### 12 those reported in the 2006 O 3 AQCD. The most significant new body of evidence since 

### 13 the 2006 O 3 AQCD comes from research on molecular mechanisms of the biochemical 

### 14 and physiological changes observed in many plant species in response to O 3 exposure. 

### 15 Recent studies have em ployed new techniques, such as those used in evaluating 

### 16 transcriptomes and proteomes to perform very comprehensive analyses of changes in 

### 17 gene transcription and protein expression in plants exposed to O 3 . These newer molecular 

### 18 studies not only provide very important information regarding the many mechanisms of 

### 19 plant responses to O 3 , they also allow for the analysis of interactions between various 

### 20 biochemical pathways which are induced in response to O 3 . However, many of these 

### 21 studies have been conducted in artificial conditions with model plants, which are 

### 22 typically exposed to very high, short doses of O 3 . Therefore, additional work remains to 

### 23 elucidate whether these plant responses are transferable to other plant species exposed to 

### 24 more realistic ambient co nditions. 

### 25
Ozone is taken up into leaves through open stomata. Once inside the substomatal cavity, 

### 26 O 3 is thought to rapidly react with the aqueous layer surrounding the cell (apoplast) to 

### 27 form breakdown products such as hydrogen peroxide (H 2 O 2 ), superoxide (O 2 ־ hydroxyl , ) 

### 28 radicals (HO · ) and peroxy radicals (HO 2 · ). Plants could be sensing the presence of O 3 

### 29 and/or its breakdown products in a variety different ways, depending upon the plant 

### 30 species and the exposure parameters. Experimental evidence suggests t hat mitogen



### 31 activated protein kinases and calcium are important components of the signal 

### 32 transduction pathways, which communicate signals to the nucleus and lead to changes in 

### 33 gene expression in response to O 3 . It is probable that there are multiple sensin g 

### 34 mechanisms and signal transduction pathways, and their activation may depend upon the 

### 35Draft

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- 37 September 2011 plant species, its developmental stage and/or O 3 exposure conditions. Initiation of signal 

### 1 transduction pathways in O 3 treated plants has also been observed in stomatal guard cells. 

### 2 Reductions in stomatal conductance in have been described for many plant species 

### 3 exposed to O 3 , and new experimental evidence suggests that this reduction may be due 

### 4 not only to a decrease in carboxylation efficiency, but also to a direct imp act of O 3 on 

### 5 stomatal guard cell function, leading to a changes in stomatal conductance. 

### 6
Alterations in gene transcription that have been observed in O 3 - treated plants are now 

### 7 evaluated more comprehensively using DNA microarray studies, which measure cha nges 

### 8 in the entire transcriptome rather than measuring the transcript levels of individual genes. 

### 9 These studies have demonstrated very consistent trends, even though O 3 exposure 

### 10 conditions (concentration, duration of exposure), plant species and sampling t imes vary 

### 11 significantly. Genes involved in plant defense, signaling, and those associated with the 

### 12 synthesis of plant hormones and secondary metabolism are generally up

- regulated in 

### 13 plants exposed to O 3 , while those related to photosynthesis and general me tabolism are 

### 14 typically down

- regulated. Proteome studies support these results by demonstrating 

### 15 concomitant increases or decreases in the proteins encoded by these genes. Transcriptome 

### 16 analysis has also illuminated the complex interactions that exist betwee n several different 

### 17 phytohormones and how they modulate plant sensitivity and response to O 3 . 

### 18 Experimental evidence suggests that while ethylene and salicylic acid are needed to 

### 19 develop O 3 - induced leaf lesions, jasmonic acid acts antagonistically to ethyle ne and 

### 20 salicylic acid to limit the spread of the lesions. Abscisic acid, in addition to its role in 

### 21 regulating stomatal aperture, may also act antagonistically to the jasmonic acid signaling 

### 22 pathway. Changes in the quantity and activity of these phytohormo nes and the 

### 23 interactions between them reveal some of the complexity of plant responses to an 

### 24 oxidative stressor such as O 3 . 

### 25
Another critical area of interest is to better understand and quantify the capacity of the 

### 26 plant to detoxify oxygen radicals using antioxidant metabolites, such as ascorbate and 

### 27 glutathione, and the enzymes that regenerate them. Ascorbate remains an important focus 

### 28 of research, and, due to its location in the apoplast in addition to other cellular 

### 29 compartments, it is regarded as a fir st line of defense against oxygen radicals formed in 

### 30 the apoplast. Most studies demonstrate that antioxidant metabolites and enzymes increase 

### 31 in quantity and activity in plants exposed to O 3 , indicating that they play an important 

### 32 role in protecting plants from oxidative stress. However, attempts to quantify the 

### 33 detoxification capacity of plants have remained unsuccessful, as high quantities of 

### 34 antioxidant metabolites and enzymes do not always translate into greater protection of the 

### 35 plant. Considerable var iation exists between plant species, different developmental 

### 36 stages, and the environmental and O 3 exposure conditions which plants are exposed to. 

### 37Draft

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- 38 September 2011 As indicated earlier, the described alterations in transcript levels of genes correlate with 

### 1 observed change s quantity and activity of the enzymes and metabolites involved in 

### 2 primary and secondary metabolism. In addition to the generalized up

- regulation of the 

### 3 antioxidant defense system, photosynthesis typically declines in O 3 treated plants. 

### 4 Declines in C fixat ion due to reductions in quantity and activity of Rubisco were 

### 5 extensively described in the 2006 O 3 AQCD. More recent studies support these results 

### 6 and indicate that declines in Rubisco activity may also result from reductions in Rubisco 

### 7 activase enzyme qu antity. Other studies, which have focused on the light reactions of 

### 8 photosynthesis, demonstrate that plant exposure to O 3 results in declines in electron 

### 9 transport efficiency and a decreased capacity to quench oxidizing radicals. Therefore, the 

### 10 overall declines in photosynthesis observed in O 3 - treated plants likely result from 

### 11 combined impacts on stomatal conductance, carbo n fixation and the light reactions. 

### 12 While photosynthesis generally declines in plants exposed to O 3 , catabolic pathways such 

### 13 as respiration have been shown to increase. It has been hypothesized that increased 

### 14 respiration may result from greater energy need s for defense and repair. Secondary 

### 15 metabolism is generally up

- regulated in a variety of species exposed to O 3 as a part of a 

### 16 generalized plant defense mechanism. Some secondary metabolites, such as flavonoids 

### 17 and polyamines, are of particular interest as they are known to have antioxidant 

### 18 properties. The combination of decreases in C assimilation and increases in catabolism 

### 19 and the production of secondary metabolites would negatively impact plants by 

### 20 decreasing the energy available for growth and reproduct ion. 

### 21



## 9.4 Nature of Effects on Vegetation and Ecosystems

9.4.1 Introduction

Ambient O 3 concentrations have long been known to cause visible symptoms, decreases 

### 22 in photosynthetic rates, decreases in growth and yield of plants as well as many other 

### 23 effects on ecosystem s ( U.S. EPA, 2006b
, 1996c
, 1986
, 1978a ) . Numerous studies have 

### 24 related O 3 exposure to plant responses, with most effort focused on the yiel d of crops and 

### 25 the growth of tree seedlings. Many experiments exposed individual plants grown in pots 

### 26 or soil under controlled conditions to known concentrations of O 3 for a segment of 

### 27 daylight hours for some portion of the plant ’ s life span. Information i n this section also 

### 28 goes beyond individual plant scale responses to consider effects at the broader ecosystem 

### 29 scale, including effects related to ecosystem services. 

### 30
This section will focus mainly on studies published since the release of the 2006 O 3 

### 31 AQCD . However, because much O 3 research was conducted prior to the 2006 O 3 AQCD, 

### 32Draft

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- 39 September 2011 the present discussion of vegetation and ecosystem response to O 3 exposure is largely 

### 1 based on the conclusions of the 1978, 1986, 1996, and 2006 O 3 AQCDs. 

### 2

9.4.1.1 Ecosystem Scale, Functio n, and Structure

Information presented in this section was collected at multiple spatial scales, ranging 

### 3 from the physiology of a given species to population, community, and ecosystem 

### 4 investigations. An ecological population is a group of individuals of th e same species and 

### 5 a community is an assemblage of populations of different species interacting with one 

### 6 another that inhabit an area. For this assessment, “ ecosystem ” is defined as the interactive 

### 7 system formed from all living organisms and their abiotic (physical and chemical) 

### 8 environment within a given area ( IPCC, 2007a ) . The boundaries of what could be called 

### 9 an ecosystem are somewhat arbitrary, depending on the focus of interest or study. Thus, 

### 10 the extent of an ecosystem may range from very small spatial scales to, ultimately , the 

### 11 entire Earth ( IPCC, 2 007a ) . All ecosystems, regardless of size or complexity, have 

### 12 interactions and physical exchanges between biota and abiotic factors, this includes both 

### 13 structural (e.g., soil type and food web trophic levels) and functional (e.g., energy flow, 

### 14 decomposition, ni trification) attributes. 

### 15
Ecosystems are most often defined by their structure based on the number and type of 

### 16 species present. Structure may refer to a variety of measurements including the species 

### 17 richness, abundance, community composition and biodiversity as well as landscape 

### 18

at tributes. Competition among and within species and their tolerance to environmental 

### 19 stressors are key elements of survivorship. When environmental conditions are shifted, 

### 20 for example, by the presence of anthropogenic air pollution, these competitive 

### 21 relati onships may change and tolerance to stress may be exceeded. Ecosystems may also 

### 22 be defined on a functional basis. “ Function ” refers to the suite of processes and 

### 23 interactions among the ecosystem components and their environment that involve 

### 24 nutrient and en ergy flow as well as other attributes including water dynamics and the flux 

### 25 of trace gases. Plant processes including photosynthesis, respiration, C allocation, 

### 26 nutrient uptake and evaporation, are directly related to functions of energy flow and C, 

### 27 nutrie nt and water cycling. The energy accumulated and stored by vegetation (via 

### 28 photosynthetic C capture) is available to other organisms. Energy moves from one 

### 29 organism to another through food webs, until it is ultimately released as heat. Nutrients 

### 30 and water can be recycled. Air pollution alters the function of ecosystems when elemental 

### 31 cycles or the energy flow are altered. This alteration can also be manifested in changes in 

### 32 the biotic composition of ecosystems. 

### 33Draft

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- 40 September 2011 There are at least three levels of ecosystem response to pollutants: (1) the individual 

### 1 organism and its environment; (2) the population and its environment; and (3) the 

### 2 biological community composed of many species and their environment ( Billings, 1978 )

. 

### 3 Individual organisms within a population vary in their ability to withstand th e stress of 

### 4 environmental change. The response of individual organisms within a population is based 

### 5 on their genetic constitution, stage of growth at time of exposure to stress, and the 

### 6 microhabitat in which they are growing ( Levine and Pinto, 1998 ) . The stress range within 

### 7 which organisms can exist and function determines the ability of the population to 

### 8 survive. Those best able to cope w ith environmental stressors survive and reproduce. 

### 9 Competition among different species results in succession (community change over time) 

### 10 and, ultimately, sensitive species may be progressively replaced and communities shift to 

### 11 favor those species that may have the capability to tolerate stressors such as O 3 ( Rapport 

### 12 and Whitford, 1999 ; Guderian, 1985 )

. 

### 13

9.4.1.2 Ecosystem Services

Ecosystem structure and function may be translated into ecosystem services. Ecosystem 

### 14

se rvices are the benefits people obtain from ecosystems ( UNEP, 2003 ) . Ecosystems 

### 15 provide many goods and services that are of vital importance for the functioning of the 

### 16 biosphere and provid e the basis for the delivery of tangible benefits to human society. 

### 17 Hassan et al. ( 2005 ) define these benefits to include supporting , provisioning, regulating, 

### 18 and cultural services: 

### 19

 Supporting services are necessary for the production of all other ecosystem 

### 20 services. Some examples include biomass production, production of 

### 21 atmospheric O 2 , soil formation and retention, nutrient cycling , water cycling, 

### 22 and provisioning of habitat. Biodiversity is a supporting service that is 

### 23 increasingly recognized to sustain many of the goods and services that humans 

### 24 enjoy from ecosystems. These provide a basis for three higher

- level categories 

### 25 of servi ces. 

### 26

 Provisioning services, such as products ( Gitay et al., 2001 ) , i.e., food 

### 27 (including game, roots, seeds, nuts and other fruit, spices, fodder), water, fiber 

### 28 (including wood, textiles), and medicinal and cosmeti c products (such as 

### 29 aromatic plants, pigments). 

### 30

 Regulating services that are of paramount importance for human society such 

### 31 as (1) C sequestration, (2) climate and water regulation, (3) protection from 

### 32 natural hazards such as floods, avalanches, or rock

- f all, (4) water and air 

### 33 purification, and (5) disease and pest regulation. 

### 34Draft

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- 41 September 2011  Cultural services that satisfy human spiritual and aesthetic appreciation of 

### 1 ecosystems and their components including recreational and other nonmaterial 

### 2 benefits. 

### 3

In the sections t hat follow, available information on individual, population and 

### 4 community response to O 3 will be discussed. Effects of O 3 on productivity and 

### 5 C sequestration, water cycling, below

- ground processes, competition and biodiversity, 

### 6 and insects and wildlife are considered below and in the context of ecosystem services 

### 7 where appropriate. 

### 8

9.4.2 Visible Foliar Injury and Biomonitoring

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

### 9 documented over several decades on many tree, shrub, herbaceous, and crop species 

### 10 ( U.S. EPA, 2006b
, 1996b
, 1984
, 1978a ) . Visible foliar injury symptoms are considered 

### 11 diagnostic as they have been verified experimentally in exposure

- response studies, using 

### 12 exposure methodologies such as CSTRs, OTCs, and f ree

- air fumigation (see Section 9.2 

### 13 for more detail on exposure methodologies). Several pictorial atlases and guides have 

### 14 been published, providing details on diagnosis and identification of O 3 - induced visible 

### 15 foliar injury on many plant species throughout North America ( Flagler, 1998 ; NAPAP, 

### 16 1987 ) and Europe ( Innes et al., 2001 ; Sánchez et al., 2001 ) . Typical visibl e injury 

### 17 symptoms on broad

- leaved plants include: stippling, flecking, surface bleaching, bifacial 

### 18 necrosis, pigmentation (e.g., bronzing), chlorosis, and/or premature senescence. Typical 

### 19 visible injury symptoms for conifers include: chlorotic banding, tip burn, flecking, 

### 20 chlorotic mottling, and/or premature senescence of needles. Although common patterns 

### 21 of injury develop within a species, these foliar lesions can vary considerably between and 

### 22 within taxonomic groups. Furthermore, the degree and extent of visible foliar injury 

### 23 development varies from year to year and site to site ( Orendovici

- Best et al., 2008 ; 

### 24 Chappelka et al., 2007 ; Smith et al., 2003 ) , even among co

- members of a population 

### 25 exposed to similar O 3 level s, due to the influence of co

- occurring environmental and 

### 26 genetic factors. Nevertheless, Chappelka et al. ( 2007 ) reported that the average incidence 

### 27 of O 3 - induced foliar injury was 73% on milkweed in the Great Smoky Mountains 

### 28 National Park in the years 1992

- 1996. 

### 29
Although the majority of O 3 - induced visible foliar injury occurrence has been observed 

### 30 on seedlings and small plants, many studies have reported visible injury of mature 

### 31 coniferous trees, primarily in the western U.S. ( Arbaugh et al., 1998 ) and to mature 

### 32 deciduous trees in eastern North America ( Schaub et al., 2005 ; Vollenweider et al., 2003 ; 

### 33Draft

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- 42 September 2011 Chappelka et al., 1999a ; Chappelka et al., 1999b ; Somers et al., 1998 ; Hildebrand et al., 

### 1 1996 )

. 

### 2
It is important to note that visible foliar injury occurs only when sensitive plants are 

### 3
expos ed to elevated O 3 concentrations in a predisposing environment. A major modifying 

### 4 factor for O 3 - induced visible foliar injury is the amount of soil moisture available to a 

### 5 plant during the year that the visible foliar injury is being assessed. This is beca use lack 

### 6 of soil moisture generally decreases stomatal conductance of plants and, therefore, limits 

### 7 the amount of O 3 entering the leaf that can cause injury ( Matyssek et al., 2006 ; Panek, 

### 8 2004 ; Grulke et al., 2003a ; Panek and Goldstein, 2001 ; Temple et al., 1992 ; Temple et 

### 9 al., 1988 ) . Consequently, many studies have shown that dry periods in local areas tend to 

### 10 decrease the incidence and severity of O 3 - induced vis ible foliar injury; therefore, the 

### 11 incidence of visible foliar injury is not always higher in years and areas with higher O 3 , 

### 12 especially with co

- occurring drought ( Smith et al., 2003 ) . Other factors such as leaf age 

### 13 influence the severity of symptom expression with older leaves showing greater injury 

### 14 severity as a result of greater seasonal exposure ( Zhang et al., 2010a )

. 

### 15
Although visible injury is a valuable indicator of the presence of phytotoxic 

### 16 concentrations of O 3 in ambient air, it is not always a reliable indicator of other negative 

### 17 effects on vegetation. The significance of O 3 injury at the leaf and whole plant levels 

### 18 depends on how much of the total leaf area of the pl ant has been affected, as well as the 

### 19 plant ’ s age, size, developmental stage, and degree of functional redundancy among the 

### 20 existing leaf area. Previous O 3 AQCDs have noted the difficulty in relating visible foliar 

### 21 injury symptoms to other vegetation effec ts such as individual plant growth, stand 

### 22 growth, or ecosystem characteristics ( U.S. EPA, 2006b
, 1996b ) . As a result, it is not 

### 23 presently possible to determine, with consistency across sp ecies and environments, what 

### 24 degree of injury at the leaf level has significance to the vigor of the whole plant. 

### 25 However, in some cases, visible foliar symptoms have been correlated with decreased 

### 26 vegetative growth ( Somers et al., 1998 ; Karnosky et al., 1996 ; Peterson et al., 1987 ; 

### 27 Benoit et al., 1982 ) and with impaired reproductive function ( Chappelka, 2002 ; Black et 

### 28 al., 2000 ) . Conversely, the lack of visible injury does not always indicate a lack of 

### 29 phytotoxic concentrations of O 3 or a lack of non

- visible O 3 effects ( Gregg et al., 2006
, 

### 30 2003 )

. 

### 31

9.4.2.1 Biomonitoring

The use of biological indicators to detect phytotoxic levels of O 3 is a longstanding and 

### 32 effective methodology ( Chappelka and Samuelson, 1998 ; Manning and Krupa, 1992 ) . A 

### 33 plant bioindicator can be defined as a vascular or nonvascular plant exhibiting a typical 

### 34Draft

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- 43 September 2011 and verifiable response when exposed to a plant stress such as an air pollutant ( Manning, 

### 1 2003 ) . To be considered a good indicator species, plants must (1) exhibit a distinct, 

### 2 verified response; (2) have few or no confounding disease or pest problems; and (3) 

### 3 exhibit genetic stability ( U.S. EPA, 2006b ) . Such sensitive plants can b e used to detect 

### 4 the presence of a specific air pollutant such as O 3 in the ambient air at a specific location 

### 5 or region and, as a result of the magnitude of their response, provide unique information 

### 6 regarding specific ambient air quality. Bioindicators c an be either introduced sentinels, 

### 7 such as the widely used tobacco ( Nicotiana tabacum ) variety Bel W3 ( Calatayud et al., 

### 8 2007b ; Laffray et al., 2007 ; Nali et al., 2007 ; Gombert et al., 2006 ; Kostka

- Rick and 

### 9 Hahn, 2005 ; Heggestad, 1991 ) or detectors, which are sensitive native plant species 

### 10 ( Chappelka et al., 2007 ; Souza et al., 2006 ) . The approach is especially useful in areas 

### 11 where O 3 monitors are not operated ( Manning, 2003 ) . For example, in remote wilderness 

### 12 areas where instrument monitoring is generally not available, the us e of bioindicator 

### 13 surveys in conjunction with the use of passive samplers ( Krupa et a l., 2001 ) may be a 

### 14 useful methodology ( Manning, 2003 ) . However, it requ ires expertise in recognizing those 

### 15 signs and symptoms uniquely attributable to exposure to O 3 as well as in their 

### 16 quantitative assessment. 

### 17

Since the 2006 O 3 AQCD, new sensitive plant species have been identified from field 

### 18 surveys and verified in controll ed exposure studies ( Kline et al., 2009 ; Kline et al., 2008 )

. 

### 19 Several multiple

- year field surveys have also been conducted at National Wildlife 

### 20 Refuges in Maine, Michigan, New Jersey, and South Carolina ( Davis, 2009
, 2007a
, b ; 

### 21
Da vis and Orendovici, 2006 )

. 

### 22
The USDA Forest Service through the Forest Health Monitoring Program (FHM) (1990

- 

### 23 2001) and currently the Forest Inventory and Analysis (FIA) Program has been collecting 

### 24 data regarding the incidence and severity of visible foliar injury on a variety of O 3 

### 25 sensitive plant species throughout the U.S. ( Coulston et al., 2003 ; Smith et al., 2003 ) . The 

### 26 plots where these data are taken are known as biosites. These biosites are located 

### 27 throughout the country and analysis of visible foliar injury within these sites follows a set 

### 28 of established protocols. For more details, see http://www.nrs.fs.fed.us/fia/topics/ozone/ 

### 29 ( USDA, 2011 ) . The network has provided evidence of O 3 concentrations high enough to 

### 30 induce visible symptoms on sensitive vege tation. From repeated observations and 

### 31 measurements made over a number of years, specific patterns of areas experiencing 

### 32 visible O 3 injury symptoms can be identified. Coulston et al. ( 2003 ) used information 

### 33 gathered over a 6

- year period (1994

- 1999) from the network to identify several species 

### 34 that were sensitive to O 3 over a regional scale including sweetgum ( Liquidambar 

### 35 styraciflua ), loblolly pine ( Pinus taeda ), and black cherry ( Prunus serotina ). In a study of 

### 36 the west coast of the U.S, Campbell et al. ( 2007 ) reported O 3 injury in 25

- 37% of biosites 

### 37 in California forested ecosystems from 2000

- 2005. 

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- 44 September 2011 A study by Kohu t ( 2007 ) assessed the risk of O 3 - induced visible foliar injury on 

### 1 bioindicator plants ( NPS, 2006 ) in 2 44 national parks in support of the National Park 

### 2 Service ’ s Vital Signs Monitoring Network ( NPS, 2007 ) . The risk assessment was based 

### 3

on a simple model relating response to the interaction of species, level of O 3 exposure, 

### 4 and exposure environment. Kohut ( 2007 ) concluded that the risk o f visible foliar injury 

### 5 was high in 65 parks (27%), moderate in 46 parks (19%), and low in 131 parks (54%). 

### 6 Some of the well

- known parks with a high risk of O 3 - induced visible foliar injury include 

### 7 Gettysburg, Valley Forge, Delaware Water Gap, Cape Cod, Fi re Island, Antietam, 

### 8 Harpers Ferry, Manassas, Wolf Trap Farm Park, Mammoth Cave, Shiloh, Sleeping Bear 

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

### 10
Lichens have also long been used as biomonitors of air pollution effec ts on forest health 

### 11 ( Nash, 2008 ) . It has been suspected, based on field surveys in the San Bernardino 

### 12 Mountains surrounding the Los Angeles air basin, that declines in lichen diversity and 

### 13 abundance were correlated with measured O 3 gradients ( Gül et al., 2011 ) . Several recent 

### 14 studies in North America ( Geiser a nd Neitlich, 2007 ; Gombert et al., 2006 ; Jovan and 

### 15 McCune, 2006 ) and Europe ( Nali et al., 2007 ; Gombert et al., 2006 ) have used lichens as 

### 16 biomonitors of atmospheric deposition (e.g., N and S) and O 3 exposure. Nali et al. ( 2007 ) 

### 17 found that epiphytic lichen bio diversity was not related to O 3 geographical distribution. 

### 18 In addition, a recent study by Riddell et al. ( 2010 ) found that lichen species, Ramalina 

### 19 menziesii
, showed no decline in physiological response to low and moderate 

### 20 concentrations of O 3 and may not be a good indicator for O 3 pollution. Mosses have also 

### 21 been used as biomonitors of air pollution; however, there remains a knowledge gap in the 

### 22 understanding o f the effects of ozone on mosses as there has been very little information 

### 23 available on this topic in recent years. 

### 24

9.4.2.2 Summary

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

### 25 documented over several decades of research on m any tree, shrub, herbaceous, and crop 

### 26 species ( U.S. EPA, 2006b
, 1996b
, 1984
, 1978a ) . Ozone

- induced visible foliar injury 

### 27 symptoms on certain bioindicator plant species are considered diagnostic as they have 

### 28 been verified experimentally in exposure

- response studies, using exposur e methodologies 

### 29 such as continuous stirred tank reactors (CSTRs), OTCs, and free

- air fumigation. 

### 30 Experimental evidence has clearly established a consistent association of visible injury 

### 31 with O 3 exposure, with greater exposure often resulting in greater and more prevalent 

### 32 injury. Since the 2006 O 3 AQCD, several multi

- year field surveys of O 3 - induced visible 

### 33 foliar injury have been conducted at National Wildlife Refuges in Maine, Michigan, New 

### 34Draft

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- 45 September 2011 Jersey, and South Carolina. New sensitive species showing visible foliar injury continue 

### 1 to be identified from field surveys and verified in controlled exposure studies. 

### 2
The use of biological indicators in field surveys to detect phytotoxic levels of O 3 is a 

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

### 4 Health Monitoring (FHM) Program (1990

- 2001) and currently the Forest Inventory and 

### 5 Analysis (FIA) Program has been collecting data regarding the incidence and severity of 

### 6 visible foliar injury on a variety of O 3 sensitive plant species throughout the U.S. The 

### 7 network has provided evidence that O 3 concentrations were high enough to induce visible 

### 8 symptoms on sensitive vegetation. From repeated observations and measurements made 

### 9 over a number of years, specific patterns of areas experienci ng visible O 3 injury 

### 10 symptoms can be identified. As noted in the preceding section, a study of 244 national 

### 11 parks indicated that the risk of visible foliar injury was high in 65 parks (27%), moderate 

### 12 in 46 parks (19%), and low in 131 parks (54%). 

### 13

Evidence is sufficient to conclude that there is a causal relationship between 

### 14 ambient O 3 exposure and the occurrence of O 3 - induced visible foliar injury on 

### 15 sensitive vegetation across the U.S. 

### 16

9.4.3 Growth, productivity and carbon storage in natural ecosystems

Ambient O 3 concentrations have long been known to cause decreases in photosynthetic 

### 17 rates, decreases in growth, and decreases in yield ( U.S. EPA, 2006b
, 1996c
, 1986
, 

### 18 1978a ) . The O 3 - induced damages at the plant scale may translate to the ecosystem scale, 

### 19 and cause changes in productivity and C storage. This sect ion focuses on the responses of 

### 20 C cycling to seasonal or multi

- year exposures to O 3 from the plant to ecosystem scale. 

### 21 Quantitative responses include changes in plant growth, plant biomass allocation, 

### 22 ecosystem production and ecosystem C sequestration. Bec ause of the available 

### 23 information, most of discussion at the plant scale focuses on the response of individual 

### 24 plants, especially tree seedlings and crops, with limited discussion of mixtures of 

### 25 herbaceous species. Changes at the ecosystem scale are diffic ult to evaluate directly due 

### 26 to the complexity and the large spatial and temporal scale. The discussion of ecosystem 

### 27 effects focuses on the new studies at the large

- scale FACE experiments and on ecological 

### 28 model simulations. 

### 29

9.4.3.1 Plant growth and biomass alloca tion

The previous O 3 AQCDs concluded that there is strong evidence that exposure to O 3 

### 30 decreases photosynthesis and growth in numerous plant species ( U.S. EPA, 2006b
, 

### 31Draft

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- 46 September 2011 1996b
, 1984
, 1978a ) . Studies published sinc e the last review support those conclusions 

### 1 and are summarized below. 

### 2
In general, research conducted over several decades has indicated that exposure to O 3 

### 3 alters stomatal conductance and reduces photosynthesis in a wide variety of plant species. 

### 4
In a rev iew of more than 55 studies, Wittig et al. ( 2007 ) reported that current O 3 

### 5 concentrations in the northern hemisphere are decreasing stomatal conductance (13%) 

### 6 and photosynthesis (11%) across tree species. It was also found that younger trees (<4 

### 7 year) were affected less by O 3 than older trees. Further, the authors also found that 

### 8 decreases in photosynthesis are consistent with the cumulative uptake of O3 into the leaf. 

### 9 In contrast, several studies reported that O 3 exp osure may result in loss of stomatal 

### 10 control, incomplete stomatal closure at night and a decoupling of photosynthesis and 

### 11 stomatal conductance, which may have implications for whole

- plant water use (Section 

### 12 9.4.5). 

### 13
In a recently published meta

- analysis, W ittig et al. ( 2009 ) quantitatively compiled peer 

### 14 reviewed studies from the past 40 years on the effect of current and future O 3 exposures 

### 15 on the physiology and growth of forest species. Wittig et al. ( 2009 ) reported that current 

### 16 ambient O 3 concen trations as reported in those studies (~40 ppb) significantly decreased 

### 17 annual total biomass growth (7%) across 263 studies. However, this effect could be 

### 18 greater (11 to 17%) in areas that have higher O 3 concentrations and as background O 3 

### 19 increases in the future ( Wittig et al., 2009 ) . This meta

- analysis demonstrates the 

### 20 coherence of O 3 effects across numerous studies and species that used a variety of 

### 21 experimental techniques, and these results support the conclusion of the previous AQCD. 

### 22
In two compani on papers, McLaughlin et al. ( 2007a ; 2007b ) investigated the effects of 

### 23 ambient O 3 on tree growth and hydrology at forest sites in the southern Appalachia n 

### 24 Mountains. The authors reported that the cumulative effects of ambient levels of O 3 

### 25 decreased seasonal stem growth by 30

- 50% for most trees species in a high O 3 year in 

### 26 comparison to a low O 3 year ( McLaughlin et al., 2007a ) . The authors also report that 

### 27 high ambient O 3 concentrations can disrupt whole

- tree water use and in turn reduce late



### 28

season stre amflow ( McLaughlin et al., 2007b ) ; see Section 9.4.5 for more on water 

### 29 cyclin g. 

### 30

Since the 2006 O 3 AQCD, several new studies based on the Aspen FACE “ free air ” O 3 

### 31 and CO 2 exposure experiment in a forest in Wisconsin were published ( Darbah et al., 

### 32 2008 ; Riikonen et al., 20 08 ; Darbah et al., 2007 ; Kubiske et al., 2007 ; Kubiske et al., 

### 33 2006 ; King et al., 2005 ) . Over the first seven years of stand development , Kubiske et al. 

### 34 ( 2006 ) observed that elevated O 3 decreased tree heights, diameters, and main stem 

### 35 volumes in the aspen community by 11, 16, and 20%, respectively. In addition, Kubiske 

### 36

et al. ( 2007 ) reported that elevated O 3 may change the intra

- and inter

- species 

### 37Draft

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- 47 September 2011 competition. For example, O 3 treatments increased the rate of conversion from a mixed 

### 1
aspen

- birch community to a birch dominated community. In a compariso n presented in 

### 2 Section 9.6.3 of this document, EPA found that effects on biomass accumulation in aspen 

### 3 during the first seven years closely agreed with the exposure

- response function based on 

### 4 data from earlier OTC experiments. 

### 5
Several studies at the Aspen FACE site also considered other growth

- related effects of 

### 6 elevated O 3 . Darbah et al. ( 2008 ; 2007 ) reported that O 3 treatments decreased paper birch 

### 7 seed weight and seed germination and that this would likely lead to a negative impact of 

### 8 regeneration for that species. Riikonen et al

. ( 2008 ) found that elevated O 3 decreased the 

### 9 amount of starch in birch buds by 16%, and reduced aspen bud size, which may have 

### 10 been related to the observed delay in spring leaf development. The results suggest that 

### 11

e levated O 3 concentrations have the potential to alter C metabolism of overwintering 

### 12 buds, which may have carry

- over effects in the subsequent growing season ( Riikonen et 

### 13 al., 2008 )

. 

### 14
Effects on growth of understory ve getation were also investigated at Aspen FACE. 

### 15 Bandeff et al. ( 2006 ) found that the effects of elevated CO 2 and O 3 on understory species 

### 16 composition, total and individual species biomass, N content, and 15 N recovery were a 

### 17 result of overstory community responses to those treatments; however, there were no 

### 18 apparent direct t reatment e ff ects due to high variability of the data. Total understory 

### 19 biomass increased with increasing light and was greatest under the open canopy of the 

### 20 aspen/maple community, as well as the more open canopy of the elevated O 3 treatments 

### 21 ( Bandeff et al., 2006 ) . Similarly, data from a study by Awmack et al. ( 2007 ) suggest that 

### 22 elevated CO 2 and O 3 may have indirect growth effects on red ( Trifolium pratense ) and 

### 23 white ( Trifolium repens ) clover in the understory via overstory community effects; 

### 24 however, no direct effects of elevated O 3 were observed. 

### 25
Overall, the studies at the Aspen FACE experiment are consistent with many of the OTC 

### 26 studies that were evaluated in previous O 3 AQCDs. These results strengthen our 

### 27 understanding of O 3 effects on forests and demonstrate the relevance of the knowledge 

### 28 gained from trees grown in open

- top chamber studies. 

### 29

For s ome annual species, particularly crops, the endpoint for an assessment of the risk of 

### 30 O 3 exposure is yield or growth, e.g., production of grain. For plants grown in mixtures 

### 31 such as hayfields, and natural or semi

- natural grasslands (including native nonagr icultural 

### 32 species), endpoints other than production of biomass may be important. Such endpoints 

### 33 include biodiversity or species composition, and effects may result from competitive 

### 34 interactions among plants in mixed

- species communities. Most of the availab le data on 

### 35

non

- crop herbaceous species are for grasslands, with many of the recent studies 

### 36Draft

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- 48 September 2011 conducted in Europe. See Section 9.4.7 for a review of the recent literature on O 3 effects 

### 1 on competition and biodiversity in grasslands. 

### 2

Root Growth
Although O 3 doe s not penetrate soil, it could alter root development by decreasing 

### 3 C assimilation via photosynthesis leading to less C allocation to the roots ( Andersen, 

### 4 2003 ) . The response of root development to O 3 exposure depends on available 

### 5 photosynthate within the plant and could vary over time. Many biotic and abiotic factors, 

### 6 such as community dynamics and drought stress, have been found to alter root 

### 7 development under elevated O 3 . An earlier study at the Aspen FACE experiment found 

### 8 that elevated O 3 reduced coarse root and fine roots biomass in young stands of paper 

### 9 birch and trembling aspen ( King et al., 2001 ) . However, this reduction disappeared 

### 10 several years later. Ozone significantly increased fine

- root (<1.0 mm) in the aspen 

### 11 community ( Pregitzer et al., 2008 ) . This increase in fine root production was due to 

### 12 changes in community composition, such as better survival of the O 3 - tolerant aspen 

### 13 genot ype, birch, and maple, rather than changes in C allocation at the individual tree level 

### 14 ( Pregitzer et al., 2008 ; Zak et al., 2007 ) . In an adult European beech/Norway spruc e 

### 15 forest in Germany, drought was found to nullify the O 3 - driven stimulation of fine root 

### 16 growth. Ozone stimulated fine

- root production of beech during the humid year, but had 

### 17 no significant impact on fine root production in the dry year ( Matyssek et al., 2010 ; 

### 18 Nikolova et al., 2010 )

. 

### 19
Using a non

- destructive method, Vollsnes et al. ( 2010 ) studied the in vivo root 

### 20 develo pment of subterranean clover ( Trifolium subterraneum ) before, during and after 

### 21 short

- term O 3 exposure. It was found that O 3 reduced root tip formation, root elongation, 

### 22 the total root length, and the ratios between below

- and above

- ground growth within 

### 23

one week after exposure. Those effects persisted for up to three weeks; however, biomass 

### 24 and biomass ratios were not significantly altered at the harvest five weeks after exposure. 

### 25

Several recent meta

- analyses have generally indicated that O 3 reduced C alloca ted to 

### 26
roots. In one meta

- analysis, Grantz et al. ( 2006 ) estimated the effect of O 3 on the 

### 27 root:sho ot allometric coefficient (k), the ratio between the relative growth rate of the root 

### 28 and shoot. The results showed that O 3 reduced the root:shoot allometric coefficient by 

### 29 5.6%, and the largest decline of the root:shoot allometric coefficient was observed in 

### 30 slow

- growing plants. In another meta

- analysis including 263 publications, Wittig et al. 

### 31 ( 2009 ) found that current O 3 exposure had no significant impacts on root biomass and 

### 32 root:shoot ratio when compared to pre

- industrial O 3 exposure. However, if O 3 

### 33 concentrations rose to 81

- 101 ppb (projected O 3 levels in 2100), both root biomass and 

### 34 root:shoot ratio were found to significantly decrease. Gymnosperms and angiosperms 

### 35Draft

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- 49 September 2011 differed in their responses, with gymnosperms being less sensitive to elevated O 3 . In two 

### 1 other meta

- analyses, Wang et al. ( 2010 ) found elevated O 3 reduced biomass allocation to 

### 2 roots by 8.3% at ambient CO 2 and 6.0% at elevated CO 2 , and Morgan et al. ( 2003 ) found 

### 3 O 3 reduced root dry weight of soybean. While there is clear evidence that O 3 reduced C 

### 4 allocation to roots, results of recent individual studies have been mixed, showing negative 

### 5 ( Jones et al., 2010 ) , non

- significant ( Andersen et al., 2010 ; Phillips et al., 2009 ) and 

### 6 positiv e effects ( Pregitzer et al., 2008 ; Grebenc and Kraigher, 2007 ) on root biomass and 

### 7 root: shoot ratio. 

### 8

9.4.3.2 Summary

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

### 9 ambient concentrations of O 3 decrease photosynthesis and growth in numerous plant 

### 10 species across the U.S. Studies published since the last review continue to support that 

### 11 conclusion. 

### 12

The meta

- analysis by Wittig et al. ( 2007 ) and ( 2009 ) demonstrates the coherence of O 3 

### 13 effects on plant photosynthesis and growth across numerous studies and species using a 

### 14 variety of experimental techniques. Since the 2006 O 3 AQCD, several studies were 

### 15 published based on the Aspen FACE experiment using “ free air, ” O 3 , and CO 2 exposures 

### 16 in a forest in Wisconsin. Overall, the studies at the Aspen FACE experiment were 

### 17 consistent with many of the open

- top chamber (OTC) studies tha t were the foundation of 

### 18 previous O 3 NAAQS reviews. These results strengthen our understanding of O 3 effects 

### 19 on forests and demonstrate the relevance of the knowledge gained from trees grown in 

### 20
open

- top chamber studies. 

### 21

In recent studies, O 3 was shown to h ave either negative, non

- significant, or positive 

### 22 effects on root biomass and root:shoot ratio. While the findings of individual studies were 

### 23 mixed, recent meta

- analyses have generally indicated that O 3 reduced C allocated to roots 

### 24 ( Wittig et al., 2009 ; Grantz et al., 2006 )

. 

### 25

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

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

### 27

9.4.3.3 Reproduction

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

### 28 reproduction. The impacts of O 3 on reproductive developme nt, as reviewed by Black et 

### 29 al. ( 2000 ) , can occur by influencing (1) age at which flowering occurs, particularly in 

### 30Draft

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- 50 September 2011 long

- lived trees that often have long juvenile periods of early growth without flower and 

### 1 seed production; (2) flower bud initiation and development; (3) pollen germination and 

### 2 pollen tube growth; (4) seed, fruit, or cone yields; and (5) s eed quality (Table 9

- 1 ) ( U.S. 

### 3 EPA, 2006b ) . Several recent studies since the 2006 O 3 AQCD further demonstrate the 

### 4 effects of O 3 on reproductive processes in herbaceous and woody plant species. Although 

### 5 there have been documented effects of ozone on reproductive processes, a knowledge gap 

### 6 still exists pertaining to the exact mechanism of these responses. 

### 7

Rämö et al. ( 2007 ) exposed several meadow species to elevated O 3 (40

- 50 ppb) and CO 2 

### 8 (+100 ppm), both individually and combined, over three growing seasons in ground



### 9 planted mesocosms, using O TCs. Elevated O 3 delayed flowering of Campanula 

### 10 rotundifolia and Vicia cracca . Ozone also reduced the overall number of produced 

### 11 flowers and decreased fresh weight of individual Fragaria vesca berries. 

### 12

Black et al. ( 2007 ) exposed Brassica campestris to 70 ppb for two days during late 

### 13 vegetative growth or ten days during most of the vegetative phase. The two

- day exposure 

### 14 had no effect on growth or reproductive characteristics, while the 10 day exposure 

### 15 reduced vegetative growth and reproductive site number on the terminal raceme, 

### 16 emphasizing the importance of exposure duration and timing. Mature seed number and 

### 17

w eight per pod were unaffected due to reduced seed abortion, suggesting that, although 

### 18 O 3 affected reproductive processes, indeterminate species such as B. campestris possess 

### 19 enough compensatory flexibility to avoid reduced seed production ( Black et al., 2007 )

. 

### 20
In the determinate species, Plantago major , Black et al. ( 2010 ) found that O 3 may have 

### 21 direct effects on reproductiv e development in populations of differing sensitivity. Only 

### 22 the first flowering spike was exposed to 120 ppb O 3 for 7 hours per day on 9 successive 

### 23 days (corresponding to flower development) while the leaves and second spike were 

### 24 exposed to charcoal

- filter ed air. Exposure of the first spike to O 3 affected seed number 

### 25 per capsule on both spikes even though spike two was not exposed. The combined seed 

### 26 weight of spikes one and two was increased by 19% in the two resistant populations, 

### 27 suggesting an overcompens ation for injury; whereas, a decrease of 21% was observed in 

### 28 the most sensitive population ( Black et al., 2010 ) . The question remains as to whether 

### 29 these effects are true direct ozone

- induced effects or compensatory responses. 

### 30
A study by Darbah et al. ( 2008 ; 2007 ) of paper birch ( Betula pap yrifera ) trees at the 

### 31 Aspen FACE site in Rhinelander, WI investigated the effects of elevated O 3 and/or CO 2 

### 32 on reproductive fitness. Elevated O 3 increased flowering, but decreased seed weight and 

### 33 germination success rate of seeds from the exposed trees. Th ese results suggest that O 3 

### 34 can dramatically affect flowering, seed production, and seed quality of paper birch, 

### 35 ultimately affecting its reproductive fitness ( Darbah et al., 2008 ; Darbah et al., 2007 )

. 

### 36Draft

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### Table 9

- 1 Ozone effects on plant reproductive processes (derived from Table AX9

- 22 o f the 2006 ozone AQCD)

Species Condition Measures References Apocynun androsaemifolium Flowering time Bergweiler and Manning ( 1999 )

Buddleia davidii Flowering time Findley et al. ( 1997 ) Rubus cuneifolius Pollen germination Chappelka ( 2002 ) Plantago major Pollen tube elongation Stewart ( 1998 ) Fragaria × ananassa Fruit yield Drogoudi and Ashmore ( 2001 ) ; Dr ogoudi and Ashmore ( 2000 )
Plantago major Seed yield Lyons and Barnes ( 1998 ) ; Pearson et al. ( 1996 ) ; Reiling and Davison ( 1992 ) ; Whitfield et al. ( 1997 ) Understory herbs Seed yield Harward and Treshow ( 1975 )

Source: Adapted from 2006 O 3 AQCD



### 9.4.3.4 Ecosystem Productivity and Carbon Sequestration

During the previous NAAQS review, there were limited studies that investigated the 

### 1 effect of O 3 exposure on ecosystem productivity and C sequestration. Recent studies 

### 2 from long

- term FACE experiments provide more evidence of the association of O 3 

### 3 exposure and changes in productivity at the ecosystem scale. In addition to experimental 

### 4 studies, model s tudies also assessed the impact of O 3 exposure on productivity and 

### 5 C sequestration from stand to global scales. 

### 6
Two types of models are most often used to study the ecological consequences of O 3 

### 7 exposure: (1) single plant growth models such as TREGRO and PnET

– II ( Hogsett et al., 

### 8 2008 ; Martin et al., 2001 ; Ollinger et al., 1997b ) , and (2) process

- based ecosystem 

### 9 models such as PnET

- CN, Dynamic Land Ecosystem Model (DLEM), Terrestrial 

### 10 Ecosystem Model (TEM), or Met Office Surface Exchange Scheme

- Top

- down 

### 11 Representation of Interactive Foliage and Flora Including Dynamics (MOSES

- TRIFFID) 

### 12 ( Felzer et al., 2009 ; Ren et al., 2007a ; Sitch et al., 2007 ; Ollinger et al., 2002 ) (Table 9

- 2 ). 

### 13 In these models, carbon uptake is simulated through photosynthesis (TREGRO, PnET

– 

### 14 II, PnET

- CN, DLEM and MOSES

- TRIFFID) or gross primary production (TEM). 

### 15 Phot osynthesis rate at leaf level is modeled by a function of stomatal conductance and 

### 16 other parameters in TREGRO, PnET

– II, PnET

- CN, DLEM and MOSES

- TRIFFID. 

### 17 Photosynthesis at canopy level is calculated by summing either photosynthesis of 

### 18 different leaf types (TREGRO, DLEM, and MOSES

- TRIFFID) or photosynthesis of 

### 19 different canopy layers (PnET

– II, PnET

- CN). The detrimental effect of O 3 on plant 

### 20 growth is often simulated by multiplying photosynthesis rate by a coefficient that is 

### 21 dependent on stomatal conducta nce and cumulative O 3 uptake (Table 9

- 2 ). Different 

### 22Draft

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- 52 September 2011 plant functional groups (PFTs, such as deciduous trees, coniferous trees or crops) show 

### 1 different responses to O 3 exposure. PnET

- II, PnET

- CN, TEM, DLEM and MOSES



### 2 TRIFFID estimate this difference by modify ing net photosynthesis with coefficients that 

### 3 represent the O 3 induced fractional reduction of photosynthesis for each functional group. 

### 4 The coefficients used in PnET

- II, PnET

- CN, TEM, DLEM are derived from the functions 

### 5 of O 3 exposure (AOT40) versus photo synthesis reduction from Reich ( 1987 ) and 

### 6 Tjoelker et al. ( 1995 )

. The coefficients use d in MOSES

- TRIFFID are derived from the 

### 7 O 3 dose

- photosynthesis response function from Pleijel et al. ( 2004a ) and Karlsson et al. 

### 8 ( 2004 ) , where O 3 dose is estimated by a metric named CUOt (cumulati ve stomatal uptake 

### 9 of O 3 ). The O 3 threshold of CUOt is 1.6 nmol/m 2 /s for woody PFT and 5 nmol/m 2 /s for 

### 10 grass PFT, and is different from AOT40, which has an O 3 threshold level of 40 ppb for 

### 11 all PFTs. Experimental and model studies on ecosystem productivity and C sequestration 

### 12 at the forest stand scale as well as regional and global scales are reviewed in the 

### 13 following section. 

### 14Draft

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### Table 9

- 2 Comparison of models used to simulate the ecological consequences of O 3 

### exposure

Model Model feature Carbon uptake Ozone ef fect Reference

TREGRO Hourly or daily step, single plant model simulating vegetation growth process Leaf: leaf photosynthesis is a function of stomatal conductance, mesophyll conductance and the gradient of CO 2 from atmosphere to the mesophyll cells Canopy: Leaf is divided into different ages. The canopy photosynthesis rate is the sum the photosynthesis of all foliage groups The effect of O 3 on photosynthesis is simulated by reducing mesophyll conductance, and increasing respiration. The degree of O 3 damage is determined by ambient O 3 exposure, and the threshold O 3 concentration below which O 3 does not affect mesophyll conductance and respiration Hogsett et al. ( 2008 ) ; Weinstein et al. ( 2005 ) ; Tingey et al. ( 2004 )

PnET

- II and PnET

- CN PnET

- II:
mo nthly time

step, single plant model PnET

– CN: monthly time

step, ecosystem mode Leaf: Maximum photosynthesis rate is determined by a function of foliar N concentration, and stomatal conductance is determined by a function of the actual rate of the photosy nthesis. Canopy: canopy is divided into multiple,
even
- mass layers and photosynthesis is simulated by a multilayered canopy submodel The effect of O 3 on photosynthesis is simulated by an equation of stomatal conductance and O 3 dose (AOT40). The model assum es that photosynthesis and stomatal conductance remain coupled under O 3 exposure, with a reduction in photosynthesis for a given month causing a proportion reduction in stomatal conductance. Ollinger et al. ( 2002 ; 1997b ) ; Pan et al. ( 2009 )

TEM monthly time

step, ecosystem mode Ecosystem: TEM is run at a 0.5*0.5 degree resolution . Each grid cell is classified by vegetation type and soil texture, and vegetations and detritus are assumed to distribute homogeneously within grid cells. Carbon flows into ecosystem via gross primary production, which is a function of maximum rate of ass imilation, photosynthetically active radiation, the leaf area relative to the maximum annual leaf area, mean monthly air temperate, and nitrogen availability. The direct O 3 reduction on GPP is simulated by multiplying GPP by f(O 3 )t, where f(O 3 )t is determined by evapotranspiration, mean stomatal conductance, ambient AOT40, and empirically O 3 response coefficient derived from previous publications. Felzer et al. ( 2005 ; 2004 )

DLEM daily time

step ecosystem model Leaf: photosynthesis is a function of 6 parameters: photosynthetic photon flux density, stomatal conductance, daytime temperature, the atmospheric CO 2 concentration, the leaf N content and the length of daytime. Canopy: Photosynthetic rates for sunlit leaf and shaded leaf scale up to the canopy level by multiplying the estimated leaf area index Ecosystem: GPP is the sum of gross C fixatio n of different plant function groups The detrimental effect of O 3 is simulated by multiplying the rate of photosynthesis by O 3 eff, where O 3 eff is a function of stomatal conductance, ambient AOT40, and O 3 sensitive coefficient. Ozone ’ s indirect effect on stomatal conductance is also simulated, with a reduction in photosynthesis for a given month causing a reduction in stomatal conductance, and therefore canopy conductance. Ren et al. ( 2007a ; 2007b ) ; Zhang et al. ( 2007a )

MOSES

TRIFFID 30 minutes time

- step, dyn amic global vegetation model Leaf: photosynthesis is a function of environmental and leaf parameters and stomatal conductance; Stomatal conductance is a function of the concentration of CO 2 and H 2 O in air at the leaf surface and the current rate of photosynthesis of the leaf Canopy: Photosynthetic rates scale up to the canopy level by multiplying a function of leaf area index and PAR extinction coefficient Ecosystem: GPP is the sum of gross C fixation of different plant function groups The effect of O 3 is simulated by multiplying the rate of photosynthesis by F, where F depends upon stomatal conductance, O 3 exposure, a critical threshold for O 3 damage, and O 3 sensitive coefficient (functional type dependent) Sitch et al. ( 2007 )Draft

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- 54 September 2011 Local Scale

The above

- and below

- ground biomass and net primary production (NPP) were measured 

### 1 at the Aspen FACE site after 7 years of O 3 exposure. Elevated O 3 caused 23, 13 and 14% 

### 2 reductions in total biomass relative to the control in the aspen, aspen

– birch and aspen

– 

### 3 maple communities, respectively ( King et al., 2005 ) . At the Kranzberg Forest FACE 

### 4 experiment in Germany, O 3 reduced annual volume growth by 9.5 m 3 /ha in a mixed 

### 5 mature stand of Norway spruce and European beech ( Pretzsch et al., 2010 ) . At the 

### 6 grassland FACE experiment at Alp Flix, Switzerland, O 3 reduced the seasonal mean rates 

### 7 of ecosystem respiration and GPP by 8%, but had no significant impacts on aboveground 

### 8 dry matte r productivity or growing season net ecosystem production (NEP) ( Volk et al., 

### 9 2011 ) . Ozone also altered C accumulation and turnover in soil, as discussed in Section 

### 10 9.4.6. 

### 11
Changes in forest stand productivity under elevated O 3 were assessed by several model 

### 12 studies. TREGRO (Table 9

- 2 ) has been widely used to simulate the effects of O 3 on the 

### 13 growth of several species in different regions in the U.S. Hogsett et al. ( 2008 ) used 

### 14 TREGRO to evaluate the effectiveness of various forms and levels of air quality 

### 15 standards for protecting tree growth in the San Bernardino Mountains of California. They 

### 16 found that O 3 exposures at the Crestline site resulted in a mean 20.9% biomass reduction 

### 17 from 1980 to 1985 and 10.3% biomass reduction from 1995 to 2000, compared to the 

### 18

‘‘ background ’’ O 3 concentrations ( O 3 concentration in Crook County, Oregon). The 

### 19 level of vegetation protection projected was different depending on the air quality 

### 20 scenarios under consideration. Specifically, when air quality was simulated to just meet 

### 21 the California 8

- h average maximum o f 70 ppb and the maximum 3 months 12

- h SUM06 

### 22 of 25 ppm

- h, annual growth reductions were limited to 1% or less, while air quality that 

### 23 just met a previous NAAQS (the second highest 1

- h max [125 ppb]) resulted in 6

- 7% 

### 24 annual reduction in growth, resulting in the least protection relative to background O 3 

### 25 ( Hogsett et al., 2008 )

. 

### 26
ZELIG is a forest succession gap model, and has been used to evaluate the dynamics of 

### 27 natural stand succession. Combining TREGRO with ZELIG, Weinstein et al. ( 2005 ) 

### 28 simulated the effects of different O 3 levels ( 0. 5, 1.5, 1.75, and 2 times ambient) on the 

### 29 growth and competitive interactions of white fir and ponderosa pine at three sites in 

### 30 California: Lassen National Park, Yosemite National Park, and Crestline. Their results 

### 31 suggested that O 3 had little impact on wh ite fir, but greatly reduced the growth of 

### 32 ponderosa pine. If current O 3 concentrations continue over the next century, ambient O 3 

### 33 exposure (SUM06 of 110 ppm

- h) at Crestline was predicted to decrease individual tree 

### 34 C budget by 10% and decrease ponderosa p ine abundance by 16%. Effects at Lassen 

### 35Draft

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- 55 September 2011 National Park and Yosemite National Park sites were found to be smaller because of 

### 1 lower O 3 exposure levels ( Weinstein et al., 2005 )

. 

### 2

The effects of O 3 on stand productivity and dynamics were also s tudied by other tree 

### 3 growth or stand models, such as ECOPHYS, INTRASTAND and LINKAGES. 

### 4 ECOPHYS is a functional

- structural tree growth model. The model used the linear 

### 5 relationship between the maximum capacity of carboxylation and O 3 dose to predict the 

### 6 rel ative effect of O 3 on leaf photosynthesis ( Martin et al., 2001 ) . Simulations with 

### 7 ECOPHYS found that O 3 decreased stem dry matter production, stem diameter and leaf 

### 8 dry matter production, in duced earlier leaf abscission, and inhibited root growth ( Martin 

### 9 et al., 2001 ) . INTRASTAND is an hourly time step model for forest stand carbon and 

### 10 water budgets. LINKAGES is a monthly time step model simulating forest growth and 

### 11 community dynamics. Linking INTRASTAND with LINKAGES, Hanson et al. ( 2005 ) 

### 12 found that a simulated increase in O 3 concentration in 2100 (a mean 20

- ppb increase over 

### 13 the current O 3 concentration) yields a 35% loss of net ecosystem C exchange (NEE) with 

### 14 respect to the current conditions (174 g C/m 2 /year). 

### 15

Regional and Global Scales
Since the publication of the 2006 O 3 AQCD, there is additional evidence suggesting that 

### 16 O 3 exposure alters ecosystem productivity and biogeochemical cycling at the regional 

### 17 and continental scale. Most of those studies were conducted by using process

- based 

### 18 ecosystem models (Table 9

- 2 ) and are briefly reviewed in the following sections. 

### 19
Ollinger et al. ( 1997a ) simulated the effect of O 3 on hardwood f orest productivity of 64 

### 20 hardwood sites in the northeastern U.S. with PnET

- II (Table 9

- 2 ). Their simulations 

### 21 indicated that O 3 caused a 3

- 16% reduction in NPP from 1987 to 1992 (Table 9

- 3 ). The 

### 22 interactive effects of O 3 , N deposition, elevated CO 2 and land use history on C dynamics 

### 23 were estimated by PnET

- CN (Table 9

- 2 ) ( Ollinger et al., 2002 ) . The results indicated that 

### 24 O 3 offset the increase in net C exchange caused by elevated CO 2 and N deposition by 

### 25 13% (25.0 g C/m 2 /year) under agriculture site history, and 23% (33.6 g C/m 2 /y ear) under 

### 26 timber harvest site history. PnET

- CN was also used to assess changes in C sequestration 

### 27 of U.S. Mid

- Atlantic temperate forest. Pan et al. ( 2009 ) designed a factorial modeling 

### 28 experiment to separate the effects of changes in atmospheric composition, historical 

### 29 climatic variability and land

- distur bances on the C cycle. They found that O 3 acted as a 

### 30 negative factor, partially offsetting the growth stimulation caused by elevated CO 2 and N 

### 31 deposition in U.S. Mid

- Atlantic temperate forest. Ozone decreased NPP of most forest 

### 32 types by 7

- 8%. Among all the forest types, spruce

- fir forest was most resistant to O 3 

### 33 damage, and NPP decreased by only 1% ( Pan et al., 2009 )

. 

### 34Draft

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- 56 September 2011 Felzer et al. ( 2004 ) developed TE M 4.3 (Table 9

- 2 ) to simulate the effects of O 3 on plant 

### 1 growth and estimated effects of O 3 on NPP and C sequestration of deciduous trees, 

### 2 conifers and crops in the conterminous U.S. The results indicated that O 3 reduced NPP 

### 3 and C sequestration in the U.S. (Table 9

- 3 ) with the largest decreases (over 13% in some 

### 4 locations) in NPP occurring in the Midwest agricultural lands during the mid

- summer. 

### 5 TEM was also used to evaluate the magnitude of O 3 damage at the global scale (Table 9

- 

### 6 3 ) ( Felzer et al., 2005 ) . Simulations for the perio d 1860 to 1995 show that the largest 

### 7 reductions in NPP and net C exchange occurred in the mid western U.S., eastern Europe, 

### 8 and eastern China ( Felzer et al., 2005 ) . DLEM (Table 9

- 2 ) was developed to simulate the 

### 9 detrimental effect of O 3 on ecosystems, and has been used to examine the O 3 damage on 

### 10 NPP and C sequestration in Great Smoky Mountains National Park ( Zhang et al., 2007a )
, 

### 11 grassland ecosystems and terrestrial ecosystems in China ( Ren et al., 2007a ; Ren et al., 

### 12 2007b ) . Results of those simulations are listed in Table 9

- 3

. 

### 13
Instead of using AOT40 as their O 3 exposure metric as PnET, TEM and DLEM did, 

### 14 Sitch et al. ( 2007 ) incorporated a different O 3 metric named CUOt (cumulative stomatal 

### 15 uptake of O 3 ), derived from Pleijel et al. ( 2004a ) , into the MOSES

- TRIFFID coupled 

### 16 model (Table 9

- 2 ). In the CUOt metric, the fractional reduction of plant production is 

### 17 dependent on O 3 uptake by stomata over a crit ical threshold for damage with this 

### 18 threshold level varying by plant functional type. Consistent with previous studies, their 

### 19 model simulation indicated that O 3 reduced global gross primary production (GPP), 

### 20 C exchange rate and C sequestration (Table 9

- 3 ). The largest reductions in GPP and land



### 21 C storage were projected over North America, Europe, China and India. In the model, 

### 22 reduced ecosystem C uptake due to O 3 damage, results in additional CO 2 accumulation 

### 23 in the atmosphere and an indirect radiative forc ing of climate change. Their simulations 

### 24 indicated that the indirect radiative forcing caused by O 3 (0.62

- 1.09 W/m 2 ) could have 

### 25 even greater impact on global warming than the direct radiative forcing of O 3 

### 26 (0.89 W/m 2 ) ( Sitch et al., 2007 )

. 

### 27
Results from the various model studies presented in Table 9

- 3 are difficult to compare 

### 28 because of the various spatial and temporal scales used in these studies. However, all the 

### 29 studies showed that O 3 exposure decreased ecos ystem productivity and C sequestration. 

### 30 These results are consistent and coherent with experimental results from the leaf, plant 

### 31 and ecosystem scales ( Sitch et al., 2007 ; Felzer et al., 2005 ) . Many of the models use the 

### 32 same underlying function to simulate the effect of O 3 exposure to C uptake. For example 

### 33 the functions of O 3 exposure (AOT40) versus photosynthesis reduction for PnET

- II, 

### 34
PnET

- CN, TEM, DLEM were all from Reich ( 1987 ) and Tjoelker et al. ( 1995 )

. 

### 35 Therefore, it is not surprising that the results are similar. While these models can be 

### 36 improved and more evaluation with experimental data can be done, these models 

### 37Draft

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- 57 September 2011 represent the state of the science for estimating the effect of O 3 exposure on productivity 

### 1 and C sequestration. 

### 2

9.4.3.5 Summary

Durin g the previous NAAQS reviews, there were very few studies that investigated the 

### 3 effect of O 3 exposure on ecosystem productivity and C sequestration. Recent studies 

### 4 from long

- term FACE experiments, such as Aspen FACE, SoyFACE and the Kranzberg 

### 5 Forest (Germa ny), provided evidence of the association of O 3 exposure and reduced 

### 6 productivity at the ecosystem level. Studies at the leaf and plant scales showed that O 3 

### 7 reduced photosynthesis and plant growth, which provided coherence and biological 

### 8 plausibility for the decrease in ecosystem productivity. Results across different ecosystem 

### 9 models, such as TREGRO, PnET, TEM and DLEM, were consistent with the FACE 

### 10 experimental evidence, which showed that O 3 reduced ecosystem productivity. 

### 11
Although O 3 generally causes n egative effects on plant growth, the magnitude of the 

### 12 response varies among plant communities. For example, O 3 had little impact on white fir, 

### 13 but greatly reduced growth of ponderosa pine in southern California ( Weinstein et al., 

### 14 2005 ) . O zone decreased net primary production (NPP) of most forest types in Mid



### 15 Atlantic region, but had small impacts on spruce

- fir forest ( Pan et al., 2009 )

. 

### 16
In addition to plant growth, other indicators that are typically estimated by model studies 

### 17 include net ecosystem CO 2 exchange (NEE), C sequestration, and cro p yield. Model 

### 18 simulations consistently found that O 3 exposure caused negative impacts on those 

### 19 indicators, but the severity of these impacts was influenced by multiple interactions of 

### 20 biological and environmental factors. The suppression of ecosystem C si nks results in 

### 21 more CO 2 accumulation in the atmosphere. Globally, the indirect radiative forcing caused 

### 22 by O 3 exposure through lowering ecosystem C sink could have an even greater impact on 

### 23 global warming than the direct radiative forcing of O 3 ( Sitch et al., 2007 ) . Ozone could 

### 24 also affect regional C budgets through interacting with multiple factors, such as N 

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

### 26 partial ly offset the growth stimulation caused by elevated CO 2 and N deposition in both 

### 27 Northeast

- and Mid

- Atlantic

- region forest ecosystems of the U.S. ( Pan et al., 2009 ; 

### 28 Ollinger et al ., 2002 )

. 

### 29

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

### 30 exposure and reduced productiv ity, and a likely causal relationship between O 3 

### 31 exposure and reduced carbon sequestration in terrestrial ecosystems. 

### 32Draft

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- 58 September 2011 

### Table 9

- 3 Modeled effects of ozone on primary production, C exchange, and C sequestration

Scale Model Index Ozone Impacts Reference

GPP Global MOSES

TRIFFID CUOta Decreased by 14

- 23% over the period 1901

- 2100 Sitch et al. ( 2007 )

NPP Global TEM AOT40 Decreased by 0.8% without agricultural management and a decrease of 2.9% with optimal agricultural management Felzer et al. ( 2005 )

U.S. TEM AOT40 Reduced by 2.3% without optimal N fertilization and 7.2% with optimal N fertilization from 1983

- 1993 Felzer et al. ( 2005 )

U.S. TEM AOT40 Reduced by 2.6

– 6.8% during the late 1980s

- early 1990s. Felzer et al. ( 2004 ) N ortheastern U.S. PnET AOT40 A reduction of 3

- 16% from 1987

- 1992 Ollinger et al. ( 1997a ) U.S. Mid

Atlantic PnET AOT40 Decreased NPP of most forest types by 7

- 8% Pan et al. ( 2009 )

China DLEM AOT40 Reduced NPP of grassland in China by 8.5 Tg C from 1960s to 1990s Ren et al. ( 2007b )

C exchange Global TEM AOT40 Reduced net C exchange (1950

– 1995) by 0.1 Pg C/yr without agricultural management and 0.3 Pg C/yr with optimal agricultural management Felzer et al. ( 2005 )

Global MOSES

TRIFFID CUOt Decreased global mean land

– atmosphere C fluxes by 1.3 Pg C/yr and 1.7 Pg C/yr for the ‘ high ’ and

‘ low ’ plant O 3 sensitivity models, respectively Sitch et al. ( 2007 )

C sequestration Global MOSES

TRIFFID CUOt Reduced land

- C storage accumulation by between 143 Pg C/yr and 263 Pg C/yr from 1900

– 2100 Sitch et al. ( 2007 )

U.S. TEM AOT40 Reduced C sequestration by 18

– 38 Tg C/yr from 1950 to 1995 Felzer et al. ( 2004 )

GSM National Park DLEM AOT40 Decreased the ecosystem C storage of deciduous forests by 2.5% and pine forest by 1.4% from 1971 to 2001 Zhang et al. ( 2007a )

China DLEM AOT40 Reduced total C storage by 0.06% in 1960s and 1.6% in 1990s in China ’ s terrestrial ecosystems Ren et al. ( 2007a )

China DLEM AOT40 O3 exposure reduced the net C sink of China ’ s terrestrial ecosystem by 7% from 1961 to 2005 Tian et al. ( 2011 )

China DLEM AOT40 Ozone induced net carbon exchange reduction ranged from 0.4

- 43.1% , depending on different forest type Ren et al. ( 2011 )

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.

d Pg equals 1 × 10 15 grams.



### 1

9.4.4 Crop yield and quality in agricultural systems

The detrimental effect of O 3 on crop production has been recognized since the 1960s and 

### 2 a large body of research has stemmed from that recognition. Previous O 3 AQCDs have 

### 3 extensively reviewed this body of literature. Table 9

- 4 summarizes recent experimental 

### 4 studies of O 3 effects on agricultural crops, exclusive of growth and yield. Growth and 

### 5 yield results are summarized in Table 9

- 1 7

. 

### 6Draft

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- 59 September 2011 The actual concentration and duration threshold for O 3 damage varies from species to 

### 1 species and sometimes even among genotypes of the same species ( Guidi et al., 2009 ; 

### 2 Sawada and Ko hno, 2009 ; Biswas et al., 2008 ; Ariyaphanphitak et al., 2005 ; Dalstein and 

### 3 Vas, 2005 ; Keutgen et al., 2005 ) . A number of comprehensive reviews and meta

- analyses 

### 4 have recently been published discussing both the current understanding of the 

### 5 quantitative effects of O 3 concentration o n a variety of crop species and the potential 

### 6 focus areas for biotechnological improvement to a future growing environment that will 

### 7 include higher O 3 concentrations ( Bender and Weigel, 2011 ; Booker et al ., 2009 ; 

### 8
Van Dingenen et al., 2009 ; Ainsworth, 2008 ; Feng et al., 2008b ; Hayes et al., 2007 ; Mills 

### 9 et al., 2007b ; Grantz et al., 2006 ; Morgan et al., 200 3 ) . Since the 2006 O 3 AQCD ( U.S. 

### 10 EPA, 2006b ) , exposure

- response indices for a variety of crops have been suggested 

### 11 ( Mills et al., 2007b ) and many reports have investigated the effects of O 3 concentration 

### 12 on seed or fruit quality to extend the knowledge base beyond yield quantity. This section 

### 13 will outline the key findings from these papers as well as highlight some of the recent 

### 14

r esearch addressing the endpoints such as yields and crop quality. 

### 15
This section will also highlight recent literature that focuses on O 3 damage to crops as 

### 16 influenced by other environmental factors. Genetic variability is not the only factor that 

### 17 determine s crop response to O 3 damage. Ozone concentration throughout a growing



### 18 season is not homogeneous and other environmental conditions such as elevated CO 2 

### 19 concentrations, drought, cold or nutrient availability may alleviate or exacerbate the 

### 20 oxidative stress response to a given O 3 concentration. 

### 21

9.4.4.1 Yield

It is well known that yield is negatively impacted in many crop species in response to 

### 22 high O 3 concentration. However, the concentrations at which damage is observed vary 

### 23 from species to species. Numerous analy ses of experiments conducted in OTCs and with 

### 24 naturally occurring gradients demonstrate that the effects of O 3 exposure also vary 

### 25 depending on the growth stage of the plant; plants grown for seed or grain are often most 

### 26 sensitive to exposure during the see d or grain

- filling period ( Soja et al., 2000 ; Pleijel et 

### 27 al., 1998 ; Younglove et al., 1994 ; Lee et al., 1988a ) . AX9.5.4.1 of the 2006 O 3 AQCD 

### 28 summarized many previous studies on crop yield. 

### 29

Field studies and meta

- analyses

The effect of O 3 exposure on U.S. crops remains an important area of research and 

### 30 several studies have been published on this topic since the 2006 O 3 AQCD ( U.S. EPA, 

### 31 2006b ) (Table 9

- 4 and 9

- 1 7 ). For example, one study with cotton in a crop

- weed 

### 32Draft

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- 60 September 2011 interaction study ( Grantz and Shres tha, 2006 ) utilizing OTCs suggests that 12

- hour 

### 1 average O 3 concentrations of 79.9 ppb decreased cotton biomass by 25% and 12

- hour 

### 2 average O 3 concentration of 122.7 ppb decreased cotton biomass by 75% compared to 

### 3 charcoal filtered control (12

- h avg: 12.8 ppb). Further, this study suggests that the weed, 

### 4 yellow nutsedge, was less sensitive to increasing O 3 concentration, which would increase 

### 5 weed competition ( Grantz and Shrestha, 2006 ) . In a study of peanuts in Nort h Carolina, 

### 6 near ambient and elevated exposures of O 3 reduced photosynthesis and yield compared to 

### 7 very low O 3 conditions ( Booker et al., 2007 ; Burkey et al., 2007 ) . In another study, 

### 8 Grantz a nd Vu ( 2009 ) reported that sugarcane biomass growth significantly declined 

### 9 under O 3 exposure. 

### 10
The average yield loss reported across a number of meta

- analytic studie s have been 

### 11 published recently for soybean ( Morgan et al., 2003 ) , wheat ( Feng et al., 2008b ) , rice 

### 12 ( Ainsworth, 2008 ) , semi

- natural vegetation ( Hayes et al., 2007 ) , potato, bean and barley 

### 13 ( Feng and Kobayashi, 2009 ) . Meta

- a nalysis allows for the objective development of a 

### 14 quantitative consensus of the effects of a treatment across a wide body of literature. 

### 15 Further, this technique allows for a compilation of data across a range of O 3 fumigation 

### 16 techniques, durations and concentrations in order to assemble the existing literature in a 

### 17 meaningful manner. 

### 18
Morgan et al. ( 2003 ) reported an average seed yield loss for soybean of 24% compared to 

### 19 charcoal filtered air across all O 3 concentrations used in the 53 compiled studies. The 

### 20 decrease in seed yield appeared to be the product of nearly equal decreases (7

- 12%) in 

### 21 seed weight, seed number and pod number. As would be expected, the lowest O 3 

### 22 concentration (30

- 59 ppb) resulted in the smallest yield losses, appro ximately 8%, while 

### 23 the highest O 3 concentration (80

- 120 ppb ) resulted in the largest yield losses, 

### 24 approximately 35% ( Morgan et al., 2003 ) . Further, the oil/protein ratio within the 

### 25 soybean seed was altered due to growth at elevated O 3 concentrations, with a decrease in 

### 26 oil c ontent. The studies included in this meta

- analysis all used enclosed fumigation 

### 27 systems or growth chambers which may have altered the coupling of the atmosphere to 

### 28 the lower plant canopy ( McLeod and Long, 1999 ) , although the results of Morgan et al. 

### 29 ( 2006 )
, Betzelberger et al. ( 2010 ) , and the comparisons presented in Section 9.6.3 

### 30 strongly suggest that decreases in yield between ambient and elevated exposures are not 

### 31 affected by exposure method. Utilizing the Soybean Free Air gas Concentration 

### 32 Enrichment Facility (SoyFACE; www.soyface.illinois.edu ), Morgan et al. ( 2006 ) report a 

### 33 20% seed yield loss due to a 23% increase in average dayti me O 3 concentration 

### 34 (56

- 69 ppb) within a single soybean cultivar across two growing seasons in Illinois, 

### 35 which lies within the range predicted by the meta

- analysis. A further breakdown of the 

### 36 effects of current O 3 concentrations (AOT40 of 4.7 ppm

- h) on bea n seed quality 

### 37 ( Phaseolus vulgaris ) has identified that growth at current O 3 concentrations compared to 

### 38Draft

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- 61 September 2011 charcoal

- filtered air raised total lipids, total crude protein and dietary fiber content ( Iriti et 

### 1 al., 2009 ) . An increase in total phenolics was also observed, however the individual 

### 2 phenolics compounds responded differently, with significant decreases in anthocyan in 

### 3 content. The seeds from ambient O 3 exposed plants also displayed increased total 

### 4 antioxidant capacity compared to charcoal

- filtered air controls ( Iriti et al., 2009 )

. 

### 5 Betzelberger et al. ( 2010 ) has recently utilized the SoyFACE facility to compare the 

### 6 impact of elevated O 3 concentrations across 10 soybea n cultivars to investigate 

### 7 intraspecific variability of the O 3 response to find physiological or biochemical markers 

### 8 for eventual O 3 tolerance breeding efforts ( Betzelberger et al., 2010 )

. They report an 

### 9 average 17% decrease in yield across all 10 cultivars across two growing seasons due to a 

### 10 doubling of ambient O 3 concentrations, with the individual cultivar responses ranging 

### 11 from

- 7% to

- 36%. The exposure

- response functions derived for the se 10 current 

### 12 cultivars were similar to the response functions derived from the NCLA N studies 

### 13 conducted in the 1980 s ( Heagle, 1989 ) suggesting there has not been any selection for 

### 14 increased tolerance to O 3 in more recent cultivars. More complete comparisons between 

### 15 yield predictions based on data from cultivars used in NCLAN studies, and yield data for 

### 16 modern cultivars from SoyFACE are reported in Section 9.6.3 of this document. They 

### 17 confirm that the resp onse of soybean yield to O 3 exposure has not changed in current 

### 18 cultivars. 

### 19

A meta

- analysis has also been performed on studies investigating the effects of O 3 

### 20 concentrations on wheat ( Feng et al., 2008b ) . Across 23 studies included, elevated O 3 

### 21 concentrations (ranging from a 7

- h daily average of 31

- 200 ppb) decreased grain yield by 

### 22 29%. Winter wheat and spring wheat did not differ in their responses; however the 

### 23 response in both varieties to increasing O 3 concentrations resulted in successively larger 

### 24 decreases in yield, from a 20% decrease in 42 ppb to 60% in 153 ppb O 3 . These yield 

### 25 losses were mainly caused by a combination of decreases in individual grain weight (

- 

### 26 18%), ear number per plant (

- 16%), and grain number per ear (

- 11%). Further, the grain 

### 27 starch concentration decreased by 8% and the grain protein yield decreased by 18% due 

### 28 to growth at elevated O 3 concentrations as well. However, increases in grain calcium and 

### 29 potassium levels were reported ( Feng et al., 2008b )

. 

### 30

A recent meta

- analysis found that growth at elevated O 3 concentrations negatively 

### 31 impacts nearly every a spect of rice performance as well ( Ainsworth, 2008 ) . While rice is 

### 32 not a major crop in the U.S., it provides a staple food for over h alf of the global 

### 33 population ( IRRI, 2002 ) and the effects of rising O 3 concentrations on rice yields merits 

### 34 consideration. On average, rice yields decreased 14% in 62 ppb O 3 compared to charcoal



### 35 filtered air. This yield loss was largely driven by a 20% decrease in grain number 

### 36 ( Ainsworth, 2008 )

. 

### 37Draft

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- 62 September 2011 Feng and Kobayashi ( 2009 ) have recently compiled yield data for six major crop species, 

### 1 potato, barley, wheat, rice, bean and soybean and grouped the O 3 treatments used in those 

### 2 studies into t hree categories: baseline O 3 concentrations (<26 ppb), current ambient 7

- or 

### 3
12

- h daily O 3 concentrations (31

- 50 ppb), and future ambient 7

- or 12

- h daily O 3 

### 4 concentrations (51

- 75 ppb). Using these categories, they have effectively characterized 

### 5 the effect s of current O 3 concentrations and the effects of future O 3 concentrations 

### 6 compared to baseline O 3 concentrations. At current O 3 concentrations, which ranged 

### 7 from 41

- 49 ppb in the studies included, soybean (

- 7.7%), bean (

- 19.0%), barley (

- 8.9%), 

### 8 wheat (

- 9. 7%), rice (

- 17.5%) and potato (

- 5.3%) all had yield losses compared to the 

### 9 baseline O 3 concentrations (<26 ppb). At future O 3 concentrations, averaging 63 ppb, 

### 10 soybean (

- 21.6%), bean (

- 41.4%), barley (

- 14%), wheat (

- 28%), rice (

- 17.5%) and potato 

### 11 (

- 11.9%) all had significantly larger yield losses compared to the losses at current O 3 

### 12 concentrations (<26 ppb) ( Feng and Kobayashi, 2009 )

. 

### 13
A review of OTC studies has determined the AOT40 critical level that causes a 5% yield 

### 14 reduction across a variety of agricultural and horticultural species ( Mills et al., 2007b )

. 

### 15 The authors classify the species studied into three groups: sensitive, mod erate and 

### 16 tolerant. The sensitive crops, including watermelon, beans, cotton, wheat, turnip, onion, 

### 17 soybean, lettuce, and tomato, respond with a 5% reduction in yield under a 3

- month 

### 18 AOT40 of 6 ppm

- h. Watermelon was the most sensitive with a critical level of 

### 19 1.6 ppm

- h. The moderately sensitive crops, including sugar beet, oilseed rape, potato, 

### 20 tobacco, rice, maize, grape and broccoli, responded with a 5% reduction in yield between 

### 21 8.6 and 20 ppm

- h. The crops classified as tolerant, including strawberry, pl um and barley, 

### 22 responded with a 5% yield reduction between 62

- 83.3 ppm

- h ( Mills et al., 2007b )

. 

### 23
Feng and Kobayashi ( 2009 ) com pared their exposure

- response results to those published 

### 24 by Mills et al. ( 2007b ) and found the ranges of yield loss to be similar for soybean, rice 

### 25 and bean. However, Feng and Kobayasi ( 2009 ) reported smaller yield losses for potato 

### 26 and wheat and larger yield losses for barley compared to the dose

- response functions 

### 27 published by Mills et al. ( 2007b ) , which they attributed to their more lenient criteria for 

### 28 literature inclusion. 

### 29
While the studies investigating the impact of various O 3 concentrations on yield are 

### 30 important and aid in determining the vulnerability of various crops to a variety of O 3 

### 31 concentrations, there is still uncer tainty as to how these crops respond under field 

### 32 conditions with interacting environmental factors such as temperature, soil moisture, CO 2 

### 33 concentration, and soil fertility ( Booker et al., 2009 ) . Further, there appears to be a 

### 34 distinct developmental and genotype dependent influence on plant sensitivity to O 3 that 

### 35 has yet to be fully investigated across O 3 concentrations in a field setting. The potentially 

### 36 mitigating effect of breeding selection for O 3 resistance has received very little attention 

### 37Draft

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- 63 September 2011 in the published scientific literature. Anecdotal reports suggest that such selection may 

### 1 have occurred in recent decades for some crops in areas of the country with high ambient 

### 2
exposures. However, the only published literature available is on soybean and these 

### 3 studies indicate that sensitivity has not changed in cultivars of soybean between the 

### 4 1980s and the 2000s ( Betzelberger et al., 2010 ) . This conclusion for soybeans is 

### 5 confirmed by comparisons presented in Section 9.6.3 of this document. 

### 6

Yield loss at regional and global scales

Because O 3 is heterogeneous in both time and space and O 3 monitoring stations are 

### 7 predominant ly near urban areas, the impacts of O 3 on current crop yields at large spatial 

### 8 scales are difficult to estimate. Fishman et al. ( 2010 ) have used satellite observations to 

### 9 estimate O 3 concentrations in the contiguous tri

- state area of Iowa, Illinois and Indiana 

### 10 and have combined that information with other measured environmental variables to 

### 11 model the historical impact of O 3 concentrations on soybean yield across the 2002

- 2006 

### 12 growin g seasons. When soybean yield across Iowa, Indiana and Illinois was modeled as a 

### 13 function of seasonal temperature, soil moisture and O 3 concentrations, O 3 had the largest 

### 14 contribution to the variability in yield for the southern

- most latitudes included in the 

### 15 dataset. Fishman et al. ( 2010 ) determined that O 3 concentrations significantly reduced 

### 16 soybean yield by 0.38 to 1.63% for every additional ppb of exposure across the 5 years. 

### 17
T his value is consistent with previous chamber studies ( Heagle, 1989 ) and results from 

### 18 SoyFACE ( Morgan et al., 2006 ) . Satellite estimates of tropospheric O 3 concentrations 

### 19 exist globally ( Fishman et al., 2008 )
, therefore utilizing this historical modeling approach 

### 20 is feasible across a wider geographical area, longer time

- span and perhaps for more crop 

### 21 species. 

### 22

The detrimental effects of O 3 on crop production at regional or global scales were also 

### 23 assessed by seve ral model studies. Two large scale field studies were conducted in the 

### 24 U.S. (NCLAN) and in Europe (European Open Top Chamber Programme, EOTCP) to 

### 25 assess the impact of O 3 on crop production. Ozone exposure

- response regression models 

### 26 derived from the two pro grams have been widely used to estimate crop yield loss 

### 27 ( Avnery et al., 2011a
, b ; Van Dingenen et al., 2009 ; Tong and Mauzerall, 2008 ; Wang 

### 28 and Mauzerall, 2004 ) . Those studies found that O 3 generally reduced crop yield and that 

### 29 different crops showed different sensitivity to O 3 pollution (Table 9

- 5 ). Ozone was 

### 30 calculated to induce a possible 45

- 82 million metric tons loss for wheat globally. 

### 31 Production losses for rice, maize and soybean were on the order of 17

- 23 million metric 

### 32 tons globally ( Van Dingenen et al., 2009 ) . The largest yield losses occur in high



### 33 production areas exposed to high O 3 concentrations, such the Midwest and the 

### 34 Mississippi Valley regions in the U.S., Europe, China and India ( Van Dingenen et al., 

### 35 2009 ; Tong et al., 2007 )

. 

### 36Draft

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- 64 September 2011 

### 9.4.4.2 Crop Quality

In general, it a ppears that increasing O 3 concentrations above current ambient 

### 1 concentrations can cause species

- dependent biomass losses, decreases in root biomass 

### 2 and nutritive quality, accelerated senescence and shifts in biodiversity. A study conducted 

### 3 with highbush bl ackberry has demonstrated decreased nutritive quality with increasing 

### 4 O 3 concentration despite no change in biomass between charcoal

- filtered control, 

### 5 ambient O 3 and 2 × ambient O 3 exposures ( Ditchkoff et al., 2009 ) . A study conducted 

### 6 with sedge using control (30 ppb), low (55 ppb), medium (80 ppb) and high (105 ppb) O 3 

### 7 treatments has demonstrated decreased root biomass and accelerated senescence in the 

### 8 medium and high O 3 treatments ( Jones et al., 2010 ) . Alfalfa showed no biomass changes 

### 9

across two years of double ambient O 3 concentrations (AOT40 of 13. 9 ppm

- h) using 

### 10 FACE fumigation ( Maggio et al., 2009 ) . However a modeling study has demonstrated 

### 11 that 84% of the variability in the relative feed value in high

- yielding alfalfa was due to 

### 12 the variability in mean O 3 concentration from 1998

- 2002 ( Lin et al., 2007 ) . Further, in a 

### 13 managed grassland FACE system, the reduction in total biomass harvest over five years 

### 14 decreased twice as fast in the elevated treatment (AOT40 of 13

- 59 ppm

- h) compare d to 

### 15 ambient (AOT40 of 1

- 20.7 ppm

- h). Compared with the ambient control, loss in annual 

### 16 dry matter yield was 23% after 5 year. Further, functional groups were differentially 

### 17 affected, with legumes showing the strongest negative response ( Volk et al., 2006 )

. 

### 18 However, a later study by Stampfli and Fuhrer ( 2010 ) at the same site suggested that 

### 19 Volk et al. ( 2006 ) was likely overestimated the eff ects of O 3 on yield reduction because 

### 20 the overlapping effects of species dynamics caused by heterogeneous initial conditions 

### 21 and a change in management were not considered in Volk et al. ( 2006 ) . An OTC study 

### 22 conducted with Trifolium subterraneum exposed to filtered ( <15 ppb), ambient, and 

### 23
40 ppb above ambient O 3 demonstrates decreases in biomass in the highest O 3 treatment 

### 24 as well as 10

- 20% decreased nutritive quality which was mainly attributed to accelerated 

### 25

senescence ( Sanz et al., 2005 ) . A study conducted with Eastern gamagrass and big 

### 26 bluestem in OTCs suggested that big bluestem is not sensitive to O 3 , but gamagrass 

### 27 displayed decreased nut ritive quality in the 2 × ambient O 3 treatment, due to higher 

### 28 lignin content and decreased N, ( Lewis et al., 2006 )

. 

### 29

9.4.4.3 Summary

The detrimental effect of O 3 on crop production has been recognized since the 1960 ’ s 

### 30 and a large body of research has subsequently stemmed from those initial findings. 

### 31 Previous O 3 AQCDs have extensively reviewed this body of literature ( U.S. EP A, 

### 32 2006b ) . Current O 3 concentrations across the U.S. are high enough to cause yield loss for 

### 33 a variety of agricultural crops including, but not limited to, soybean, wheat, potato, 

### 34Draft

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- 65 September 2011 watermelon, beans, turnip, onion, lettuce, and tomato. Continued increase s in O 3 

### 1 concentration may further decrease yield in these sensitive crops. Despite the well



### 2 documented yield losses due to increasing O 3 concentration, there is still a knowledge 

### 3 gap pertaining to the exact mechanisms of O 3 - induced yield loss. Research has linked 

### 4 increasing O 3 concentration to decreased photosynthetic rates and accelerated 

### 5 senescence, which are related to yield. 

### 6
New research is beginning to consider the mechanism of damage caused by prolonged, 

### 7 lower O 3 concentration (so

- called chronic expos ure) compared to short, very high O 3 

### 8 concentration (so

- called acute exposure). Both types of O 3 exposure cause damage to 

### 9 agricultural crops, but through very different mechanisms. Historically, most research on 

### 10 the mechanism of O 3 damage used acute exposure studies. During the last decade, it has 

### 11 become clear that the cellular and biochemical processes involved in the response to 

### 12 acute O 3 exposure are not involved in response to chronic O 3 exposure, even though both 

### 13 cause yield los s in agriculturally important crops. 

### 14
In addition, new research has highlighted the effects of O 3 on crop quality. Increasing O 3 

### 15 concentration decreases nutritive quality of grasses, decreases macro

- and micro

- nutrient 

### 16 concentrations in fruits and vegetable crops, and decreases cotton fiber quality. These 

### 17 areas of research require further investigation to determine mechanisms and exposure



### 18 response relationships. 

### 19
During the previous NAAQS reviews, there were very few studies that estimated O 3 

### 20 impacts on crop yields at large spatial scales. Recent modeling studies found that O 3 

### 21 generally reduced crop yield, but the impacts varied across regions and crop species. For 

### 22 example, the largest O 3 - induced crop yield losses occurred in high

- production areas 

### 23 exposed to h igh O 3 concentrations, such the Midwest and the Mississippi Valley regions 

### 24 of the U.S. ( Van Dingenen et al., 2009 ) . Among crop species, the estimated yield loss for 

### 25 wheat and soybean were higher than for rice and maize ( Van Dingenen et al., 2009 )

. 

### 26 Using satellite air

- column observations with direct air

- sampling O 3 data, Fishman et al. 

### 27 ( 2010 ) modeled the yield

- loss due to O 3 over the continuous tri

- state area of Illinois, 

### 28 Iowa and Wisconsin. They determined that O 3 concentrations significantly reduced 

### 29 soybean yield, which further reinforces previous results from FACE

- type experiments 

### 30 and OTC experiments. 

### 31

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

### 32 exposure and reduced yield and quality of agricultural crops. 

### 33Draft

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- 66 September 2011 

### Table 9

- 4 Summary 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

Alfalfa ( Medicago sativa cv. Beaver) Growth chambers 1, 2 or 4 days 3, 5 or

- h/day 85 ppb (Exposure duration) Relative feed value n.s. *high variability among treatment groups (N/A) Muntifering et al. ( 2006 )

Bean ( Phaseolus vulgari s I. cv Borlotto) OTC, ground

- planted Curno, Italy 4 months 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 )

Big Blue Stem ( Andropogon gerardii ) OTC Alabama, U.S. 4 months 12

- h avg: CF = 14 ppb; Ambient = 29 ppb; Elevated = 71 ppb (N/A) Relative feed value n.s. (n.s.) Lewis et al. ( 2006 )

Brassica napus Growth chambers Belgium 4 days CF & 176 ppb for 4 h/day (N/A) Glucosinolates −41 (N/A) Gielen et al. ( 2006 )

Brassica napus cv. Westar 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. ( 2009b )

Eastern Gamagrass ( Tripsacu m dactyloides ) OTC Alabama, U.S. 4 months 12

- h avg: CF = 14 ppb; Ambient = 29 ppb; Elevated = 71 ppb (N/A) Relative feed value −17 (−12) Lewis et al. ( 2006 )

Lettuce ( Lactuca sativa ) OTC Carcaixent Experimental Station, Spain 30 days 12

- h mean: CF = 10.2 ppb; NF = 30.1 ppb; NF+O 3 = 62.7 ppb (4 cultivars) Lipid peroxidation; Root length +77 (+38) −22 (−14) Calatayud et. al. ( 2002 )

Peanut ( Arachis hypogaea ) OTC Raleigh, NC; U.S. 3 yr 12

- h avg: CF = 22 ppb; Ambient = 46 ppb; Elevated = 75 ppb (CO 2 : 375 ppm; 548 ppm; 730 ppm) Harvest biomass −40 (−10) Booker et al. ( 2007 )

Poa pratensis OTC Braunschweig, Germany 3 yr; 4

- 5 wk in the spring 8

- h avg: CF+25 = 21.7 ppb; NF+50 = 73.1 ppb (Competition) Relative feed value N/A (n.s.; −8) Bender et al. ( 2 006 )

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 )

Potato (Solanum tuberosum cv. Indira) 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. ( 2007 )

Soybean OTC Italy 3 yr AOT40: CF = 0 ppm

- h; Ambient = 3.4 ppm

- h; Elevated = 9.0 ppm
- h (Well

- watered & water

- stressed) Daily evapotranspiration −28 (−14) Jaude et al. ( 2008 )Draft

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- 67 September 2011 Species Facility Location Exposure Duration Ozone Exposure a (Additional treatment) Variable(s) measured percent change from CF b (percent change from ambient) Reference

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 )

Soybean ( Glycine max cv. 93B15) SoyFACE Urbana, IL; U.S. 4 months 8

- h avg: Ambient = 38.5 ppb; Elevated = 52 ppb (Herbivory) Herbivory defense

- related genes N/A (N/A) Casteel et al. ( 2008 )

Soybean ( Glycine max cv. Essex) OTC, ground

- planted 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 residue N/A (−15.46) Booker et al. ( 2005 )

Soybean ( Glycine max cv. Essex) OTCs, 21 L pots Raleigh, NC; U.S. 2×3 months 12

- h avg: CF = 18 ppb); Elevated = 72 ppb) (CO 2 : 367 & 718) Water

- use efficiency n.s. (N/A) Booker et al. ( Booker et al., 2004a )

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. ( 2010 )

Spring Wheat ( Triticum aestivum cv. Minaret; Satu; Drabant; Dragon) OTCs Belgium, Finland, & Sweden 7 yr Seasonal AOT40s ranged from 0 to16 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. ( 2008a )

Strawberry ( Fragaria x ananassa Duch. Cv. Korona & Elsanta) Growth chambers Bonn, Germany 2 months 8

- h avg: CF = 0 ppb; Elevated = 78 ppb (N/A) Total leaf area −16 (N/A) Keutgen et al. ( 2005 )

Sweet Potato Growth Chambers Bonn, Germany 4 wk 8

- h avg: CF = 0 ppb; Ambient < 40 ppb; Elevated = 255 ppb (N/A) Tuber weight −14 (−11.5) Keutgen et al. ( 2008 )

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 harv est) Brix degree −7.2 (−3.6) Calvo, et al. ( 2005 )

Trifolium repens & Trifolium pretense Aspen FACE Rhinelander, WI; U.S. 3 months 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 )

a Ozone exposure in ppb unless otherwise noted.
b CF = Carbon

- filtered air. NF = Non

- filtered air.Draft

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- 68 September 2011 

### Table 9

- 5 Modeled effects of ozone on crop yield loss at regional and global scales

Scale Index Ozone Impacts Reference
Global M7a; M12b; AOT40 Reduced by 7.3% to 12.3% for wheat, 5.4% to 15.6% for soybean, 2.8% to 3.7% for rice, and 2.4% to 4.1% for maize in year 2000. Van Dingenen et al. ( 2009 ) Global M12b; AOT40 O 3 - induced global yield reductions ranged from 8.5

- 14% for soybean, 3.9

- 15% for wheat, and 2.2

- 5.5% for maize in year 2000. Global crop production losses totaled 79

- 121 million metric tons, worth $11

- 18 billion annually (USD2000). Avnery et al. ( 2011a )

U.S. M7; M12; AOT40 Reduced by 4.1% to 4.4% for wheat, 7.1% to 17.7% for soybean, 2.6% to 3.2% for rice, and 2.2% to 3.6% for maize in year 20 00. Van Dingenen et al. ( 2009 ) U.S. SUM06 Caused a loss of 53.8 million to 438 million bushels in soybean production, which account for 1.7

– 14.2% of total U.S. soybean production in 2005 Tong et al. ( 2007 )

East Asia M7; M12 Reduced the yield of wheat, rice and corn by 1

– 9% and soybean by 23

– 27% in China, Japan and South Korea in 1990 Wang and Mauzerall ( 2004 )

a M7 is defined as 7

- h mean O 3 concentration (ppb).

b M12 is defined as 12

- h mean O 3 concentration (ppb).



### 9.4.5 Water Cycling

Ozone can affect water use in plants and ecosystems through several mechanisms 

### 1 including damage to stomatal functioning and loss of leaf area. Section 9.3.2 reviewed 

### 2 possible mechanisms for effects of O 3 exposure on stomatal functioning including build



### 3 up of CO 2 in substomatal cavity, impacts on signal transduction pathways, and direct O 3 

### 4 impact on guard cells. Regardless of the mechanism, O 3 exposure has been shown to alter 

### 5 stomatal performance, which ma y affect plant and stand transpiration and therefore could 

### 6 affect hydrological cycling (Figure 9

- 7). 

### 7Draft

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- 69 September 2011 

### Figure 9

- 7 The potential effects of ozo ne exposure on watering cycling.

In the literature, there is not a clear consensus on the nature of leaf

- level st omatal 

### 1 conductance response to O 3 exposure. At the leaf level, O 3 exposure is known to result in 

### 2 stomatal patchiness ( Paoletti and Grulke, 2005 ; Omasa et al., 1987 ; Ellenson and 

### 3 Amundson, 1982 ) , i.e., the heterogeneous aperture of stomata on the leaf surface, and, as 

### 4 a result, the collective response of groups of stomata on leav es and canopies determines 

### 5 larger

- scale responses to O 3 . When measured at steady

- state high light conditions, leaf



### 6 level stomatal conductance is often found to be reduced when exposed to O 3 . For 

### 7 example, a meta

- analysis of 55 studies found that O 3 reduced stomatal conductance by 

### 8 11% ( Wittig et al., 2007 )

. However, these steady

- state measurements were generally 

### 9 taken at saturating light conditions and steady

- state vapor pressure deficit (VPD). 

### 10 Saturating light and steady

- state VPD conditions are not common in the field since many 

### 11 parts of the plant canopy are shaded throughout the day. When studied under varying 

### 12 environmental conditions, many studies have reported incomplete stomatal closure with 

### 13 elevated O 3 exposure duri ng the day ( Mills et al., 2009 ; Grulke et al., 2007b ; Matyssek et 

### 14 al., 1995 ; Wieser and Havranek, 1995 ) or at night ( Grulke et al., 2004 )

. This may be due 

### 15 to sluggish stomatal response. Sluggish stomatal response, defined as a delay in stomatal 

### 16 response to changing environmental factors relative to controls ( Paoletti and Grulke, 

### 17 2010 ) ha s also been documented by several researchers ( Grulke et al., 2007c ; Maty ssek et 

### 18 O 3 exposure

Altered canopy water loss

Soil moisturestem sap flow Decrease stomatal conductance or sluggish stomatal response

Altered
run off

Stream flowsDraft

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- 70 September 2011 al., 1995 ; Pearson and Mansfield, 1993 ; Wallin and Skärby, 1992 ; Lee et al., 1990 ; 

### 1 Skarby et al., 1987 ; Keller and Häsler, 1984 ; Reich and Lassoie, 1984 )

. Sluggish stomatal 

### 2 response associated with O 3 exposure suggests an uncoupling of the normally tight 

### 3 relationship between carbon assimilation and stomatal conductance as measured under 

### 4 steady

- state conditions ( Gregg et al., 2006 ; Paoletti and Grulke, 2005 )

. Several tree and 

### 5 ecosystem models, such as TREGRO, PnET and DLEM, rely on this tight relationship to 

### 6 simulate water and carbon dynamics. The O 3 - induced impairment of stomatal control 

### 7 may be more pronounced for plants growing under water stress ( Wilkinson and Davies, 

### 8 2010 ; Grulke et al., 2007a ; Paoletti and Grulke, 2005 ; Bonn et al., 2004 ; Kellomaki and 

### 9 Wang, 1997 ; Tjoelker et al., 1995 ; Reich and Lassoie, 1984 )

. Since leaf

- level stomatal 

### 10 regulation is usually assesse d in a steady state rather than as a dynamic response to 

### 11 changing environmental conditions, steady state measurements cannot detect sluggish 

### 12 stomatal response. Because of sluggish stomatal responses, water loss from plants may be 

### 13 greater under dynamic environmental conditions over days and months. 

### 14
In addition to the impacts on stomatal performance, O 3 - induced physiological changes, 

### 15 such as reduced leaf area index and accelerated leaf senescence could alter water use 

### 16 efficiency. It is well established f rom chamber and field studies that O 3 exposure is 

### 17 correlated with lower foliar retention ( Karnosky et al., 2003 ; Topa et al., 2001 ; Pell et al., 

### 18 1999 ; Grulke and Lee, 1997 ; Karnosky et al., 1996 ; Miller et al., 1972 ; Miller et al., 

### 19 1963 )

. However, Lee et al. ( 2009a ) did not find changes in needle area of ponderosa pine 

### 20 and reported that greater canopy conductance followed by water stress under elevated O 3 

### 21 may h ave been caused by stomatal dysfunction. At the Aspen FACE experiment, stand



### 22 level water use, as indicated by sap flux per unit ground area, was not significantly 

### 23 affected by elevated O 3 despite a 22% decrease in leaf area index and 20% decrease in 

### 24 basal a rea ( Uddling et al., 2008 ) . The lack of negative effect of elevated O 3 on stand 

### 25 water use may be due to the substantially increased whole plant hydraulic conductance 

### 26 per unit leaf area under elevated O 3 , as indicated by the sap flux per unit total leaf area 

### 27 (kl) ( Uddling et al., 2009 ) . The increased kl may be caused by the sluggish of stomatal 

### 28 response. In pure aspen stands, the stomatal closure response to increasing vapor pressure 

### 29 deficit was less sensitive and mid

- day leaf water potential was lower under elevated O 3 , 

### 30 suggesting O 3 impaired stomatal control over transpiration ( Uddling et al., 2009 ) . Other 

### 31 potential factors contributing to the unchanged stand

- level water use included the higher 

### 32 proportion of sun leaves, and similar or even increased fine root biomass under elevated 

### 33 O 3 ( Uddling et al., 2008 ) . Elevated O 3 could also affect evapotranspiration by altering 

### 34 tree crown interception of precipitation. Ozone has been shown to change branch 

### 35

arc hitectural parameters, and the effects were species

- dependent at the Aspen FACE 

### 36 experiment ( Rhea et al., 2010 ) . The authors found that there was a significant correlation 

### 37 between canopy architecture parameters and stem flow for birch but not aspen. 

### 38Draft

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- 71 September 2011 It is difficult to scale up physiology measurements from leaves to ecosystems. Thus, the 

### 1 current understanding of how stomatal response at leaf scale is integrated at the scale of 

### 2 whole forest canopies, and therefore how it influences tree and forest stand water use is 

### 3 limited. Field studies by McLaughlin et al. ( 2007a ; 2007b ) provided valuable insight into 

### 4 the possible consequences of stomatal sluggishness for ecosystem water cycling. 

### 5 McLaughlin et al. ( 2007a ; 2007b ) indicated that O 3 increased water use in a mixed 

### 6 deciduous forest in eastern Tennessee. McLaughlin et al. ( 2007a ; 2007b ) found that O 3 , 

### 7 with daily maximum levels ranging from 69.2 to 82.9 ppb, reduced stem growth by 30

- 

### 8 50% in the high

- O 3 yea r 2002. The decrease in growth rate was caused in part by 

### 9 amplification of diurnal cycles of water loss and recovery. Peak hourly O 3 exposure 

### 10 increased the rate of water loss through transpiration as indicated by the increased stem 

### 11 sap flow. The authors su ggested that a potential mechanism for the increased sap flow 

### 12 could be altered stomatal regulation from O 3 exposure, but this was inferred through sap 

### 13 flow measurements and was not directly measured. The increased canopy water loss 

### 14 resulted in higher water uptake by the trees as reflected in the reduced soil moisture in the 

### 15 rooting zone. The change in tree water use led to further impacts on the hydrological 

### 16 cycle at the landscape level. Increased water use under high O 3 exposure was reported to 

### 17 reduce late

- season modeled streamflow in three forested watersheds in eastern Tennessee 

### 18 ( Mc Laughlin et al., 2007b )

. 

### 19

Felzer et al. ( 2009 ) used TEM

- Hydro to assess the interactions of O 3 , climate, elevated 

### 20 CO 2 and N limitation on the hydrological cycle in the eastern U.S. They found that 

### 21 elevated CO 2 decreased evapotranspiration by 2

- 4% and increased runoff by 3

- 7%, as 

### 22 compared to th e effects of climate alone. When O 3 damage and N limitation were 

### 23 included, evapotranspiration was reduced by an additional 4

- 7% and runoff was increased 

### 24 by an additional 6

- 11% ( Felzer et al., 2009 ) . Based upon simulation with INTRAST and 

### 25 LINKAGES, Hanson et al. ( 2005 ) found that increasing O 3 concentration by 20 ppb 

### 26 above the current ambient level yields a modest 3% reduction in water use. Those 

### 27 ecological models were generally built on the assumption that O 3 induces stomatal 

### 28 closure and have not inco rporated possible stomatal sluggishness due to O 3 exposure. 

### 29 Because of this assumption, results of those models normally found that O 3 reduced 

### 30

water use. 

### 31

9.4.5.1 Summary

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

### 32 showe d an association between O 3 exposure and alteration of water use and cycling in 

### 33 vegetation and at the ecosystem level. There is not a clear consensus on the nature of 

### 34 leaf

- level stomatal conductance response to O 3 exposure. When measured under steady

- 

### 35Draft

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- 72 September 2011 state high light conditions, leaf

- level stomatal conductance is often found to be reduced 

### 1 when plants are exposed to O 3 . However, measurements of stomatal conductance under 

### 2 dynamic light and VPD conditions indicate sluggish responses under elevated O 3 

### 3
exposure, which could potentially lead to increased water loss from vegetation. Field 

### 4 studies conducted by McLaughlin et al. ( 2007a ; 2007b ) suggested that peak hourly O 3 

### 5 exposure increased the rate of water loss from several tree species, and led to a reduction 

### 6 in the late

- season modeled stream flow in three forested watersheds in eastern Tennessee. 

### 7 Sluggish stomatal responses during O 3 exposure was suggested as a possible mechanism 

### 8 for increased water loss during peak O 3 exposure. Currently, the O 3 - induced reduction in 

### 9 stomatal aperture is the biological assumption for most process

- based models. Because of 

### 10 this assumption, results of those models normally found t hat O 3 reduced water loss. For 

### 11 example, Felzer ( 2009 ) found that O 3 damage and N limitation together reduced 

### 12 evapotranspiration and increased runoff. 

### 13
Although the direction of the response differed among studies, the evidence is 

### 14 sufficient to conclude that there is likely to be a causal relations hip between O 3 

### 15 exposure and the alteration of ecosystem water cycling. 

### 16

9.4.6 Below

- Ground Processes

Above

- ground and below

- ground processes are tightly interconnected. Because roots and 

### 17 soil organisms are not exposed directly to O 3 , below

- ground processes are af fected by O 3 

### 18 through alterations in the quality and quantity of C supply from photosynthates and 

### 19 litterfall ( Andersen, 2003 ) . Ozone can decrease leaf C uptake by redu cing photosynthesis 

### 20 (Section 9.3). Ozone can also increase metabolic costs by stimulating the production of 

### 21 chemical compounds for defense and repair processes, and by increasing the synthesis of 

### 22 antioxidants to neutralize free radicals (see Section 9.3), both of which increase the 

### 23 consumption of carbon for above

- ground processes. Therefore, O 3 could significantly 

### 24 reduce the amount of C available for allocation to below

- ground by decreasing C uptake 

### 25 while increasing C consumption of above

- ground processes ( Andersen, 2003 )

. 

### 26

Since the 2006 O 3 AQCD, there is additional evidence for O 3 effects on below

- ground 

### 27 processes. Ozone has been found to alter root growth, soil food web structure, 

### 28 decomposer activities, C turnover, water cycling and nutrient flow (Figure 9

- 8). Ozone 

### 29 effects on root development and root biomass production and soil food web structure are 

### 30 reviewed in sections 9.4.3.1 and 9.4.9.2, respectively. The focus in this section is on the 

### 31 response of litter input, decomposer activities, soil respiration, soil C formation and 

### 32 nutrient cycling. 

### 33Draft

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- 73 September 2011 Source: Modified from Andersen ( 2003 ) Arrows denote C flux pathways that are affected by ozone. Dashed lines indicate where the impact of ozone is suspected but unknown.


### Figure 9

- 8 Conceptual diagram showing where ozone alters C, water and nutrient flo w in a tree

- soil system, including transfer between biotic and abiotic components below ground that influence soil physical and chemical properties.

9.4.6.1 Litter Carbon Chemistry, Litter Nutrient and Their Ecosystem Budgets

Consistent with previous findings, rec ent studies show that, although the responses are 

### 1 often species

- dependent, O 3 tends to alter litter chemistry ( U.S. EPA, 2006b ) .Alterations 

### 2 CO 2 , H 2 O

Litter production and chemistry Allocation of C

CO 2 release

Roots/symbionts CO 2 , H 2 O

Altered stomatal function

Nutrients
Soil foodweb

• Bacteria

• Fungi

• Micro & marco invertebrates

Soil physical & chemical properties Organic matter Root, leaf litter exudation Soil moisture
Run off Altered species competition retentionDraft

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- 74 September 2011 in chemical parameters, such as changes in C chemistry and nutrient concentrations, were 

### 1 observed in both leaf and root litter (9

- 7). 

### 2
At the Aspen FACE site, several studies investigated litter chemistry changes ( Parsons et 

### 3 al., 2008 ; Johnson and Pregitzer, 2007 ; Chap man et al., 2005 ; Liu et al., 2005 ) . In both 

### 4 aspen and birch leaf litter, elevated O 3 increased the concentrations o f soluble sugars, 

### 5 soluble phenolics and condensed tannins ( Parsons et al., 2008 ; Liu et al., 2005 )

. 

### 6 Compared to other treatments, aspen litter under elevated O 3 had the highest fiber 

### 7 concentration, with the lowest concentra tion associated with the birch litter under the 

### 8 same conditions ( Parsons et al., 2008 ) . Chapman et al. ( 2005 ) measured chemical 

### 9 changes in fine root litter and found that elevated O 3 decreased lignin concentration. O 3 - 

### 10 induced chemistry changes were also reported from other experimental sites. Results 

### 11 from an OTC study in Finland sugg ested that elevated O 3 increased the concentration of 

### 12 acid

- soluble lignin, but had no significant impact on other chemicals such as total sugars, 

### 13 hemicelluloses, cellulose or total lignin in the litter of silver birch ( Kasurinen et al., 

### 14 2006 ) . Results from the free air canopy O 3 exposure experiment at Kranzberg Forest 

### 15 showed that O 3 increased starch co ncentrations but had no impact on cellulose and lignin 

### 16 in beech and spruce leaf litter ( Aneja et al., 2007 ) . The effect of O 3 on three antioxidants 

### 17 (ascorbate, glutathione and α

- tocopherol) in fine roots of beech was also assessed at 

### 18 Kranzberg Forest. The results indicated that O 3 had no significant effect on α

- tocopherol 

### 19 and ascorbate concentrations, but decreased glutathione concentrations in fine roots 

### 20 ( Haberer et al., 2008 ) . In addition to changing C chemistry, O 3 also altered nutrient 

### 21 concentrations in green leaves and litter (Table 9

- 6 ). 

### 22

The combined effects of O 3 on biomass productivity and chemistry changes may alter 

### 23 C chemicals and nutrient contents at the canopy or ecosystem level. For example, 

### 24 although O 3 had different impacts on their concentrations, annual fluxes of C chemicals 

### 25 (soluble sugar, soluble phenol ics, condensed tannins, lipid and hemicelluloses), macro 

### 26 nutrients (N, P, K and S) and micro nutrients (Mg, B, Cu and Zn) to soil were all reduced 

### 27 due to lower litter biomass productivity at Aspen FACE ( Liu et al., 2007a ; Liu et al., 

### 28 2005 ) . At the Kranzberg Forest, N content of spruce canopy in a mixed culture and Ca 2+ 

### 29 content of beech canopy in a monoculture increased due to elevated O 3 increased leaf 

### 30 concentrations of those nutrients although leaf production was not significantly altered by 

### 31 O 3 ( Rodenkirchen et al., 2009 )

. 

### 32Draft

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- 75 September 2011 

### Table 9

- 6 The effect of elevated ozone on leaf/litter nutrient concentrations

Study Site Species Ozone Concentration Response Reference
Suonenjoki Research Station, Finland Silver birch Ambient: 10

- 60 ppb Elevated: 2×ambient Decreased the concentration of P, Mn, Zn and B in leaf litter Kasurinen et al. ( 2006 ) Aspen FACE Aspen and birch Ambient: 50

- 60 ppb Elevated: 1.5×ambient Decreased the concentrations of P, S, Ca and Zn, but had no impact on the concentrations of N, K, Mg, Mn, B and Cu in leaf litter. Liu et al. ( 2007a )

Aspen FACE Birch Ambient: 50

- 60 ppb Elevated: 1.5×ambient Incr ease N concentration in birch litter Parsons et al. ( 2008 )

Kranzberg Forest, Germany Beech and spruce Ambient: 9

- 41 ppb Elevated: 2×ambient Increased N concentration in beach leaf, but not in spruce needle Kozovits et al. ( 2005 )

Kranzberg Forest, Germany Beech an d spruce Ambient: 9

- 41 ppb Elevated: 2×ambient 1) Had no significant effects on spruce needle chemistry; 2) increased Ca concentration in beech leaves in monoculture, but had no impacts on other nutrients Rodenkirchen et al. ( 2009 )

Salerno, Italy Holm oak Non

- filtered OTC: 29 ppb Filtered OTC: 17ppb Ozone had no significant impacts on litter C, N, lignin and cellulose concentrations Baldantoni et al. ( 2011 ) Kuopio University Research Garden, Finland Red Clover Ambient: 25.7 ppb Elevated: 1.5×ambient increased the total phenolic content of leaves and had minor effects on the concentrations of individual phenolic compounds Saviranta et al. ( 2010 )



### 9.4.6.2 Decomposer Metabolism and Litter Decomposition

The above

- and below

- ground physiological changes caused by O 3 exposure cascade 

### 1 through the ecosystem and affect soil food webs. In the 2006 O 3 AQCD, there were very 

### 2 few studies on the effect of O 3 on the structure and function of soil food webs, except 

### 3 two studies conducted by Larson et al. ( 2002 ) and Phillips et al. ( 2002 ) . Since the last O 3 

### 4 AQCD, new studies have provided more information on how O 3 affects the metabolism 

### 5 of soil microbes and soil fauna. 

### 6
Chung et al. ( 2006 ) found that the activity of the cellulose

- degrading enzyme 1,4

- β



### 7 glucosidase w as reduced by 25% under elevated O 3 at Aspen FACE. The decrease in 

### 8 cellulose

- degrading enzymatic activity was associated with the lower cellulose 

### 9 availability under elevated O 3 ( Chung et al., 2006 ) . However, a later study at the same 

### 10 site, which was conducted in the 10th year of the experiment, found that O 3 had no 

### 11 impact on cellulolytic activity in soil ( Edwards and Zak, 2011 ) . In a lysimeter study of 

### 12 beech trees ( Fagus sylvatica ) in Germany, soil enzyme activity was found to be 

### 13 suppressed by O 3 exposure ( Es perschutz et al., 2009 ; Pritsch et al., 2009 ) . Except for 

### 14 xylosidase, enzyme activities involved in plant cell wall degradation (cellobiohydrolase, 

### 15 beta

- glucosidase and glucuronidase) were decreased in rhizosphere soil samples un der 

### 16 elevated O 3 (2 × ambient level) ( Pritsch et al., 2009 ) . Similarly, Chen et al. ( 2009 ) found 

### 17Draft

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- 76 September 2011 O 3 exposure, with a 3

- month AOT40 of 21.4

- 44.1 ppm

- h, decreased the microbial 

### 1 metabolic capability in the rhizosphere and bulk soil of wheat, although the observed 

### 2 reduction in bulk soil was not significant. 

### 3

Ozone

- induced cha nge in soil organisms ’ activities could affect litter decomposition 

### 4

rates. Results of recent studies indicated that O 3 slightly reduced or have no impacts on 

### 5 litter decomposition ( Liu et al., 2009b ; Parsons et al., 2008 ; Kasurinen et al., 2006 ) 

### 6 ( Baldantoni et al., 2011 ) . The responses varied among species, sites a nd exposure length. 

### 7
Parsons et al. ( 2008 ) collected litter from aspen and birch seedlings at Aspen FACE site, 

### 8 and conducted a 23

- month field litter incubation starting in 1999. They found that 

### 9 elevated O 3 had different impacts on the decomposition of aspen and birch litter. 

### 10 Elevated O 3 was fou nd to reduce aspen litter decomposition. However, O 3 accelerated 

### 11 birch litter decomposition under ambient CO 2 , but reduced it under elevated CO 2 

### 12 ( Parsons et al., 2008 ) . Liu et al. ( 2009b ) conducted another litter decomposition study at 

### 13 Aspen FACE from 2003 to 2006, when stand leaf area index (LAI) reached its maximum. 

### 14 During the 935

- day field incubation, elevated O 3 was shown to reduce litter mass loss in 

### 15 the first year, but not in the second year. They suggested that higher initial tannin and 

### 16 phenolic concentrations under elevated O 3 reduced microbial activity in the first year 

### 17 ( Liu et al., 2009b ) . In an OTC experiment, Kasurinen et al. ( 2006 ) collected silver birch 

### 18 leaf litter from three consecutive growing seasons and conducted three separate litter

- bag 

### 19 incubation experiments. Litter decomposition was not affected by O 3 exposure in the first 

### 20 two incubations, but a slower decomposition rate was found in the third incubation. Their 

### 21 principle component analysis indicated that the litter chemistry changes caused by O 3 

### 22 (decreased Mn, P, B and increased C :N) might be partially responsible for the decreased 

### 23 mass loss of their third incubation. In another OTC experiment, Baldatoni et al. ( 2011 ) 

### 24 found that O 3 significantly reduced leaf litter decomposition of Quercus ilex L, although 

### 25 litter C, N, lignin and cellulose concentrations were not altered by O 3 exposure. 

### 26

9.4.6.3 Soil re spiration and carbon formation

Ozone could reduce the availability of photosynthates for export to roots, and increase 

### 27 root mortality and turnover rates. Ozone has also been shown to reduce above

- ground 

### 28 litter productivity and alter litter chemistry, whic h would affect the quality and quantity 

### 29 of the C supply to soil organisms (Section 9.4.6.1). The complex interactions among 

### 30 those changes make it difficult to predict the response of soil C cycling under elevated 

### 31 O 3 . The 2006 O 3 AQCD concluded that O 3 had no consistent impact on soil respiration 

### 32 ( U.S. EPA, 2006b ) . Ozone could increase or decrease soil respiration, depending on the 

### 33 approach and timing of the measurements. Ozone may also alter soil C formation. 

### 34 However, very few experiments directly measured changes in soil organic matter content 

### 35Draft

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- 77 September 2011 under O 3 fumigation ( U.S. EPA, 2006b ) . Recent studies on soil respiration and soil 

### 1 C content also found mixed responses. Most importantly, recent results from long

- term 

### 2 fumigation experiments, such as the Aspen FACE experiment, suggest that ecosystem 

### 3
respo nse to O 3 exposure can change over time. Observations made during the late 

### 4 exposure years can be inconsistent with those during the early years, highlighting the 

### 5 need for caution when assessing O 3 effects based on short

- term studies (Table 9

- 7 ). 

### 6

Table 9

- 7 The temporal variation of ecosystem responses to ozone exposure at Aspen FACE site

Endpoint Period of Measurement Response Reference

Litter decomposition 1999

- 2001 O 3 reduced 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 )

2003

- 2006 O 3 reduced litter mass loss in the first year, but not in the second year. Liu et al. ( 2009b )

Fine root production 1999 O 3 had no significant impact on fine root biomass King et al. ( 2001 ) 2002, 2005 O 3 increased fine root biomass Pregitzer et al. ( 2008 ) Soil respiration 1998

- 1999 Soil respiration under +CO 2 +O 3 treatment was lower than that under +CO 2 treatment King et al. ( 2001 )

2003

- 2007 Soil respiration under +CO 2 +O 3 treatment was 5

- 25% higher than under elevated CO 2 treatment. Pregitzer et al. ( 2006 ) ( 2008 )

Soil C formation 1998

- 2001 O 3 reduced the formation rates of total soil C by 51% and acid

insoluble soil C by 48% Loya et al. ( 2003 )

2004

- 2008 No significant effect of O 3 on the new C formed under elevated CO 2 Talhelm et al. ( 2009 )

Soil Respiration
Ozone has shown inconsistent impacts on soil respiration. A sun

- lit controlled



### 7 environment chamber study found that O 3 had no significant effects on soil respiration, 

### 8 fine root biomass or any of the soil organisms in a reconstructed ponderosa pine/soil

- litter 

### 9 system ( Tingey et al., 2006 ) . In an adult European beec h/Norway spruce forest at 

### 10 Kranzberg Forest, the free air O 3 fumigation (AOT40 of 10.2

- 117 ppm

- h) increased soil 

### 11 respiration under both beech and spruce during a humid year ( Nikolova et al., 2010 ) . The 

### 12 increased soil respiration under beech has been accompanied by the increase in fine root 

### 13 biomass and ectomycorrhizal fungi diversity and turnover ( Grebenc and Kraigher, 2007 )

. 

### 14
Th e stimulating effect on soil respiration disappeared under spruce in a dry year, which 

### 15 was associated with a decrease in fine root production in spruce under drought. This 

### 16 finding suggested that drought was a more dominant stress than O 3 for spruce ( Nikolova 

### 17 et al., 2010 ) . Andersen et al. ( 2010 ) labeled the canopies of European beech and Norway 

### 18Draft

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- 78 September 2011 spruce with CO 2 depleted in 13 C at the same site. They did not observe any significant 

### 1 changes in soil respiration for either species. 

### 2
The nearly 10 year long studies at Aspen FACE indica ted that the response of soil 

### 3 respiration to O 3 interacted with CO 2 exposure and varied temporally (Table 9

- 7 ) 

### 4 ( Pregitzer et al., 2008 ; Pregitzer et al., 2006 ; King et al., 2001 ) . Ozone treatment alone 

### 5 generally had the lowest mean soil respiration rates, although those differences between 

### 6 control and elevated O 3 were usually not significant. However, soil respiration rates were 

### 7 different with O 3 alone and when acting in combination with elevated CO 2 . In the first 

### 8 five years (1998

- 2002), soil respiration under +CO 2 +O 3 treatment was similar to that 

### 9 under control and lower than that under +CO 2 treatment ( Pregitzer et al., 2006 ; King et 

### 10 al., 2001 ) . Since 2003, +CO 2 +O 3 treatment started to show the greatest impact on soil 

### 11 respiration. Compared to elevated CO 2 , soil respiration rate under +CO 2 +O 3 treatment 

### 12 was 15

- 25% higher from 2003

- 2004, and 5

- 10% higher from 2005

- 2007 ( Pregitzer et al., 

### 13 2008 ; Pregitzer et al., 2006 ) . Soil respiration was highly correlated with the biomass of 

### 14 roots with diameters of <2 mm and <1 mm, across plant community and atmospheric 

### 15 treatments. The authors suggested that the increase in soil respiration rate may be due to 

### 16 +CO 2 +O 3 increased fine root (<1.0 mm) biomass production ( Pregitzer et al., 2008 )

. 

### 17

C hanges in leaf chemistry and productivity due to O 3 exposure have been shown to affect 

### 18 herbivore growth and abundance (See Section 9.4.9.1). Canopy insects could affect soil 

### 19 carbon and nutrient cycling through frass deposition, or altering chemistry and qu antity 

### 20 of litter input to the forest floor. A study at the Aspen FACE found that although elevated 

### 21 O 3 affected the chemistry of frass and greenfall, these changes had small impact on 

### 22 microbial respiration and no effect on nitrogen leaching ( Hillstrom et al., 2010a )

. 

### 23 However, respiratory carbon loss and nitrate immobilization were nearly double in 

### 24 microcosms receiving herbivore inputs than those receiving no herbivore inputs 

### 25 ( Hillstrom et al., 2010a )

. 

### 26

Soil Ca rbon Formation

Ozone

- induced reductions in plant growth can result in reduced C input to soil and 

### 27 therefore soil C content ( Andersen, 2003 ) . The simulations of most e cosystem models 

### 28 support this prediction ( Ren et al., 2007a ; Zhang et al., 2007a ; Felzer et al., 2004 )

. 

### 29 However, very few studies have directly measured soil C dynamics under elevated O 3 . 

### 30 After the first four years of fumigation (from 1998 t o 2001) at the Aspen FACE site, 

### 31 Loya et al. ( 2003 ) found that forest stands exposed to both elevated O 3 and CO 2 

### 32 accumulated 51% less total soil C, and 48% less acid

- insoluble soil C compared to stands 

### 33 exposed only to elevated CO 2 . Soil organic carbon (SOC) was continuously monitored at 

### 34 the Aspen FACE site, and the later data showed that the initial reduction in new 

### 35Draft

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- 79 September 2011 C formation (soil C derived from plant litter since the start of the experiment) by O 3 

### 1 under elevated CO 2 is only a temporary effect (Table 9

- 7 ) ( Talhelm et al., 2009 ) . The 

### 2 amount of new soil C in the elevated CO 2 and the combined elevated CO 2 and O 3 

### 3 treatments has converged since 2002. There was no significant effect of O 3 on the new C 

### 4 formed under elevated CO 2 ov er the last four years of the study (2004

- 2008). Talhelm 

### 5

et al. ( 2009 ) suggested the observed reduction in the early years of the experiment might 

### 6 be driven by a suppression of C allocated to fine root biomass. During the early exposure 

### 7 years, O 3 had no significant impact on fine root production ( King et al., 2001 ) . However, 

### 8 the effect of O 3 on fine root biomass was observed later in the experiment. Ozone 

### 9 increased fine root production and the highest fine root biomass was observed under t he 

### 10 combined elevated CO 2 and O 3 treatment in the late exposure years (Table 9

- 7 ) 

### 11 ( Pregitzer et al., 2006 ) . This increase in fine root production was due to changes in 

### 12

commun ity composition, such as better survival of O 3 - tolerant aspen genotype, birch and 

### 13 maple, rather than changes in C allocation at the individual tree level ( Pregitzer et al., 

### 14 2008 ; Zak et al., 2007 )

. 

### 15

9.4.6.4 Nutrient cycling

Ozone can affect nutrient cycling by changing nutrient release from litter, nutrient uptake 

### 16 by plants, and soil microbial activity. Nitrogen is the limiting nutrient for most temperate 

### 17 ecosystems, and several st udies examined N dynamics under elevated O 3 . Nutrient 

### 18 mineralization from decomposing organic matter is important for sustaining ecosystem 

### 19 production. Holmes et al. ( 2006 ) found that elevated O 3 decreased gross N mineralization 

### 20 at the Aspen FACE site, indicating that O 3 may reduce N availability. Other N cycling 

### 21 processes, such as NH 4 + immobilization, gross nitrification, microbial biomass N and soil 

### 22 organic N, were not affected by elevated O 3 ( Holmes et al., 2006 ) . Similarly, Kanerva 

### 23

et al. ( 2006 ) found total N, NO 3 - , microbial biomass N, potential nitrification and 

### 24 denitrification in t heir meadow mesocosms were not affected by elevated O 3 (40

- 50 ppb). 

### 25 Ozone was found to decreased soil mineral N content at SoyFACE, which was likely 

### 26 caused by a reduction in plant material input and increased denitrification ( Pujol Pereira 

### 27 et al., 2011 )

. Ozone also sh owed small impact on other micro and macro nutrients. Liu 

### 28

et al. ( 2007a ) assessed N, P, K, S, Ca, Mg, Mn, B, Zn and Cu release dynamics at Aspen 

### 29 FACE, and they found that O 3 had no effects on most nutrients, except to decrease N and 

### 30 Ca release from litter. These studies reviewed above suggested that s oil N cycling 

### 31 processes were not affected or slightly reduced by O 3 exposure. However, in a lysimet er 

### 32 study with young beech trees Stoelken et al. ( 2010 ) found that elevated O 3 stimulated N 

### 33 release from litter which was largely attributed to an enhanced mobilization of inert 

### 34 nitrogen fraction. 

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- 80 September 2011 Using the SImple NItrogen Cycle mode l (SINIC), Hong et al. ( 2006 ) evaluated the 

### 1 impacts of O 3 exposure on soil N dynamics and streamflow nitrate flux. The detrimental 

### 2 effect of O 3 on plant growth was found to reduce plant uptake of N and therefore increase 

### 3 nitrate leaching. Their model simulation indicated that ambient O 3 exposure increased the 

### 4 mean annual stream flow nitrate export by 12% (0.042 g N/m 2 /year) at the Hubbard 

### 5 Brook E xperimental Watershed from 1964

- 1994 ( Hong et al., 2006 )

. 

### 6

9.4.6.5 Dissolved Organic Carbon and Biogenic Trace Gases Emission

The O 3 - induced changes in plant gr owth, C and N fluxes to soil and microbial 

### 7 metabolism can alter other biogeochemical cycling processes, such as soil dissolved 

### 8 organic carbon (DOC) turnover and trace gases emission. 

### 9

Jones et al. ( 2009 ) collected fen cores from two peatlands in North Wales, UK and 

### 10 exposed them to one of four levels of O 3 (AOT40 of 0, 3.69, 5.87 and 13.80 ppm

- h for 

### 11
41 days). They found the concentratio n of porewater DOC in fen cores was significantly 

### 12 decreased by increased O 3 exposure. A reduction of the low molecular weight fraction of 

### 13 DOC was concurrent with the observed decrease in DOC concentration. Their results 

### 14 suggested that O 3 damage to overlyin g vegetation may decrease utilizable C flux to soil. 

### 15 Microbes, therefore, have to use labile C in the soil to maintain their metabolism, which, 

### 16 the authors hypothesized, leads to a decreased DOC concentration with a shift of the 

### 17 DOC composition to more aro matic, higher molecular weight organic compounds. 

### 18

Several studies since the 2006 O 3 AQCD have examined the impacts of O 3 on nitrous 

### 19 oxide (N 2 O) and methane (CH 4 ) emission. Kanerva et al. ( 2007 ) measured the fluxes of 

### 20
N 2 O and CH 4 in meadow mesocosms, which were exposed to elevated CO 2 and O 3 in 

### 21 OTCs in south

- western Finland. They found that the daily N 2 O fluxes were decreased in 

### 22 the NF+O 3 (non

- filtered air + elevated O 3 , 40

- 50 ppb) after three seasons of exposure. 

### 23 Elevated O 3 alone or combined with CO 2 did not have any significant effect on the daily 

### 24 fluxes o f CH 4 ( Kanerva et al., 2007 ) . In another study conducted in central Finland, the 

### 25
4 year open air O 3 fumigation (AOT40 of 20.8

- 35.5 ppm

- h for growing season) slightly 

### 26 increased potential CH 4 oxidation by 15% in the peatland microcosms, but did not affect 

### 27 the rate of potential CH 4 production or net CH 4 e missions, which is the net result of the 

### 28 potential CH 4 production and oxidation ( Morsky et al., 2008 ) . However, several studies 

### 29 found that O 3 could significantly reduce CH 4 emiss ion. Toet et al. ( 2011 ) exposed 

### 30 peatland mesocosms to O 3 in OTCs for two years, and found that CH 4 emissions were 

### 31 significa ntly reduced by about 25% during midsummer periods of both years. In an OTC 

### 32 study of rice paddy, Zheng et al. ( 2011 ) found that the daily mean CH 4 emissions were 

### 33Draft

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- 81 September 2011 signif icantly lower under elevated O 3 treatments than those in charcoal

- filtered air and 

### 1 nonfiltered air treatments. They found that the seasonal mean CH 4 emissions were 

### 2 negatively related with AOT40, but positively related to the relative rice yield, 

### 3 abovegroun d biomass and underground biomass. 

### 4

9.4.6.6 Summary

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

### 5 often site specific, O 3 altered the quality and quantity of litter input to soil, microbial 

### 6 community composition, and C and nutrient cycling. Biogeochemical cycling of below



### 7 ground processes is driven by C input from plants. Studies at the leaf and plant level have 

### 8 provided bio logically plausible mechanisms, such as reduced photosynthetic rates, 

### 9 increased metabolic cost, and reduced root C allocation for the association of O 3 

### 10 exposure and the alteration of below

- ground processes. 

### 11
Results from Aspen FACE and other experimental s tudies consistently found that O 3 

### 12 reduced litter production and altered C chemistry, such as soluble sugars, soluble 

### 13 phenolics, condensed tannins, lignin, and macro/micro nutrient concentration in litter 

### 14 ( Parsons et al., 2008 ; Kasurinen et al., 2006 ; Liu et al., 2005 ) . The changes in substrate 

### 15 quality and quantity could alter microbial metabolism under elevated O 3 , and therefore 

### 16 soil C and nutrient cycling. Sev eral studies indicated that O 3 suppressed soil enzyme 

### 17 activities ( Pritsch et al., 2009 ; Chung et al., 2006 ) . However, the impact of O 3 on litter 

### 18 decomposition was inconsistent and varied among species, sites and e xposure length. 

### 19 Similarly, O 3 had inconsistent impacts on dynamics of micro and macro nutrients. 

### 20
Studies from the Aspen FACE experiment suggested that the response of below

- ground 

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

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

- 2003), O 3 had 

### 23 no impact on soil respiration but reduced the formation rates of total soil C under 

### 24 elevated CO 2 . However, after 10

- 11 yr of exposure, O 3 was found to i ncrease soil 

### 25 respiration but have no significant impact on soil C formation under elevated CO 2 . 

### 26

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

### 27 exposure and the alteration of below

- ground biogeochemical cycles. 

### 28

9.4.7 Community composition

The effects of O 3 on species competition (AX9.3.3.4) and community composition 

### 29 (AX9.6.4) were summarized in the 2006 O 3 AQCD. Plant species differ in their 

### 30Draft

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- 82 September 2011 sensitivity to O 3 . Fast growing plants with high stomatal conductance and high specific 

### 1 leaf area (SLA) were more likely to be sensitive to O 3 exposure. Further, different 

### 2 genotypes of a given species also vary in their sensitivity. This differential sensitivity 

### 3 could change the competitive interactions that lead to loss in O 3 sensitive spec ies or 

### 4 genotypes. In addition, O 3 exposure has been found to alter reproductive processes in 

### 5 plants (See Section 9.4.3.3). Changes in reproductive success could lead to changes in 

### 6 species composition. However, since ecosystem

- level responses result from th e 

### 7 interaction of organisms with one another and with their physical environment, it takes 

### 8 longer for a change to develop to a level of prominence at which it can be identified and 

### 9 measured. A shift in community composition in forest and grassland ecosystem s noted in 

### 10 the 2006 O 3 AQCD has continued to be observed from experimental and gradient studies. 

### 11 Additionally, research since the last review has shown that O 3 can alter community 

### 12 composition and diversity of soil microbial communities. 

### 13

9.4.7.1 Forest

In the San Bernardino Mountains in southern California, O 3 pollution caused a 

### 14 significant decline in ponderosa pine ( Pinus ponderosa ) and Jeffrey pine ( Pinus jeffreyi ) 

### 15 ( U.S. EPA, 2006b ) . Pine trees in the young mature age class group exhibited hig her 

### 16 mortality rates compared with mature trees at a site with severe O 3 visible foliar injury. 

### 17 The vulnerability of young mature pines was most likely caused by the fact that trees in 

### 18 this age class were emerging into the canopy, where higher O 3 concentrat ions were 

### 19 encountered ( McBride and Laven, 1999 ) . Because of the loss of O 3 - sensitive pines, 

### 20 mixed forests of ponderosa pine, Jeffery Pine and white fir ( Abies c oncolor ) shifted to 

### 21 predominantly white fir ( Miller, 1973 ) . Ozone may have indirectly caused the decline in 

### 22 understory diversity in coniferous forests in the San Bernardino Mountains through an 

### 23 increase in pine litterfall. This increase in litterfall from O 3 exposure results in an 

### 24 understory layer that may prohibit the establishment of native herbs, but not e xotic annual 

### 25 Galium aparine ( Allen et al., 2007 )

. 

### 26
Ozone damage to conifer forests has also been observed in several other regions. In the 

### 27 Valley of Mexico, a widespread mortality of sacred fir ( Abies religiosa ) was observed in 

### 28 the heavily polluted area of the Desierto de los Leones National Park in the early 1980s 

### 29 ( de Lourdes de Bauer and Hernandez

- Tejeda, 2007 ; Fenn et al., 2002 ) . Ozone damage 

### 30 was widely believed to be an important causal factor in the dramatic decline of sacred fir. 

### 31 In alpine regions of southern France and the Carpathians Mountains, O 3 wa s also 

### 32 considered as the major cause of the observed decline in cembran pine ( Pinus 

### 33 cembra ) ( Wieser et al., 2006 ) . However, many environmental factors such as light, 

### 34 temperature, nutrient and soil moisture, and climate extremes such as unusual dry and 

### 35Draft

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- 83 September 2011 wet periods could interact with O 3 and alter the response of forest to O 3 exposure. For 

### 1 those pollution gradient studies, several confounding factors, such as drought, insect 

### 2 outbreak and forest management, may also contribute to or even be the dominant factors 

### 3 causing the mortality of trees ( de Lourdes de Bauer and Hernandez

- Tejeda, 2007 ; Wieser 

### 4 et al., 2006 )

. 

### 5
New evidence from long

- term free O 3 fumigation experiments provided additional 

### 6 support for the potential impacts of O 3 on specie s competition and community 

### 7 composition changes in forest ecosystems. At the Aspen FACE site, community 

### 8 composition at both the genetic and species levels was altered after seven years of 

### 9 fumigation with O 3 ( Kubiske et al., 2007 ) . In the pure aspen community, O 3 fumigation 

### 10 reduced growth and increased mortality of sensitive clone 259, while the O 3 tolerant 

### 11 clone 8L em erged as the dominant clone. Growth of clone 8L was even greater under 

### 12 elevated O 3 compared to controls, probably due to O 3 alleviated competitive pressure on 

### 13 clone 8L by reducing growth of other clones. In the mixed aspen

- birch and aspen

- maple 

### 14 communities , O 3 reduced the competitive capacity of aspen compared to birch and maple 

### 15 ( Kubiske et al., 2007 ) . In a phytotron study, O 3 fumigation reduced growth of beech but 

### 16 not spruce in mixed culture, suggesting a higher susceptibility of beech to O 3 under 

### 17 interspecific competition ( Kozovits et al., 2005 )

. 

### 18

9.4.7.2 Grassland and Agricultural Land

The respons e of managed pasture, often cultivated as a mixture of grasses and clover, to 

### 19 O 3 pollution has been studied for many years. The tendency for O 3 - exposure to shift the 

### 20 biomass of grass

- legume mixtures in favor of grass species, reported in the previous O 3 

### 21 AQ CD has been generally confirmed by recent studies. In a mesocosm study, Trifolium 

### 22

repens and Lolium perenne mixtures were exposed to an episodic rural O 3 regime within 

### 23 solardomes for 12 weeks. T. repens showed significant changes in biomass but not L. 

### 24

pere nne , and the proportion of T. repens decreased in O 3 - exposed mixtures compared to 

### 25 the control ( Hayes et al., 2009 ) . The changes in community composition of grass

- legume



### 26 forb mixtures were also observed at the Le Mouret FACE experiment, Switzerland. 

### 27 During the 5

- year O 3 fumigation (AOT40 of 13.3

- 59.5 ppm

- h ), the dominance of 

### 28 legumes in fumigated plots declined more quickly than those in the control plots ( Volk et 

### 29 al., 2006 ) . However, Sta mpfli and Fuhrer ( 2010 ) re

- analyzed the species and soil data and 

### 30 suggested that Volk et al. ( 2006 ) overestimated the O 3 effect. Stampfli and Fuhrer ( 2010 ) 

### 31 found that the difference in the species dynamics between control and O 3 treatment was 

### 32 more caused by heterogeneous initial conditions than O 3 exposure

. Several studies also 

### 33 suggested the mature/species

- rich ecosystems were more resilient to O 3 exposure. At 

### 34 another FACE experiment, located at Alp Flix, Switzerland, O 3 fumigation (AOT40 of 

### 35Draft

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- 84 September 2011 15.2

- 64.9 ppm

- h ) showed no significant impact on communi ty composition of this 

### 1 species

- rich pasture ( Bassin et al., 2007b ) . Although most studies demonstrated an 

### 2 increase in grass:forb ration with O 3 exposure ( Hayes et al., 2009 ; U.S. EPA, 2006b ) , a 

### 3 study on a simulated upland grassland community O 3 reduce grass:forb ratio ( Felicity et 

### 4 al., 2010 ) , which may be due to grass species in this community, such as Anthoxanthum 

### 5 odoratum, was more sensitive to O 3 than other most studied grass species such as L. 

### 6
perenne ( Hayes et al., 2009 ) . Pfleeger et al. ( 2010 ) collected seed bank soil from an 

### 7 agricultural field and examin ed how the plant community responded over several 

### 8 generations to elevated O 3 exposures. Sixty plant species from 22 families emerged in the 

### 9 chambers over their four year study. Overall, they found that O 3 appeared to have small 

### 10 impacts on seed germination and only a minor effect on species richness of pioneer plant 

### 11 communities. 

### 12
Several review papers have discussed the physiological and ecological characteristics of 

### 13 O 3 - sensitive herbaceous plants. Hayes et al. ( 2007 ) assessed species traits associated with 

### 14 O 3 sensitivity by the changes in biomass caused by O 3 exposure. Plants of the therophyte 

### 15 (e.g., annual) li fe form were particularly sensitive to O 3 . Species with higher mature leaf 

### 16 N concentration tended to be more sensitive than those with lower leaf N concentration. 

### 17 Plants growing under high oxidative stress environments, such as high light or high 

### 18 saline, w ere more sensitive to O 3 . Using the same dataset from Hayes et al. ( 2007 ) , Mills 

### 19

et al. ( 2007a ) identified the O 3 sensitive communities. They found that the largest number 

### 20 of these O 3 sensitive communities were associated with grassland ecosystems. Among 

### 21 grassland ecosystems, alpine grassland, sub

- alpine grassland, woodland fringe, and dry 

### 22 grassland were identified as the most sensitive communities. 

### 23

9.4.7.3 Microbes

Several methods ha ve been used to study microbial composition changes associated with 

### 24 elevated O 3 . Phospholipid fatty acid (PLFA) analysis is widely used to determine 

### 25 whether O 3 elicits an overall effect on microbial community composition. However, 

### 26 since PLFA markers cover a broad range of different fungi, resolution of this method 

### 27 may be not fine enough to detect small changes in the composition of fungal 

### 28 communities. Methods, such as microscopic analyses and polymerase chain reaction

– 

### 29 denaturing gradient gel electrophoresis (PCR

– DGGE), have better resolution to 

### 30 specifically analyze the fungal community composition. The resolution differences 

### 31 among those methods needs to be considered when assessing the O 3 impact on microbial 

### 32 community composition. 

### 33Draft

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- 85 September 2011 Kanerva et al. ( 2008 ) found that elevated O 3 (40

- 50 ppb) decreased total, bacterial, 

### 1 actinobacterial and fungal PLFA biomass values as well as fungal:bacterial PLFA 

### 2 biomass ratio in their meadow mesocosms in south

- western Finland. The relative 

### 3 proportions of individual PLFAs between the control and elevated O 3 treatments were 

### 4 significantly different, suggesting that O 3 modified the structure of the microbial 

### 5 community. Morsky et al. ( 2008 ) exposed boreal peatland microcosm s to elevated O 3 , 

### 6 with growing season AOT40 of 20.8

- 35.3 ppm

- h, in an open

- air O 3 exposure field in 

### 7 Central Finland. They also found that microbial composition was altered after three 

### 8 growing seasons with O 3 fumigation, as measured by PLFA. Ozone tended to increase 

### 9 the presence of Gram

- positive bacteria and the biomass of fungi in the peatland 

### 10 microcosms. Ozone also resulted in higher microbial biomass, which co

- occurred with 

### 11 the increases in concentrations of organic acids and leaf density of sedges ( Morsky et al., 

### 12 2008 ) . In a lysimeter experiment in Germany, O 3 was found to alter the PLFA profiles in 

### 13 the upper 0

- 20 cm rhizosphere soil of European beech. Elevated O 3 reduced bacter ial 

### 14 abundance but had no detectable effect on fungal abundance ( Pritsch et al., 2009 ) . Using 

### 15 microscopic analyses, Kasurinen et al. ( 2005 ) found that elevated O 3 , with 5 or 6 months 

### 16 of AOT40 of 20.6

- 30.9 ppm

- h, decreased the proportions of black and liver

- brown 

### 17 mycorrhizas and increased that of light brown/orange mycorrhizas. In an herbaceous 

### 18 plant study, SSCP (single

- strand conformation polymorphism) profiles indicated that O 3 

### 19 stress (about 75 ppb) had a very small effect on the structural diversity of the bacterial 

### 20 community in rhizospheres ( Dohrmann and Tebbe, 2005 ) . At the Aspen FACE site, O 3 

### 21 had no significant effect on fungal relative abundance, as indicated by PLFA profile. 

### 22 However, elevated O 3 altered fungal community compositio n, according to the 

### 23 identification of 39 fungal taxonomic units from soil using polymerase chain reaction

– 

### 24 denaturing gradient gel electrophoresis (PCR

- DGGE) ( Chung et al., 2006 ) . In another 

### 25 study at Aspen FACE, phylogenetic analysis suggested that O 3 exposure altered 

### 26 agaricomycete community. The ectomycorrhizal communities developing under elevated 

### 27 O 3 had higher proportions of Cortinarius and Inocybe species, and lower proportions of 

### 28 Laccaria and Tomentella ( Edwards and Zak, 2011 )

. Ozone was found to change 

### 29 microbial community composition in an agricultural system. Chen et al. ( 2010b ) found 

### 30 elevated O 3 (100

- 150 ppb) had significant effects on soil microbial composition 

### 31 expressed as PLFA percentage in a rice paddy in China. 

### 32

9.4.7.4 Summary

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

### 33 composition was assessed. Ozone was found to cause a significant decline in ponderosa 

### 34 and Jeffrey pine in the San Bernardino Mountains in southern California. Ozone exposure 

### 35Draft

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- 86 September 2011 also tended to shift the grass

- legume mixtures in favor of grass species ( U.S. EPA, 

### 1 2006b ) . Since the 2006 O 3 AQCD, more evidence has shown that O 3 exposure changed 

### 2 the competitive interactions and could lead to loss of O 3 sensitive species or genotypes. 

### 3 Studies at plant level found that the severity of O 3 damage on growth, reproduction and 

### 4 foliar inju ry varied among species, which provided the biological plausibility for the 

### 5 alteration of community composition. Additionally, research since the last review has 

### 6 shown that O 3 can alter community composition and diversity of soil microbial 

### 7 communities. 

### 8

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

### 9 regions. Ozone damage was believed to be an important causal factor in the dramatic 

### 10 decline of sacred fir in the valley of Mexico ( de Lourdes de Bauer and Hernandez



### 11 Tejeda, 2007 ) , as well as cembran pine in southern France and Carpathian Mountains 

### 12 ( Wieser et al., 2006 ) . Results from the Aspen FACE site indicated that O 3 could alter 

### 13 community compositi on of broadleaf forests as well. At the Aspen FACE site, O 3 

### 14 reduced growth and increased mortality of a sensitive aspen clone, while the O 3 tolerant 

### 15 clone emerged as the dominant clone in the pure aspen community. In the mixed aspen



### 16 birch and aspen

- maple c ommunities, O 3 reduced the competitive capacity of aspen 

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

. 

### 18

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

- legume mixtures in favor of 

### 19 grass species, was reported in the 2006 O 3 AQCD and has been generally confirmed by 

### 20 recent studies. However, in a high elevation mature/species

- rich grass

- legume pasture, O 3 

### 21 fumigation showed no significant impact on community composition ( Bassin et al
., 

### 22 2007b )

. 

### 23
Ozone exposure not only altered community composition of plant species, but also 

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

### 25 observed in both natural and agricultural ecosystems, although no general patte rns could 

### 26 be identified ( Kanerva et al., 2008 ; Morsky et al., 2008 ; Kasurinen et al., 2005 )

. 

### 27

The evidence is sufficient to conclude that there is likely a causal relationship 

### 28 between O 3 exposure and the alteration of community composition. 

### 29

9.4.8 Factors that Modify Functional and Growth Response

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

### 30 temperature, water and nutrient availability, and other air pollutants, as well as elevated 

### 31 CO 2 , influence or alter plant response to O 3 . These modifying factors were 

### 32 comprehensively reviewed in AX9.3 of the 2006 O 3 AQCD and thus, this section serves 

### 33Draft

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- 87 September 2011 mainly as a brief summary of the previous findings. A limited number of new studies 

### 1 published since the 2006 O 3 AQCD add to our understanding of the role of these 

### 2 interactions in modifying O 3 - induced plant responses. Many of these modifying factors 

### 3 and interactions are integrated into discussions elsewhere in this chapter and the reader is 

### 4 directed to those sections. 

### 5

9.4.8.1 Genetic s

It is well known that species vary greatly in their responsiveness to O 3 . Even within a 

### 6 given species, individual genotypes or populations can also vary significantly with 

### 7 respect to O 3 sensitivity ( U.S. EPA, 2006b ) . Therefore, caution should be taken when 

### 8 considering a species ’ degree of sensitivity to O 3 . Plant response to O 3 is determined by 

### 9 genes that are directly related to oxidant stress and to an unknown number of genes that 

### 10 are not specifically related to oxidants, but instead c ontrol leaf and cell wall thickness, 

### 11 stomatal conductance, and the internal architecture of the air spaces. It is rarely the case 

### 12 that single genes are responsible for O 3 tolerance. Studies using molecular biological 

### 13 tools and transgenic plants have positi vely verified the role of various genes and gene 

### 14 products in O 3 tolerance and are continuing to increase the understanding of O 3 toxicity 

### 15 and differences in O 3 sensitivity. See Section 9.3.3.2 of this document for a discussion of 

### 16 recent studies related to gene expression changes in response to O 3 . 

### 17

9.4.8.2 Environmental Biological Factors

As stated in the 2006 O 3 AQCD, the biological factors within the plant ’ s environment 

### 18 that may influence its response to O 3 encompass insects and other animal pests, diseases, 

### 19 weeds, and other competing plant species. Ozone may influence the severity of a disease 

### 20 or infestation by a pest or weed, either by direct effects on the causal species, or 

### 21 indirectly by affecting the ho st, or both. In addition, the interaction between O 3 , a plant, 

### 22 and a pest, pathogen, or weed may influence the response of the target host species to O 3 

### 23 ( U.S. EPA, 2006b ) . Several recent studies on the effects of O 3 on insects via their 

### 24 interactions with plants are discussed in Section 9.4.9.1. In addition, O 3 has also been 

### 25 shown to alter soil fauna communities (Section 9.4.9.2). 

### 26
In contrast to detrimental biological interactions, there are mutually beneficial 

### 27 relationships or symbioses i nvolving higher plants and bacteria or fungi. These include 

### 28 (1) the nitrogen

- fixing species Rhizobium and Frankia that nodulate the roots of legumes 

### 29 and alder and (2) the mycorrhizae that infect the roots of many crop and tree species, all 

### 30Draft

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- 88 September 2011 of which may be affected by exposure of the host plants to O 3 . Some discussion of 

### 1 mycorrhizae can be found in Section 9.4.6. 

### 2
In addition to the interactions involving animal pests, O 3 also has indirect effects on 

### 3 higher herbivorous animals, e.g., livestock, due to O 3 - indu ced changes in feed quality. 

### 4 Recent studies on the effects of O 3 on nutritive quality of plants are discussed in Sections 

### 5 9.4.4.2. 

### 6

Intra

- and interspecific competition are also important factors in determining vegetation 

### 7 response to O 3 . Plant competition involves the ability of individual plants to acquire the 

### 8 environmental resources needed for growth and development: light, water, nutrients, and 

### 9 space. Intraspecific competition involves individuals of the same species, typically in 

### 10 monoculture crop situat ions, while interspecific competition refers to the interference 

### 11 exerted by individuals of different species on each other when they are in a mixed 

### 12 culture. This topic was previously reviewed in AX9.3.3.4 of the 2006 O 3 AQCD. Recent 

### 13 studies on competition and its implications for community composition are discussed in 

### 14 Section 9.4.7. 

### 15

9.4.8.3 Physical Factors

Physical or abiotic factors play a large role in modifying plant response to O 3 , and have 

### 16 been extensively discussed in previous O 3 AQCDs. This section summariz es those 

### 17 findings as well as recent studies published since the last review. 

### 18
Although some studies have indicated that O 3 impact significantly increases with 

### 19 increased ambient temperature ( Ball et al., 2000 ; Mills et al., 2000 ) , other studies have 

### 20 indicated that temperature has little effect ( Balls et al., 1996 ; Fredericksen et al., 1996 ) . A 

### 21 recent study by Riikonen et al. ( 2009 ) at the Ruohon iemi open air exposure field in 

### 22 Kuopio, Finland found that the effects of temperature and O 3 on total leaf area and 

### 23 photosynthesis of Betula pendula were counteractive. Elevated O 3 reduced the saplings ’ 

### 24 ability to utilize the warmer growth environment by i ncreasing the stomatal limitation for 

### 25 photosynthesis and by reducing the redox state of ascorbate in the apoplast in the 

### 26 combination treatment as compared to temperature alone ( Riikonen et al., 2009 )

. 

### 27
Temperature affects the rates of all physiological processes based on enzyme catalysis 

### 28 and diffusion; each process and overall growth (the integral of all processes) has a 

### 29 distinct optimal temperature range. It is important to note that a plant ’ s response to 

### 30 changes in temperature will depend on whether it is growing near its optimum 

### 31 temperature for growth or near its maximum temperature ( Rowland

- Bamford, 2000 )

. 

### 32 However, temperature is very likely an important variable affecting plant O 3 response in 

### 33Draft

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- 89 September 2011 the presence of the elevated CO 2 levels contributing to global climate change. In contrast, 

### 1 some evidence suggests that O 3 exposure sensitizes plants to low temperature stress 

### 2 ( Colls and Unsworth, 1992 ) and, also, that O 3 decreases below

- ground carbohydrate 

### 3 reserves, which may lead to responses in perennial sp ecies ranging from rapid demise to 

### 4 impaired growth in subsequent seasons (i.e., carry

- over effects) ( Andersen et al., 1997 )

. 

### 5
Light, a component of the plant ’ s physical environment, is an essential “ resource ” of 

### 6 energy content that drives photosynthesis and C assimilation. It has been suggested that 

### 7 increased light intensi ty may increase the O 3 sensitivity of light

- tolerant species while 

### 8 decreasing that of shade

- tolerant species, but this appears to be an oversimplification with 

### 9 many exceptions. Several studies suggest that the interaction between O 3 sensitivity and 

### 10 light e nvironment is complicated by the developmental stage as well as the light 

### 11 environment of individual leaves in the canopy ( Kitao et al., 2009 ; Topa et al., 2001 ; 

### 12 Chappelka and Samuelson, 1998 )

. 

### 13
Although the relative humidity of the ambient air has generally been found to increase the 

### 14 effects of O 3 by increasing stomatal conductance (thereby increasing O 3 flux into the 

### 15 leaves), abundant evidence also indicates that the ready availability of soil moisture 

### 16 results in greater O 3 sensitivity ( Mills, 2002 ) . The partial “ protection ” against the effects 

### 17 of O 3 afforded by drought has been observed in field experiments ( Low et al., 2006 ) a nd 

### 18 modeled in computer simulations ( Broadmeadow and Jackson, 2000 ) . Conversely, 

### 19 drought may exacerbate the effects of O 3 on plants ( Pollastrini et al., 2010 ; Grulke et al., 

### 20 2003b ) . There is also some evidence that O 3 can predispose plants to drought stress 

### 21 ( Maier

- Maercker, 1998 ) . Hence, the nature of the response is largely species

- specific and 

### 22 will depend to some extent upon the sequence in which the stressors occur. 

### 23

9.4.8.4 Interactions with other Pollutants

Ozone

- Nitrogen Interactions

Elevated O 3 exposure and N deposition often co

- occur. However, the interactions of O 3 

### 24 exposure and N deposition on vegetation are complex and less well understood compared 

### 25 to their independent effects. Consistent with the conclusion of the 2006 O 3 AQCD, th e 

### 26 limited number of studies published since the last review indicated that the interactive 

### 27 effects of N and O 3 varied among species and ecosystems (Table 9

- 8 ). To better 

### 28 understand these interactions in ecosystems across the U.S., more information is neede d 

### 29 considering combined O 3 exposure and N deposition related effects. 

### 30
Nitrogen deposition could stimulate relative growth rate (RGR), and lead to increased 

### 31 stomatal conductance. Therefore, plants might become more susceptible to O 3 exposure. 

### 32Draft

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- 90 September 2011 Alternatively, N deposition may increase the availability of photosynthates for use in 

### 1 detoxification and plants could become more tolerant to O 3 ( Bassin et al., 2007a ) . Only a 

### 2 few recent studies have investigated the interactive effects of O 3 and N in the U.S. Grulke 

### 3 et al. ( 2005 ) measured stomatal conductance of California black oak ( Quercus kelloggii ) 

### 4 at a long

- term N

- enrichment site located in the San Bernardino Mountains, which is 

### 5 accompanied by high O 3 exposure (80 p pb, 24

- h avg. over a six month growing season). 

### 6 The authors found that N amendment led to poor stomatal control in full sun in 

### 7 midsummer of the average precipitation years, but enhanced stomatal control in shade 

### 8 leaves of California black oak. In an OTC st udy, Handley and Grulke ( 2008 ) found that 

### 9 O 3 lowered photosynthetic ability and water

- use efficiency, and increased leaf chlorosis 

### 10 and necrosis of California black oak. Nitrogen fertilization tended to reduce plant 

### 11 sensitivity to O 3 exposure; however, the interaction was not statistically significant. 

### 12

Studies conducted outside the U.S. are also summarized in Table 9

- 8 . Gener ally, the 

### 13 responses were species specific. The O 3 - induced reduction in photosynthetic rate and 

### 14 biomass loss were greater in the relatively high N treatment for watermelon ( Citrillus 

### 15 lanants ) ( Calatayud et al., 2006 ) and Japanese beech ( Fagus crenata ) seedlings 

### 16 ( Yamaguchi et al., 2007 ) . However, there was no significant interactive effect of O 3 and 

### 17 N on biomass production for Quercus serrata seedlings ( Watanabe et al., 2007 ) , young 

### 18 Norway spruce ( Picea abies ) trees ( Thomas et al., 2005 ) , and young European beech 

### 19 ( Fagus sylvatica ) trees ( Thomas et al., 2006 )

. 

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- 91 September 2011 

### Table 9

- 8 Response of plants to the interactive effects of elevated ozone exposure and N enrichment

Site Species Ozone exposure N addition Responses References San Bernardino Mountains, U.S. California black oak ( Quercus kelloggii ) 80 ppb 0, and 50 kg N/ ha/yr N

- amended trees had lower late summer C gain and greater foliar chlorosis in the drought year, and poor stomatal control and lower leaf water use efficiency and in midsummer of the average precipitation year. Grulk e et al. ( 2005 )

San Bernardino Mountains, U.S. California black oak ( Quercus kelloggii ) 0, 75, and 150 ppb 0, and 50 kg N/ ha/yr N fertilization tended to reduce plant sensitivity to O 3 exposure; however the interaction was not statistically significant. Handley and Grulke ( 2008 )

Switzerland S pruce trees ( Picea abies ) Filtered (19.4

- 28.1 ppb); ambient (37.6

- 47.4 ppb) 0, 20, 40 and 80 kg N/ ha/yr Higher N levels alleviated the negative impact of O 3 on root starch con centrations Thomas et al. ( 2005 )

Switzerland B eech trees ( Fagus sylvatica ) Filtered (19.4

- 28.1 ppb); ambient (37.6

- 47.4 ppb) 0, 20, 40 and 80 kg N/ ha/yr N addition amplified the negative effects of O 3 on leaf area and shoot elongation. Thomas et al. ( 2006 )

Switzerland Alpine pasture A mbient ( AOT40 of 11.1

- 12.6 ppm

- h); 1.2 ambient (AOT40 of 15.2

- 29.5 ppm

- h) and 1.6 ambient (28.4

- 64.9 ppm

- h) 0, 5, 10 ’ 25, 50 kg N/ ha/yr The positive effects of N addition on canopy greenness were counteracted by accelerated leaf senescence in the highest O 3 treatment. Bassin et al. ( 2007b )

Switzerland Alpine pasture A mbient ( AOT40 of 11.1

- 12.6 ppm

- h); 1.2 ambient (AOT40 of 15.2

- 29.5 ppm

h) and 1.6 ambient (28.4

- 64.9 ppm

- h) 0, 5, 10, 25, 50 kg N/ha/yr Only a small number of species showed significant O 3 and N interactive effects on leaf chlorophyll concentration, leaf weight and change in 18O, and the patterns were not consistent

. Bassin et al. ( 2009 )

Switzerland Alpine pasture A mbient ( AOT40 of 11.1

- 12.6 ppm

- h); 1.2 ambient (AOT40 of 15.2

- 29.5 ppm

h) and 1.6 ambient (28.4

- 64.9 ppm

- h) 0, 5, 10 ’ 25, 50 kg N/ ha/yr The positive effects of N addition on canopy greenness were counteracted by accelerated leaf senescence in the highest O 3 treatment. Bassin et al. ( 2007b )

Switzerland Alpine pasture A mbient ( AOT40 of 11.1

- 12.6 ppm

- h); 1.2 ambient (AOT40 of 15.2

- 29.5 ppm

- h) and 1.6 ambient (28.4

- 64.9 ppm

- h) 0, 5, 10 ’ 25, 50 kg N/ ha/yr Highest N addition resulted in carbon loss, but there was no interaction between O 3 and N treatments. Volk et al. ( 2011 )

Spain W atermelon ( Citrillus lanants ) O 3 free (AOT40 of 0 ppm

h), ambient (AOT40 of 5.1

- 6.3 ppm

- h) and elevated O 3 (AOT40 of 32.5

- 35.6 ppm
- h) 140, 280, and 436 kg N/ ha/yr High N concentration enhanced the detrimental effects of O 3 on Chlorophyll a fluorescence parameters, l ipid peroxidation, and the total yield. Calatayud et al. ( 2006 )

Spain Trifolium striatum Filtered (24

- h avg. of 8

- 22 ppb); ambient (29

- 34 ppb), elevated O 3 (35

- 56 ppb) 10, 30, and 60 kg N/ ha/yr O 3 reduced total aerial biomass. N fertilization counterbalanced O 3 - induced effects only when plants were exposed to m oderate O 3 levels (ambient) but not under elevated O 3 concentrations. Sanz et al. ( 2007 )

Japan Japanese beech seedlings ( Fagus crenata ) Filtered (24

- h avg. of 10.3

- 13.2 ppb); ambient (42.0

- 43.3 ppb), 1.5 ambient (62.6

- 63.9 ppb) and 2.0 ambient (82.7

- 84.7 ppb) 0, 20 and 50 kg N/ ha/yr The O 3 - induced reduction in net photosynthesis and whole

- plant dry mass were greater in the relatively high N treatment than that in the low N treatment. Yamaguchi et al. ( 2007 )

Japan Quercus serrata seedlings Filtered (24

- h avg. of 10.3

- 13.2 ppb); ambient (42.0

- 43.3 ppb), 1.5 ambient (62.6

- 63.9 ppb) and 2.0 ambient (82.7

- 84.7 ppb) 0, 20 and 50 kg N/ ha/yr No significant interactive effects of O 3 and N load on the growth and net photosynthetic rate were detected. Watanabe et al. ( 2007 )

Ozone

- Carbon Dioxide Interactions
Several decades of research has shown that exposure to elevated CO 2 increases 

### 1 photosynthetic rates ( Bernacchi et al., 2006 ; Bernacchi et al., 2005 ; Tissue et al., 1999 ; 

### 2Draft

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- 92 September 2011 Tissue et al., 1997 ; Will and Ceulemans, 1997 ) , decreases stomatal conductance 

### 1 ( Ainsworth and Rogers, 2007 ; Paoletti et al., 2007 ; Bernacchi et al., 2006 ; Leakey et al., 

### 2 2006 ; Medlyn et al., 200 1 ) and generally increases the growth of plants ( McCarthy et al., 

### 3 2009 ; Norby et al., 2005 ) . This is in contrast to the decrease in photosynthesis and growth 

### 4 in many p lants that are exposed to elevated O 3 . The interactive effects on vegetation have 

### 5 been the subject of research in the past two decades due to the implications on 

### 6 productivity and water use of ecosystems. This area of research was discussed in detail in 

### 7 AX9 .3.8.1 of the 2006 O 3 AQCD and the conclusions made then are still relevant ( U.S. 

### 8 EPA, 2006b )

. 

### 9
The bulk of the available evidence shows that, under the various experimental conditions 

### 10 used (which almost exclusively employed abrupt or “ st ep ” increases in CO 2 

### 11 concentration, as discussed below), increased CO 2 levels (ambient + 200 to 400 ppm) 

### 12 may protect plants from the adverse effects of O 3 on growth. This protection may be 

### 13 afforded in part by CO 2 acting together with O 3 in inducing stomata l closure, thereby 

### 14 reducing O 3 uptake, and in part by CO 2 reducing the negative effects of O 3 on Rubisco 

### 15 and its activity in CO 2 - fixation. Although both CO 2 - induced and O 3 - induced decreases in 

### 16 stomatal conductance have been observed primarily in short

- term studies, recent data 

### 17 show a long

- term and sustained reduction in stomatal conductance under elevated CO 2 

### 18 for a number of species ( Ainsworth and Long, 2005 ; Ellsworth et al., 2004 ; Gun derson et 

### 19 al., 2002 ) . Instances of increased stomatal conductance have also been observed in 

### 20 response to O 3 exposure, suggesting partial stomatal dysfunction after extended periods 

### 21 of exposure ( Paoletti and Grulke, 2010 ; Grulke et al., 2007a ; Maier

- Maercker, 1998 )

. 

### 22
Important caveats must be raised with regard to the findings presented in published 

### 23 research. The first caveat concerns the distinctly different na tures of the exposures to O 3 

### 24 and CO 2 experienced by plants in the field. Changes in the ambient concentrations of 

### 25 these gases have very different dynamics. In the context of climate change, CO 2 levels 

### 26 increase relatively slowly (globally 2 ppm/year) and ma y change little over several 

### 27 seasons of growth. On the other hand, O 3 presents a fluctuating stressor with 

### 28 considerable hour

- to

- hour, day

- to

- day and regional variability ( Polle and Pell, 1999 )

. 

### 29 Almost all of the evidence presented comes from experimentation involving plants 

### 30 subjected to an abrupt step increa se to a higher, steady CO 2 concentration. In contrast, the 

### 31 O 3 exposure concentrations usually varied from day to day. Luo and Reynolds ( 1999 )
, 

### 32 Hui et al. ( 2002 ) , and Luo ( 2001 ) noted the difficulties in predicting the likely effects of a 

### 33
gra dual CO 2 increase from experiments involving a step increase or those using a range 

### 34 of CO 2 concentrations. It is also important to note that the levels of elevated CO 2 in 

### 35 many of the studies will not be experienced in the field for 30 or 40 years, but elevated 

### 36 levels of O 3 can occur presently in several areas of the U.S. Therefore, the CO 2 × O 3 

### 37 interaction studies may be less relevant for current ambient conditions. 

### 38Draft

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- 93 September 2011 Ano ther caveat concerns the interactions of O 3 and CO 2 with other climatic variables, 

### 1 such as temperature and precipitation. In light of the key role played by temperature in 

### 2 regulating physiological processes and modifying plant response to increased CO 2 lev els 

### 3 ( Morison and Lawlor, 1999 ; Long, 1991 ) and the knowledge that relatively modest 

### 4 increases in temperature may lead to dramatic consequences in terms of plant 

### 5 development ( Lawlor, 1998 ) , it is important to consider that studying CO 2 and O 3 

### 6 in teractions alone may not create a complete understanding of effects on plants under 

### 7 future climate change. 

### 8

9.4.9 Insects and Other Wildlife

9.4.9.1 Insects

Insects may respond indirectly to changes to plants (i.e., increased reactive oxygen 

### 9 species, altered phytochemis try, altered nutrient content) that occur under elevated O 3 

### 10 conditions, or O 3 can have a direct effect on insect performance ( Menendez et al., 2009 )

. 

### 11 Effects of O 3 on insects occur at the species level (i.e., growth, survival, reproduction, 

### 12 development, feeding behavior) and at the population and community

- level (i.e., 

### 13 population gr owth rate, community composition). In general, effects of O 3 on insects are 

### 14 highly context

- and species

- specific ( Lindroth, 2010 ; Bidart

- Bouzat and Imeh

- Nathaniel, 

### 15 2008 ) . Furthermore, plant responses to O 3 exposure and herbivore attack have been 

### 16 demonstrated to share signaling pathways, complicating characterization of these 

### 17

stressors ( Lindroth, 2010 ; Menendez et al., 2010
, 2009 )

. Although bot h species

- level and 

### 18 population and community

- level responses to elevated O 3 are observed in field and 

### 19 laboratory studies discussed below, there is no consensus on how insects respond to 

### 20 feeding on O 3 – exposed plants. 

### 21

Species

- Level Responses
In considering insect growth, survival and reproduction in elevated O 3 conditions, several 

### 22 studies have indicated an effect while others have found no correlation. The performance 

### 23 of five herbivore species (three moths and two weevils) was assessed in an OTC 

### 24 experiment a t 2 × ambient concentration ( Peltonen et al., 2010 ) . Growth of larvae of the 

### 25 Autumnal moth, Epirrita autumna , was significantly decreased in the O 3 treatment while 

### 26 no effects were observed in the other species. In an aphid oviposition preference study 

### 27 using birch buds grown in a three year OTC experiment, O 3 had neither a stimulatory or 

### 28 deterring effect on egg

- laying ( Peltonen et al., 2006 ) . Furthermore, changes in birch bud 

### 29 phenolic compounds associated with the doubled ambient concentrations of O 3 did not 

### 30Draft

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- 94 September 2011 correlate with changes in aphid oviposition ( Peltonen et al., 2006 ) . Reproduction in 

### 1 Popillia japonica
, that were fed soybeans and grown under elevated O 3 appeared to be 

### 2 unaffected ( O'Neill et al., 2008 ) . In a meta

- analysis of effects of elevated O 3 on 22 

### 3 species of trees and 10 species of insects, the rates of survival, reproduction and food 

### 4 consumption were typically unaffected while development times were reduced and pupal 

### 5 masses were increased ( Valkama et al., 2007 )

. 

### 6
At the Aspen FACE site insect performance under elevated (50

- 60 ppb) O 3 conditions 

### 7 (approximately 1.5 × background ambient levels of 30

- 40 ppb O 3 ) have been considered 

### 8 for several species. Cumulative fecundity of aphids ( Cepegillettea betulaefoliae) , that 

### 9 were reared on O 3 - exposed paper birch ( Betula papyrifera ) trees, was lower than aphids 

### 10 from control plots ( Awmack et al., 2004 ) . No effects on growth, development, adult 

### 11 weight, embryo number and birth weight of n ewborn nymphs were observed. In a study 

### 12 conducted using three aspen genotypes, performance of the aspen beetle ( Chrysomela 

### 13 crochi ) decreased across all parameters measured (development time, adult mass and 

### 14 survivorship) under elevated O 3 ( Vigue and Lindroth, 2010 ) . There was an increase in the 

### 15 development time of male and female aspen beetle larvae although the percentages varied 

### 16 across genotypes. Decreased beetle adult mass and survivorship was observed across all 

### 17 genotypes under elevated O 3 conditions. Another study from the Aspen FACE site, did 

### 18 not find any significant effects of elevated O 3 on performance (longevity, fecundity, 

### 19 abundance) of the invasive weevil ( Polydrusus sericeus ) ( Hillstrom et al., 2010b )

. 

### 20

Since the 2006 O 3 AQCD, several studies have considered the effect o f elevated O 3 on 

### 21 feeding behavior of insects. In a feeding preference study, the common leaf weevil 

### 22 ( Phyllobius pyri ) consumed significantly more leaf discs from one aspen clone when 

### 23 compared to a second clone under ambient air conditions ( Freiwald et al., 2008 ) . In a 

### 24 moderately elevated O 3 environment (1.5 × ambient), this preference for a certain aspen 

### 25 clone was less evident, however, leaves from O 3 - exposed trees were significan tly 

### 26 preferred to leaves grown under ambient conditions. Soybeans grown under enriched O 3 

### 27 had significantly less loss of leaf tissue to herbivory in August compared to earlier in the 

### 28 growing season (July) when herbivory was not affected ( Hamilton et al., 2005 ) . Other 

### 29 plant

- herbivore interactions have shown no effects of elevated O 3 on feeding. Feeding 

### 30 behavior of Japanese beetles ( P. japonica ) appea red to be unchanged when beetles were 

### 31 fed soybean leaves grown under elevated O 3 conditions ( O'Neill et al., 2008 ) . At the 

### 32 Aspen FACE site, feeding by the invasive weevil ( Polydrusus sericeus ), as measured by 

### 33 leaf area consumption, was not significantly different between foliage that was grown 

### 34 under elevated O 3 versus ambient conditions ( Hillstrom et al., 2010b )

. 

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- 95 September 2011 Population

- Level and Community

- Level Responses

Recent data on insects provide evidence of population

- level and community

- level 

### 1 responses to O 3 . Elevated levels of O 3 can affect plant phytochemistry and nutrient 

### 2 content which in turn can alter population density and structure of the associated 

### 3 herbivorous insect communities a nd impact ecosystem processes ( Cornelissen, 2011 ; 

### 4 Lindroth, 2010 ) . In 72

- hour exp osures to elevated O 3 , mean relative growth rate of the 

### 5 aphid Diuraphis noxia increased with ozone concentration suggesting that more rapid 

### 6 population growth may occur when atmospheric O 3 is elevated (Summers et al., 1994, 

### 7 735955 ) . In a long

- term study of elevated O 3 on herbivore performance at the Aspen 

### 8 FACE site, individual performance and population

- level effects of the aphid 

### 9
C. betulaefoliae were assessed. Elevated O 3 levels had a strong positive effect on the 

### 10 population growth rates of the aphids; although effects were not detected by measuring 

### 11 growth, development, adult weight, embryo number or birth weight of newborn nymphs 

### 12 ( Awmack et al., 2004 ) . Conversely, a lower rate of population growth was obs erved in 

### 13 aphids previously exposed to O 3 in an OTC ( Menendez et al., 2010 ) . No direct effects of 

### 14 O 3 were observed; however, nymphs born from adults exposed to and feeding on O 3 

### 15 exposed plants were less capable of infesting new plants when compared to nymphs in 

### 16 the control plots ( Menendez et al., 2010 ) . Elevated O 3 reduced total arthropod abundance 

### 17 by 17% at Aspen FACE, largely as a result of the negative effects on par asitoids, 

### 18 although phloem

- feeding insects may benefit ( Hillstrom and Lindroth, 2008 ) . Herbivore 

### 19 communities affected by O 3 and N were sampled along an air pollution gradient in the 

### 20
Los Angeles basin ( Jones and P aine, 2006 ) . Abundance, diversity, and richness of 

### 21 herbivores were not affected. However, a shift in community structure, from phloem



### 22 feeding to chewing dominated communities, was observed along the gradient. No 

### 23 consistent effect of elevated O 3 on herbi vory or insect population size was detected at 

### 24 SoyFACE ( O'Neill et al., 2010 ; Dermody et al., 2008 )

. 

### 25
Evidence of modification of insect populations and communities in response to elevated 

### 26 O 3 includes genotypic and phenotypic changes. In a study conducted at the Aspen FACE 

### 27 site, elevated O 3 altered the genotype frequencies of the pea aphid ( Acyrthosiphon p isum ) 

### 28 grown on red clover ( Trifolium pratense ) over multiple generations ( Mondor et al., 

### 29 2005 ) . Aphid color was used to distinguish between the two genotypes. Ozone increased 

### 30 the genotypic frequencies of pink

- morph:green

- morph aphids from 2:1 to 9:1, and 

### 31 depressed wing

- induction responses more strongly in the pink than the green genotype 

### 32 ( Mondor et al., 2005 ) . Growth and development of individual green and pink aphids 

### 33 reared as a single genotype or mixed genotypes were unaffected by elevated O 3 ( Mondor 

### 34 et al., 2010 ) . However, growth of pea aphid populations is not readily predictable using 

### 35 individual growth rates. 

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- 96 September 2011 

### 9.4.9.2 Wildlife

Herpetofauna

Since the 2006 O 3 AQCD, direct ef fects of O 3 exposure including physiological changes 

### 1 and alterations of ecologically important behaviors such as feeding and thermoregulation 

### 2 have been observed in wildlife. These studies have been conducted in limited laboratory 

### 3 exposures, and the levels of O 3 treatment (e.g. 0.2

- 0.8 ppm) were often unrealistically 

### 4 higher than the ambient levels. Amphibians may be especially vulnerable to airborne 

### 5 oxidants due to the significant gas exchange that occurs across the skin ( Andrews et al., 

### 6 2008 ; Dohm et al., 2008 ) . Exposure to 0.2 ppm to 0.8 ppm O 3 for 4 hours resulted in a 

### 7 decrease of oxygen consumption and depressed lung ventilation in the California tree 

### 8 frog Pseudacris cadaverina ( Mautz and Dohm, 2004 ) . Following a single 4

- h exposure to 

### 9 O 3 , reduced pulmonary macrophage phagocytosis was observed at 1 and 24 hours post 

### 10 exposure in the marine toad ( Bufo marinus ) indicating an effect on immune s ystem 

### 11 function ( Dohm et al., 2005 ) . There was no difference in macrophage function at 

### 12
48 hours post exposure in exposed and control individuals. 

### 13

Behavioral effects of O 3 observed in amphibians include responses to minimize the 

### 14 surface area of the body exposed to the air and a decrease in feeding rates ( Dohm et al., 

### 15 2008 ; Mautz and Dohm, 2004 ) . The adoption of a low

- profile “ water conservation 

### 16 posture ” during O 3 exposure was observed in experiments with the California tree frog 

### 17 ( Mautz and Dohm, 2004 ) . Marine toads, Bufo marinus , exposed to 0.06 μL/L O 3 for 

### 18
4 hours ate significantly fewer mealworms at 1 hour and 48 hours post exposure than 

### 19 control toads ( Dohm et al., 2008 ) . In the same study, escape/exploratory behavior as 

### 20 measured by total distance moved was not adversely affected in the O 3 - exposed 

### 21 individuals as compared to the controls ( Dohm et al., 2008 )

. 

### 22
Water balance and thermal preference in herpetofauna are altered with elevated O 3 . 

### 23 Marine toads exposed to 0.8 ppm O 3 for 4 hours exhibited behavioral hypothermia when 

### 24 temperature selection in the toads was assessed at 1, 24 and 48 hours post exposure 

### 25 ( Dohm et al., 2001 ) . Ozone

- exposed individuals lost almost 5g more body mass on 

### 26 average than controls due to evaporative water loss. At 24 hours after exposure, the 

### 27 individuals that had lost significant body mass selected lower body temperatures ( Dohm 

### 28 et al., 2001 ) . Behavioral hypothermia was also observed in reptiles following 4

- h 

### 29 exposures to 0. 6 ppm O 3 . Exposure of the Western Fence Lizard ( Sceloporus 

### 30 occidentalis ) at 25 o C induced behavioral hypothermia that recovered to control 

### 31 temperatures by 24 hours ( Mautz and Dohm, 2004 ) . The behavioral hypothermic 

### 32 response persisted in lizards exposed to O 3 at 35 o C a t 24 hours post exposure resulting in 

### 33 a mean body temperature of 3.3 o C over controls. 

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- 97 September 2011 Soil Fauna Communities

Ozone has also been shown to alter soil fauna communities ( Meehan et al., 2010 ; 

### 1 Kasurinen et al., 2007 ; Loranger et al., 2004 ) . Abundance of Acari (mites and ticks) 

### 2 decreased by 47% under elevated O 3 at Aspen FACE site, probably due to the higher 

### 3

s econdary metabolites and lower N concentrations in litter and foliage under elevated O 3 

### 4 ( Loranger et al., 2004 )

. In another study from the Aspen FACE site, leaf litter collected 

### 5 from aspen grown under elevated O 3 conditions were higher in fibe r and lignin 

### 6 concentrations than trees grown under ambient conditions. These chemical characteristics 

### 7 of the leaves were associated with increased springtail population growth following 

### 8 10 weeks in a laboratory microcosm ( Meehan et al., 2010 ) . Consumption rates of 

### 9 earthworms fed on leaf litter for 6 weeks from trees grown u nder elevated O 3 conditions 

### 10 and ambient air did not vary significantly between treatments ( Meehan et al., 2010 ) . In 

### 11 another study on juvenile earthworms Lumbricus terrestris , individual growth was 

### 12 reduced when worms were fed high

- O 3 birch litter from trees exposed for three years to 

### 13 elevated O 3 in an OTC system ( Kasurinen et al., 2007 ) . In the same study no significant 

### 14 growth or mortality effects were observed in isopods. 

### 15

9.4.9.3 Indirect Effects on Wildlife

In addition to the direct effects of O 3 exposure on physiological and behavioral endpoints 

### 16 observed in the laboratory, there are indirec t effects to wildlife. These effects include 

### 17 changes in biomass and nutritive quality of O 3 - exposed plants (reviewed in Section 9.4.4) 

### 18 that are consumed by wildlife. Reduced digestibility of O 3 - exposed plants may alter 

### 19 dietary intake and foraging strategie s in herbivores. In a study using native highbush 

### 20 blackberry ( Rubus argutus ) relative feed value of the plants decreased in bushes exposed 

### 21 to double ambient concentrations of O 3 ( Ditchko ff et al., 2009 ) . Indirect effects of 

### 22 elevated O 3 on wildlife include changes in chemical signaling important in ecological 

### 23 interactions reviewed below. 

### 24

Chemical Signaling in Ecological Interactions
Ozone has been shown to degrade or alter biogenic VOC signals important to ecological 

### 25 interactions including; (1) attraction of pollinators and seed dispersers; (2) defense 

### 26 against herbivory; and (3) predator

- prey interactions ( Pinto et al., 2010 ; McFrederick et 

### 27 al., 2009 ; Yuan et al., 2009 ; Pinto et al., 2007a ; Pinto et al., 2007b ) . Each signal released 

### 28 by emitters has an atmospheric lifetime and a unique chemical signature comprised of 

### 29 different ratios of individual hydrocarbons that is s usceptible to atmospheric oxidants 

### 30 such as O 3 ( Yuan et al., 2009 ; Wright e t al., 2005 ) . Unde r elevated O 3 conditions, these 

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- 98 September 2011 olfactory cues may travel shorter distances before losing their specificity ( McFrederick et 

### 1 al., 2009 ; McFrederick et al., 2008 ) . Additional non

- phytogenic VOC

- mediated 

### 2 interrelationships with the potential to be modified by O 3 include territorial marking, 

### 3 pheromones for attraction of mates and various social interactions including scent trails, 

### 4 nestmate recognition and signals involved in aggregation behaviors ( McFrederick et al., 

### 5 2009 ) . For example, the alcohols, ketones and aldehydes comprising sex pheromones in 

### 6 moths could be especially vulnerable to degradation by O 3 , since some males travel >100 

### 7 m to find mates ( Carde and Haynes, 2004 ) . In general, effects of O 3 on scent

- mediated 

### 8 ecological interactions are highly context

- and species

- specific ( Lindroth, 2010 ; Bidart



### 9 Bouzat and Imeh

- Nathaniel, 2008 )

. 

### 10

Pollination and Seed Dispersal
Phytogenic VOC ’ s attract pollinators and seed dispersers to flowers and fruits ( Dudareva 

### 11 et al., 2006 ; Theis and Raguso, 2005 ) . These floral scent trails in plant

- insect interactions 

### 12 may be destroyed or transformed by O 3 ( McFrederick et al., 2008 ) . Using a Lagrangian 

### 13 model, the rate of destruction of phytogenic VOC ’ s was estimated in air parcels at 

### 14 increasing distance from a source in response to increased regional levels of O 3 , hydroxyl 

### 15 and nitrate radicals ( McFrederick et al., 2008 ) . Based on the model, the ability of 

### 16 pollinators to locate highly reactive VOCs from emitting flowers may have decreased 

### 17 from kilometers during pre

- industrial times to <200 m at current ambient conditions 

### 18 ( McFrederick et al., 2008 ) . Scents that travel shorter distances (0

- 10 m) are less 

### 19 susceptible to air pollutants, while highly reactive scents that travel longer dis tances (10 

### 20 to 100 ’ s of meters), are at a higher risk for degradation ( McFrederick et al., 2009 ) . For 

### 21 example, male euglossine bees can det ect bait stations from a distance of at least one 

### 22 kilometer ( Dobson, 1994 )

. 

### 23

Defense Against Herbivory
Ozone can alter the chemical signature of VOCs emitted by plants and these VOCs are 

### 24 subsequently detected by herbivores ( Blande et al., 2010 ; Iriti and Faoro, 2009 ; Pinto et 

### 25 al., 2007a ; Vuorinen et al., 2004 ; Jackson et al., 1999 ; Cannon, 1990 ) . These 

### 26 modifications can make the plant either more attractive or repellant to phytophagous 

### 27 insects ( Pinto et al., 2010 ) . For example, under elevated O 3 , the host plant preference by 

### 28 forest tent caterpillars increased for birch compare d to aspen ( Agrell et al., 2005 ) . Ozone



### 29 induced emissions from red spruce needl es were found to repel spruce budworm larvae 

### 30 ( Cannon, 1990 ) . Transcriptional profiles of field grown soybean ( Glycine max ) grown in 

### 31 elevated O 3 conditions were altered due to herbivory by Japanese beetles. The herbivory 

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- 99 September 2011 resulted in a higher number of transcripts in the leaves of O 3 - exposed plants and up



### 1 regulation of antioxidant metabolism associated with plant defense ( Casteel et al., 2008 )

. 

### 2
Ozone may modify signals involved in plant

- to

- plant interactions and plant defense 

### 3 against pathogens ( Blande et al., 2010 ; Pinto et al., 2010 ; McFrederick et al., 2009 ; Yuan 

### 4 et al., 2009 ) . In a recent study w ith lima beans, 80 ppb O 3 degraded several herbivore



### 5 induced VOCs, reducing the distance over which plant

- to

- plant signaling occurred 

### 6 ( Blande et al., 2010 )

. 

### 7

Predator

- Prey Interactions

Elevat ed O 3 conditions are associated with disruption of pheromone

- mediated 

### 8 interactions at higher trophic levels (e.g., predators and parasitoids of herbivores). In a 

### 9 study from the Aspen FACE site, predator escape behaviors of the aphid ( Chatophorus 

### 10
stevensis ) were enhanced on O 3 - fumigated aspen trees although the mechanism of this 

### 11 response remains unknown ( Mondor et al., 2004 ) . The predatory mite Phytoseiulus 

### 12 persimilis can distinguish between the VOC signature of ozonated lima bean plants and 

### 13 ozonated lima bean plants simultaneously damaged by T. urticae ( Vuorinen et al., 2004 ) 

### 14 however, other tritrophic interactions have shown no effect ( Pinto et al., 2007b )

. 

### 15
There are few studies that consider host location behaviors of parasites under elevated 

### 16 O 3 . In closed chambers fumigated with O 3 , the searching efficiency and proportion of the 

### 17 host larval fruit flies parasitized by Asobara tabida , declined when compared to filtered 

### 18 air controls ( Gate et al., 1995 ) . The host location behavior and rate of parasitism of the 

### 19 wasp ( Coesia plutellae ) on Plutella xylostella

- infested potted cabbage plants was tested 

### 20 under ambient and doubled O 3 conditions in an open

- air fumigation system ( Pinto et al., 

### 21 2008 ) . The number of wasps found in the field and the percentages of parasitized larvae 

### 22 were not significantly different from controls under elevated O 3 . 

### 23

Elevated O 3 has the potential to perturb specialized food

- web communication in 

### 24 transgenic crops. In insect

- resistant oilseed rape Brassica na pus grown under 100 ppb O 3 

### 25 in a growth chamber, reduced feeding damage by Putella xylostella led to deceased 

### 26 attraction of the endoparasitoid ( Costesia vestalis ), however this tritrophic interaction 

### 27 was influenced by the degree of herbivore feeding ( Himanen et al., 2009a ; Himanen et 

### 28 al., 2009b ) . Under chronic O 3 - expos ure, the insect resistance trait BT cry1Ac in 

### 29 transgenic B. napus was higher than the control ( Himanen et al., 2009c ) . There was a 

### 30 negative relative growth rate of the B t target herbivore, P. xylostella , in all O 3 treatments. 

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- 100 September 2011 

### 9.4.9.4 Summary

New information on O 3 effects on insects and other wildlife is limited to a few species 

### 1 and there is no consensus on how these organisms respond to elevated O 3. Studies 

### 2 published since the l ast review show impacts of elevated O 3 on both species

- level 

### 3 responses (reproduction, growth, feeding behavior) and community and ecosystem

- level 

### 4 responses (population growth, abundance, shift in community structure) in some insects 

### 5 and soil fauna. Changes in ecologically important behaviors such as feeding and 

### 6 thermoregulation have recently been observed with O 3 exposure in amphibians and 

### 7 reptiles, however, these responses occur at concentrations of O 3 much higher that 

### 8 ambient levels. 

### 9

New information avai lable since the last review considers the effects of O 3 on chemical 

### 10 signaling in insect and wildlife interactions. Specifically, studies on O 3 effects on 

### 11 pollination and seed dispersal, defenses against herbivory and predator

- prey interactions 

### 12 all consider the ability of O 3 to alter the chemical signature of VOCs emitted during these 

### 13 pheromone

- mediated events. The effects of O 3 on chemical signaling between plants, 

### 14 herbivores and pollinators as well as interactions between multiple trophic levels is an 

### 15

emer ging area of study that may result in further elucidation of O 3 effects at the species, 

### 16 community and ecosystem

- level. 

### 17



## 9.5 Effects

- Based Air Quality Exposure Indices and Dose Modeling

9.5.1 Introduction

Exposure indices are metrics that quantify exposure as it rela tes to measured plant 

### 18 damage (e.g., reduced growth). They are summary measures of monitored ambient O 3 

### 19 concentrations over time, intended to provide a consistent metric for reviewing and 

### 20 comparing exposure

- response effects obtained from various studies. Su ch indices may 

### 21 also provide a basis for developing a biologically

- relevant air quality standard for 

### 22 protecting vegetation and ecosystems. Effects on plant growth and/or yield have been a 

### 23 major focus of the characterization of O 3 impacts on plants for purpo ses of the air quality 

### 24 standard setting process ( U.S. EPA, 2007b
, 1996e
, 1986 ) . The relationship of O 3 and 

### 25 plant responses can be characterized quantitatively as “ dose

- r esponse ” or “ exposure



### 26
respon se. ” The distinction is in how the pollutant concentration is expressed: “ dose ” is 

### 27 the pollutant concentration absorbed by the leaf over some time period, and is very 

### 28 difficult to measure directly, whereas “ exposure ” is the ambient air concentration 

### 29Draft

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- 101 September 2011 measur ed near the plant over some time period, and summarized for that period using an 

### 1 index. Exposure indices have been most useful in considering the form of secondary O 3 

### 2 NAAQS, in large part because they only require ambient air quality data rather than more 

### 3 complex indirect calculations of dose to the plant. The attributes of exposure indices that 

### 4 are most relevant to plant damage are the weighting of O 3 concentrations and the daily 

### 5 and seasonal time

- periods. Several different types of exposure indices are di scussed in 

### 6 Section 9.5.2. 

### 7
Form a theoretical perspective, a measure of plant O 3 uptake or dose from ambient air 

### 8 (either rate of uptake or cumulative seasonal uptake) might be a better predictor of O 3 

### 9 damage to plants than an exposure index and may be usefu l in improving risk assessment. 

### 10 An uptake estimate would have to integrate all those environmental factors that influence 

### 11 stomatal conductance, including but not limited to temperature, humidity, and soil water 

### 12 status (Section 9.5.4). Therefore, uptake val ues are generally obtained with simulation 

### 13 models that require knowledge of species

- and site

- specific values for the variables 

### 14 mentioned. However, a limitation of modeling dose is that environmental variables are 

### 15 poorly characterized. In addition, it has also been recognized that O 3 detoxification 

### 16 processes and the temporal dynamics of detoxification must be taken into account in dose 

### 17 modeling ( Heath et al., 2009 ) (Section 9.5.4). Because of this, research has focused 

### 18 historically on predictors of O 3 damage to plants based only on exposure as a summary 

### 19 measure of monitored ambient pollutant concentration over some integral of time, rather 

### 20 than dose ( U.S. EPA, 1996c ; Costa et al., 1992 ; Lee et al., 1988b ; U.S. EPA, 1986 ; 

### 21 Lefohn and Benedict, 1982 ; O'Gara, 1922 )

. 

### 22

9.5.2 Description of Exposure Indices Available in the Literature

Mathematical approa ches for summarizing ambient air quality information in biologically 

### 23 meaningful forms for O 3 vegetation effects assessment purposes have been explored for 

### 24 more than 80 years ( U.S. EPA, 1996b ; O'Gara, 1922 ) . In the context of national standards 

### 25 that protect for “ known or anticipated ” effects on many plant species in a variety of 

### 26 habitats, exposure indices provide a numerical summary of very large numbers of 

### 27 ambient observations of concentr ation over extended periods. Like any summary statistic, 

### 28 exposure indices retain information on some, but not all, characteristics of the original 

### 29 observations. Several indices have been developed to attempt to incorporate some of the 

### 30 biological, environme ntal, and exposure factors that influence the magnitude of the 

### 31 biological response and contribute to observed variability ( Hogsett et al., 1988 ) . In the 

### 32 1996 O 3 AQCD, the exposure indices were arranged into five categories; (1) One event, 

### 33 (2) Mean, (3) Cumulative, (4) Concentration weighted, and (5) Multicomponent, and 

### 34 were discussed in detail ( Lee et al., 1989 ) . Figure 9

- 9 ill ustrates how several of the 

### 35Draft

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- 102 September 2011 indices weight concentration and accumulate exposure. For example, the SUM06 index 

### 1 (panel a) is a threshold

- based approach wherein concentrations below 0.06 ppm are given 

### 2 a weight of zero and concentrations above 0.06 ppm are gi ven a weight of 1.0 that is 

### 3 summed usually over 3 to 6 months . The Sigmoid approach (panel b), which is similar to 

### 4 the W126 index, is a non

- threshold approach wherein all concentrations are given a 

### 5 weight that increases from zero to 1.0 with increasing co ncentration and summed. 

### 6

Source: Used with permission from Air and Waste Management Association ( Tingey et al., 1991 ) (a) SUM06: the upper graphic illustrates an episodic exposure profile; the shaded area under some of the peaks illustrates th e concentrations greater than or equal to 0.06 ppm that are accumulated in the in dex. The insert shows the concentration weighting (0 to 1) function. The lower portion of the graphic illustrates how concentration is accumulated over the exposure period. (b) S IGMOID: the upper graphic illustrates an episodic exposure profile; the variab le shaded area under the peaks illustrates the concentration

dependent weights that are accumulated in the index. The insert shows the sigmoid concentration weighting function. This is s imilar to the W126 function. The lower portion of the graphic illustra tes how concentration is accumulated over the exposure period. (c) second HDM and M

- 7: the upper graphic illustrates an episodic exposure profile. The lower portion of the graphic illustrates that the second 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.


### Figure 9

- 9 Diagrammatic representation of several exposure indice s illustrating how they weight concentration and accumulate exposure.

Various factors with known or suspected bearing on the exposure

- response relationship, 

### 7 including concentration, time of day, respite time, frequency of peak occurrence, plant 

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- 103 September 2011 phenology, predisposition, etc., have been weighted with various functions in a l arge set 

### 1 of indices. The resulting indices were evaluated by ranking them according to the 

### 2 goodness

- of

- fit of a regression model of growth or yield response ( Lee et al., 1989 ) . The 

### 3 statistical evaluations for each of these indices were completed using growth or yield 

### 4 response data from many earlier exposure studies (e.g., NCLAN). This retrospective 

### 5 approach was necessary because there were no studies specifically designed to test the 

### 6 goodness of fit of the various indices. The goodness of fit of a set of linear and nonlinear 

### 7 models for exposure

- response was ranked as various proposed i ndices were used in turn 

### 8 to quantify exposure. This approach provided evidence for the best indices. The results of 

### 9 retrospective analyses are described below. 

### 10
Most of the early retrospective studies reporting regression approaches used data from the 

### 11 NCLAN program or data from Corvallis, Oregon or California ( Costa et al., 1992 ; Lee et 

### 12 al., 1988b ; Lefohn et al., 1988 ; Musselman et al., 1988 ; Lee et al., 1987 ; U.S. EPA, 

### 13 1986 ) . These studies were previously reviewed by the EPA ( U.S. EPA, 1996c ; Costa et 

### 14 al., 1992 ) and were in general agreement that the best fit to the data resulted from using 

### 15 cumulative concentration

- weighted exposure indices (e.g. W126, SUM06). Lee et al. 

### 16 ( 1987 ) suggested that exposure indi ces that included all the 24

- h data performed better 

### 17 than those that used only 7 hours of data; this was consistent with the conclusions of 

### 18 Heagle et al. ( 1987 ) that p lants receiving exposures for an additional 5

- h/day showed 

### 19 10% greater yield loss than those exposed for 7

- h/day. In an analysis using the National 

### 20 Crop Loss Assessment Network (NCLAN) data, Lee et al. ( 1988 ) found several indices 

### 21 which only cumulated and weighted higher concentrations (e.g., W126, SUM06, SUM08, 

### 22 and AOT40) performed very well. Am ongst this group no index had consistently better 

### 23 fits than the other indices across all studies and species ( Heagle et al., 1994b ; Lefohn et 

### 24 al., 1988 ; Musselman et al., 1988 )

. Lee et al. ( 1988 ) found that adding phenology 

### 25 weighting to the index somewhat improved the performance of the indices. The “ best ” 

### 26 exposure index was a phenologically weighted cumulative index, with sigmoid weighting 

### 27 on concentration and a gamma weigh ting function as a surrogate for plant growth stage. 

### 28 This index provided the best statistical fit when used in the models under consideration, 

### 29 but it required data on species and site conditions, making specification of weighting 

### 30 functions difficult for ge neral use. 

### 31
Other factors, including predisposition time ( Hogsett et al., 1988 ; McCool et al., 1988 ) 

### 32 and crop d evelopment stage ( Tingey et al., 2002 ; Heagle et al., 1991 ) contributed to 

### 33 variation in the biological response and suggested the need for weighting O 3 

### 34 concentrations to account for predisposition time and phenology. However, the roles of 

### 35 predisposition and p henology in plant response vary considerably with species and 

### 36 environmental conditions; therefore, specification of a weighting function for general use 

### 37 in characterizing plant exposure has not been possible. 

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- 104 September 2011 European scientists took a similar approach in developing indices describing growth and 

### 1 yield loss in crops and tree seedlings, using OTCs with modified ambient exposures, but 

### 2 many fewer species and study locations were employed in the European studies. There is 

### 3 evidence from some European studies that a lower ( Pleijel et al., 1997 ) or higher ( Finnan 

### 4 et al., 1997 ; Finnan et al., 1996 ) cutoff value in indices with a threshold may provide a 

### 5 better statistical fit to the experimental data. Finnan et al. ( 1997 ) used seven exposure 

### 6 studies of spring wheat to confirm that cumulative exposure indices emphasizing higher 

### 7 O 3 concentrati ons were best related to plant response and that cumulative exposure 

### 8 indices using weighting functions, including cutoff concentrations, allometric and 

### 9 sigmoidal, provided a better fit and that the ranking of these indices differed depending 

### 10 on the exposur e

- response model used. Weighting those concentrations associated with 

### 11 sunshine hours in an attempt to incorporate an element of plant uptake did not improve 

### 12 the index performance ( Finnan et al., 1997 ) . A more recent study using data from several 

### 13 European studies of Norway spruce, analyzed the relationship between relative biomass 

### 14 accumulation and several cumulativ e, weighted indices, including the AOT40 (area over 

### 15 a threshold of 40ppb) and the SUM06 ( Skarby et al., 2004 ) . All the indices performed 

### 16 relatively well in regressing biomass and exposure index, w ith the AOT20 and AOT30 

### 17 doing slightly better than others (r 2 = 0.46

- 0.47). In another comparative study of four 

### 18 independent data sets of potato yield and different cumulative uptake indices with 

### 19 different cutoff values, a similarly narrow range of r 2 was observed (r 2 = 0.3

- 0.4) ( Pleijel 

### 20 et al., 2004b )

. 

### 21
In Europe, the cutoff concentration

- weighted index AOT40 was selected in developing 

### 22
exposure

- response relationships based on OTC studies of a limited number of crops and 

### 23

trees ( Grunhage and Jager, 2003 ) . The United Nations E conomic Commission for Europe 

### 24 ( UNECE, 1988 ) adopted the critical levels approach for assessment of O 3 risk to 

### 25 vegetation across Europe. As used by the UNECE, the critical levels are not like the air 

### 26 quality regulatory standards used in the U.S., but rather function as planning targets for 

### 27 reductions in pollutant emissions to protect ecological resources. Critical levels for O 3 are 

### 28 intended to prevent long

- term deleterious effects on the most sensitive plant species 

### 29 under the most sensitive environmental conditions, but not intended to quantify O 3 

### 30 effects. A critical level was defined as “ the concentration of pollutant in the atmosphere 

### 31 above which direct adverse effects on receptors, such as plants, ecosystems, or materials 

### 32 may occur according to present knowledge ” ( UNECE, 1988 ) . The nature of the “ adverse 

### 33 effects ” was not specified in the ori ginal definition, which provided for different levels 

### 34 for different types of harmful effect (e.g., visible injury or loss of crop yield). There are 

### 35 also different critical levels for crops, forests, and semi

- natural vegetation. The caveat, 

### 36

“ according to pr esent knowledge ” is important because critical levels are not rigid; they 

### 37 are revised periodically as new scientific information becomes available. For example, 

### 38 the original critical level for O 3 specified concentrations for three averaging times, but 

### 39Draft

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- 105 September 2011 furt her research and debate led to the current critical level being stated as the cumulative 

### 1 exposure (concentration × hours) over a cutoff concentration of 40 ppb (AOT40) ( Fuhrer 

### 2 et al., 1997 )

. 

### 3
More recently in Europe, a decision was made to work towards a flux

- based approach 

### 4 (see section 9.5.4) for the critical levels ( “ Level II ” ), with the goal of modeling O 3 flux



### 5 effect relationships for three vegetation types: c rops, forests, and semi

- natural vegetation 

### 6 ( Grunhage and Jager, 2003 ) . Progress has been made in modeling flux ( U.S. EPA, 2006b ) 

### 7 and the Mapping Manual is being revised ( Ashmore et al., 2004a
, b ; Grennfelt, 2004 ; 

### 8 Karlsson et al., 2003 ) . The revisions may include a flux

- based approach for three crops: 

### 9 wheat, potatoes, and cotton. However, because of a lack of flux

- response data, a 

### 10 cumulative, cutoff concentration

- based (AOTx ) exposure index will remain in use for the 

### 11 near future for most crops and for forests and semi

- natural herbaceous vegetation 

### 12 ( Ashmore et al., 2004b ) 

### 13
In both the U.S. and Europe, the adequacy of these numerical summaries of exposure in 

### 14 relating biomass and yield changes have, for the most part, all been evaluated using data 

### 15 from studies not necessarily designed to compare one index to another ( Skarby et al., 

### 16 2004 ; Lee et al., 1989 ; Lefohn et al., 1988 ) . Very few studies in the U.S. have addressed 

### 17 this issue since the 2006 O 3 AQCD. McLaughlin et al. ( 2007a ) reported that the 

### 18 cumulative exposure index of AOT60 related well to reductions in growth rates at forest 

### 19 sites in the southern Appalachian Mountains. However, the authors did not report an 

### 20 analysis to compare multiple indices. Overall, given the available data from previous O 3 

### 21 AQCDs and the few recent studies, the cumulative, concentration

- weighted indices 

### 22 perform better than the peak or mean indices. It is still not possible, however, to 

### 23 distinguis h the differences in performance among the cumulative, concentration

- weighted 

### 24 indices. 

### 25

The main conclusions from the 1996 and 2006 O 3 AQCDs regarding an index based on 

### 26 ambient exposure are still valid. No information has come forth since the 2006 O 3 AQCD 

### 27 to alter those conclusions significantly. These key conclusions can be restated as follows: 

### 28

 O 3 effects in plants are cumulative; 

### 29

 higher O 3 concentrations appear to be more important than lower 

### 30 concentrations in eliciting a response; 

### 31

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

### 32 and 

### 33

 exposure indices that accumulate the O 3 hourly concentrations and 

### 34 preferentially weight the higher concentrations have better statistical fits to 

### 35 growth/yield response than do the mean and peak indi ces. 

### 36Draft

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- 106 September 2011 Following the 2006 criteria review process ( U.S. EPA, 2006b ) , the EPA proposed an 

### 1 alternative form of the secondary NAAQS for O 3 using a cumulative, concentration



### 2 weighted exposure index to protect vegetation from dam age (72 FR 37818 ) . The EPA 

### 3 considered two specific concentration

- weighted indices: the cutoff concentration 

### 4 weighted SUM06 and the sigmoid

- weighted W126 exposure index ( U.S. EPA, 2007b )

. 

### 5 These two indices performed equally well in predicting the exposure

- response 

### 6 relationships observed in the crop and tree seedlings studies ( Lee et al., 1989 ) . At a 

### 7 workshop convened to consider the science supporting these indices ( Heck and Cowling, 

### 8 1997 ) there was a consensus that these cumulative concentration

- weighted indices being 

### 9 considered were equally capable of predictin g plant response. Below are the definitions 

### 10 of the two cumulative index forms considered in the previous staff paper review ( U.S. 

### 11 EPA, 2007b ) : 

### 12

 SUM06: Sum of all hourly O 3 concentrations greater than or equal to 

### 13 0.06 ppm observed during a specified daily and seasonal time window (Figure 

### 14
9

- 9a). 

### 15

 W126: Sigmoidally weighted sum of all hourly O 3 concentrations o bserved 

### 16 during a specified daily and seasonal time window (Similar to Figure 9

- 9b). 

### 17 The sigmoidal weighting of hourly O 3 concentration is given in the equation 

### 18 below, where C is the hourly O 3 concentration in ppm: 

### 19

Equation 9

- 1

The SUM06 and W126 indices have a variety of relevant time windows that may be 

### 20 applied and are discussed in Section 9.5.3. 

### 21
It should be noted that there are some important differences between the SUM06 and 

### 22 W126. When considering the response of vegetation to ozone exposures repres ented by 

### 23 the threshold (e.g., SUM06) and non

- threshold (e.g., W126) indices, the W126 metric 

### 24 does not have a cut

- off in the weighting scheme as does SUM06 and thus it includes 

### 25 consideration of potentially damaging exposures below 60 ppb. The W126 metric al so 

### 26 adds increasing weight to hourly concentrations from about 40 ppb to about 100 ppb. 

### 27 This is unlike cut

- off metrics such as the SUM06 where all concentrations above 60 ppb 

### 28 are treated equally. This is an important feature of the W126 since as hourly 

### 29

conc entrations become higher, they become increasingly likely to overwhelm plant 

### 30 defenses and are known to be more detrimental to vegetation (See Section 9.5.3.1). 

### 31 C c 

## e w 126 

## 4403 1 1

−



## + =Draft

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- 107 September 2011 

### 9.5.3 Important Components of Exposure Indices

In the previous O 3 AQCDs it was established that higher hourly concentrations have 

### 1 greater effects on vegetation than lower concentrations ( U.S. EPA, 2006b
, 1996c )

. 

### 2 Further, it was determined that the diurnal and seasonal duration of exposure is imp ortant 

### 3 for plant response. Weighting of hourly concentrations and the diurnal and seasonal time 

### 4 window of exposure are the most important variables in a cumulative exposure index and 

### 5 will be discussed below. However, these variables must be taken in the co ntext of plant 

### 6 phenology, diurnal conductance rates, plant canopy structure, and detoxification 

### 7 mechanisms of vegetation as well as the climate and meteorology, all of which are 

### 8 determinants of plant response. These more specific factors will be discussed in the 

### 9 uptake and dose modeling section 9.5.4. 

### 10

9.5.3.1 Role of Concentration

The significant role of peak O 3 concentrations was established based on several 

### 11 experimental studies ( U.S. EP A, 1996c ) . Several studies ( Oksanen and Holopainen, 

### 12 2001 ; Yun and Laurence, 1999 ; Nussbaum et al., 1995 ) have added support for the 

### 13 important role that peak concentrations, as well as the pattern of occurrence, plays in 

### 14 plant response to O 3 . Oksanen and Holopainen ( 2001 ) found that the peak concentrations 

### 15 and the shape of the O 3 exposure (i.e., duration of the event) were important determinants 

### 16 of foliar injury in European white birch saplings, but growth reductions were found to be 

### 17 more related to total cumulative exposure. Based on air quality data from 10 U.S. cities, 

### 18 three 4

- week exposure treatments having the same SUM06 value were constructed by 

### 19 Yun and Laurence ( 1999 ) . The authors used different exposure regimes to explore effects 

### 20 of treatments with variable versus uniform peak occurrence during the exposure p eriod. 

### 21 The authors reported that the variable peak exposures were important in causing injury, 

### 22 and that the different exposure treatments, although having the same SUM06, resulted in 

### 23 very different patterns of foliar injury. Nussbaum et al. ( 1995 ) also found peak 

### 24 concentrations and the pattern of occurrence to be critical in determining the measured 

### 25 response. The authors recommended that to describe the effect on total forage yield, peak 

### 26 concentrations >0.11 ppm must be emphasized by using an AOT w ith higher threshold 

### 27 concentrations. 

### 28
A greater role for peak concentrations in effects on plant growth might be inferred based 

### 29 on air quality analyses for the southern California area ( Tingey et al., 2004 ; Lee et al., 

### 30 2003a ) . In the late 1960s and 1970s, extremely high O 3 concentration s had impacted the 

### 31 San Bernardino National Forest. However, over the past 20+ years, significant reductions 

### 32 in O 3 exposure have occurred ( Bytnerowicz et al., 2008 ; Lee et al., 2003a ; Lefohn and 

### 33Draft

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- 108 September 2011 Shadwick, 2000 ; Davidson, 1993 ) . An illustration of this improvement in air q uality is 

### 1 shown by the 37

- year history of O 3 air quality at the Crestline site in the San Bernardino 

### 2 Mountains (Figure 9

- 10) ( Lee et al., 2003a ) . Ozone exposure increased from 1963 to 

### 3 1979 concurrent with increased population and vehicular miles, followed by a decline to 

### 4 the present mirroring decreases in precursor emissions. The pattern in exposure was 

### 5 evident in various exposure indices including the cumulative concentration weighted 

### 6 (SUM06), as well as maximum peak event (1

- h peak), and the number of days having 

### 7 hourly averaged O 3 concentrations greater than or equal to 95 ppb. The number of days 

### 8 having hourly averaged O 3 concentrations greater than or equal to 95 ppb declined 

### 9 significantly from 163 days in 1978 to 103 days in 1997. The changes in ambient O 3 air 

### 10 quality for the Cre stline site were reflected in the changes in frequency and magnitude of 

### 11 the peak hourly concentration and the duration of exposure (Figure 9

- 10). Considering 

### 12 the role of exposure patterns in determining response, the seasonal and diurnal patterns in 

### 13 hourly O 3 concentration did not vary appreciably from year to year over the 37

- year 

### 14 period ( Lee et al., 2 003a )

. 

### 15
The potential importance of exposure to peak concentrations comes both from results of 

### 16 measures of tree conditions on established plots and from results of model simulations. 

### 17 Across a broad area of the San Bernardino National Forest, the Forest P est Management 

### 18 (FPM) method of injury assessment indicated an improvement in crown condition from 

### 19 1974 to 1988; and the area of improvement in injury assessment is coincident with an 

### 20 improvement in O 3 air quality ( Miller and Rechel, 1999 ) . A more recent analysis of 

### 21 forest changes in the San Bernardino National Forest, using an expanded network of 

### 22 monitoring sites, has verified significant changes in growth, mor tality rates, basal area, 

### 23 and species composition throughout the area since 1974 ( Arbaugh et al., 2003 ) . A model 

### 24 simulation of ponderosa pine growth over the 40

- year period in the San Bernardino 

### 25 National Forest showed a signi ficant impact of O 3 exposure on tree growth and indicates 

### 26 improved growth with reduced O 3 concentrations. This area has also experienced 

### 27 elevated N deposition and based on a number of environmental indicators, it appears that 

### 28 this area is experiencing N sa turation ( Fenn et al., 1996 ) . To account for this potential 

### 29 interaction, the model simulations were conducted under conditions of unlimited soil N. 

### 30 The actual interactions are not known. The improvement in growth over the years was 

### 31 attributed to improved air quality, but no distinction was made regarding the relative role 

### 32 of mid

- range and higher hourly concentrations, only that impro ved growth tracked 

### 33 decreasing SUM06, maximum peak concentration, and number of days of hourly O 3 

### 34 >95 ppb ( Tingey et al., 2004 ) . A summary of air quality data from 1980 to 2000 for the 

### 35 San Bernardino National Forest area o f the number of “ mid

- range ” hourly concentrations 

### 36 indicated no dramatic changes over this 20

- year period, ranging from about 1,500 to 

### 37 2,000 hours per year (Figure 9

- 11). There was a slow increase in the number of mid



### 38 range concentrations from 1980 to 1986, which corresponds to the period after 

### 39Draft

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- 109 September 2011 implementation of the air quality standard. Another sharper increase was observed in the 

### 1 late 1990s. This pattern of occurrence of mid

- range hourly concentrations suggests a 

### 2 lesser role for these concentration ranges compared to the higher values in either of the 

### 3 ground

- level tree injury observations of the model simulation of growth over the 40

- year 

### 4 period. 

### 5

6

7

Source: Used with permis sion from Elsevier Science Ltd. ( Lee et al., 2003a )

. Annual ROG and NO X emissions data for San Bernardino County were obtained from Alexis et al. ( 2001a ) and the California Air Resource Board ’ s emission inventory available at http://www.arb.ca.gov/aqd/aqdpage.htm ( Cal/EPA, 2010 )

. 

### Figure 9

- 10 Trends in May to September 12

- h SUM06 , peak 1

- h ozone concentration and number of daily e xceedances of 95 pp b for the Crestline site in 1963 to 1999 in relation to trends in mean daily maximum temperature for Crestline and daily reactive organic gases (ROG) and oxides of nitrogen (NO X 

### ) for San Bernardino County.Draft

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- 110 September 2011 

### Figure 9

- 11 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.

9.5.3.2 Diurnal and Seasonal Exposure

Diurnal Exposure
The diurnal patterns of maximal leaf/needle conductance and occurren ce of higher 

### 1 ambient concentrations can help determine which hours during the day over a season 

### 2 should be included in an exposure index. Stomatal conductance is species and phenology 

### 3 dependent and is linked to both diurnal and seasonal meteorological activ ity as well as to 

### 4 soil/site conditions (e.g., VPD, soil moisture). Daily patterns of leaf/needle conductance 

### 5 are often highest in midmorning, whereas higher ambient O 3 concentrations generally 

### 6 occurred in early to late afternoon when stomata were often par tially closed and 

### 7 conductances were lower. Total O 3 flux depends on atmospheric and boundary layer 

### 8 resistances, both of which exhibit variability throughout the day. Experimental studies 

### 9 with tree species demonstrated the decoupling of ambient O 3 exposure, peak occurrence, 

### 10Draft

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- 111 September 2011 and gas exchange, particularly in areas of drought ( Panek, 2004 ) . Several studies have 

### 1 suggeste d that ponderosa pine trees in the southern and northern Sierra Nevada 

### 2 Mountains may not be as susceptible to high O 3 concentrations as to lower 

### 3 concentrations, due to reduced needle conductance and O 3 uptake during the period when 

### 4 the highest concentratio ns occur ( Panek et al., 2002 ; Panek and Goldstein, 2001 ; Bauer et 

### 5 al., 2000 ; Arbaugh et al., 1998 ) . Panek et al. ( 2002 ) compared direct O 3 flux 

### 6 measurements into a canopy of ponderosa pine and demonstrated a lack of correlation of 

### 7 daily patterns of conductance and O 3 occurrence, especially in the late season drought 

### 8 period; the authors concluded that a consideration of climate or season was essential, 

### 9 especially considering the role of soil moisture and conductance/uptake. In contrast, 

### 10 Grulke et al. ( 2002 ) reported high conductance when O 3 concentrations were high in the 

### 11 same species, but under different growing site c onditions. The longer

- term biological 

### 12 responses reported by Miller and Rechel ( 1999 ) for ponderosa pine in the same region, 

### 13 and the general reduction in recent y ears in ambient O 3 concentrations, suggest that 

### 14 stomatal conductance alone may not be a sufficient indicator of potential vegetation 

### 15 injury or damage. Another consideration for the effect of O 3 uptake is the diurnal pattern 

### 16 of detoxification capacity of th e plant. The detoxification capacity may not follow the 

### 17 same pattern as stomatal conductance ( Heath et al., 2009 )

. 

### 18

The use of a 12

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

### 19 exposure was based primarily on evidence that the conditions for uptake of O 3 into the 

### 20 plant occur mainly during the daytime hours. In general, plants have the highest sto matal 

### 21 conductance during the daytime and in many areas atmospheric turbulent mixing is 

### 22 greatest during the day as well ( Uddling et al., 2010 ; U.S. EPA, 2006b ) . However, 

### 23 notable exceptions to maximum daytime conductance are cacti and other plants with 

### 24 crassulacean a cid metabolism (CAM photosynthesis) which only open their stomata at 

### 25 night. This section will focus on plants with C3 and C4 photosynthesis, which generally 

### 26 have maximum stomatal conductance during the daytime. 

### 27
Recent reviews of the literature reported tha t a large number of species had varying 

### 28 degrees of nocturnal stomatal conductance ( Caird et al., 2007 ; Dawson et al., 2007 ; 

### 29 Musselman and Minnick, 2000 ) . The reason for night

- time water loss through stomata is 

### 30 not well understood and is an area of active research (e.g. ( Christman et al., 2009 ; 

### 31 Howard et al., 2009 ) Night

- time stomata opening may be enhanced by O 3 damage that 

### 32 could result in loss of stomatal control, and less complete closure of stomata, than under 

### 33 low O 3 conditions ( Caird et al., 2007 ; Grulke et al., 2007b )

. In general, the rate of 

### 34 stomatal conductan ce at night is much lower than during the day ( Caird et al., 2007 )

. 

### 35 Atmospheric turbulence at night is also often low, which results in stable boundary layers 

### 36 and unfavorable conditions for O 3 uptake into vegetation ( Finkelstein et al., 2000 )

. 

### 37 Nevertheless, nocturnal turbulence does intermittently occur and may result in 

### 38Draft

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- 112 September 2011 nonnegligible O 3 flux int o the plants. In addition, plants might be more susceptible to O 3 

### 1 exposure at night than during the daytime, because of potentially lower plant defenses 

### 2 ( Heath et al., 2009 ; Loreto and Fares, 2007 ; Musselman et al., 2006 ; Musselman and 

### 3 Minnick, 2000 ) . For significant nocturnal stomatal flux and O 3 effects to occur, specific 

### 4 conditions must exist. A suscepti ble plant with nocturnal stomatal conductance and low 

### 5 defenses must be growing in an area with relatively high night

- time O 3 concentrations 

### 6 and appreciable nocturnal atmospheric turbulence. It is unclear how many areas there are 

### 7 in the U.S. where these con ditions occur. It may be possible that these conditions exist in 

### 8 mountainous areas of southern California, front

- range of Colorado ( Turnipseed et al., 

### 9 2009 ) and the Great Smoky Mountains of North Carolina and Tennessee. Tobiessen et al

. 

### 10 ( 1982 ) found that shade intolerant tree species showed op ening of stomata in the dark and 

### 11 did not find this in shade tolerant species. This may indicate shade intolerant trees may be 

### 12 more likely to be susceptible to O 3 exposure at night. More information is needed in 

### 13 locations with high night

- time O 3 to assess t he local O 3 patterns, micrometeorology and 

### 14 responses of potentially vulnerable plant species. 

### 15
Several field studies have attempted to quantify night

- time O 3 uptake with a variety of 

### 16 methods. However, many of these studies have not linked the night

- time flux to measured 

### 17 effects on plants. Grulke et al. ( 2004 ) showed that the stomatal conductance at night for 

### 18 ponderosa pine in the San Bernardino National Forest (CA) ranged from one tenth to one 

### 19 fourth that of maximum daytime stomatal conductance. In June, at a high

- elevation site, it 

### 20 was calculated that 11% of the total daily O 3 uptake of pole

- sized trees occurred at night. 

### 21 In late summer, however, O 3 uptake at night was negligible. However, this study did not 

### 22 consider the turbulent conditions at night. Finklestein et al. ( 2000 ) investigated O 3 

### 23 deposition velocity to forest canopies at three different sites. The authors foun d the total 

### 24 flux (stomatal and non

- stomatal) to the canopy to be very low during night

- time hours as 

### 25 compared to day

- time hours. However, the authors did note that higher nocturnal 

### 26 deposition velocities at conifer sites may be due to some degree of stomata l opening at 

### 27 night ( Finkelstein et al., 2000 ) . Work by Mereu et al. ( 2009 ) in Italy on Mediterranean 

### 28 species indicated that nocturnal uptake was from 10 to 18 % of total daily uptake during a 

### 29 weak drought and up to 24% as the drought became more pronounced. The proportion of 

### 30 night

- time uptake was greater during the drought due to decreases in daytime stomatal 

### 31 conductance ( Mereu et al., 2009 ) . In a study c onducted in California, Fares et al. ( Fares et 

### 32 al., 2011 ) reported that calculat ed mean percentages of nocturnal uptake were 5%, 12.5%, 

### 33 6.9% of total O 3 uptake for lemon, mandarin, and orange, respectively. In another recent 

### 34 study at the Aspen FACE site in Wisconsin, calculated leaf

- level stomatal O 3 flux was 

### 35 near zero from the night

- time hours of 8:00 p.m. to 5:00 a.m. ( Uddling et al., 2010 ) . This 

### 36 was likely due to low horizontal wind speed (>1 m/s) and low O 3 concentrations 

### 37 (<25 ppb) during those same night

- time hours (Figure 9

- 12). 

### 38Draft

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- 113 September 2011 Source: Used with permission from Elsevier Ltd ( Uddling et al., 2010 )

. 

### Figure 9

- 12 Mean diurnal

. (a) conductance through boundary layer and stomata (g bs 

### ), (b) Oz one concentration, and leaf

- level stomatal ozone flux without 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.

A few studies have tested the biological effects of night

- time O 3 exposure on vegetation 

### 1 in controlled chambers. Biomass of ponderosa pine seedlings was significantly reduced 

### 2 when seedlings were exposed to either daytime or nighttime episodic profiles ( Lee and 

### 3 Hogsett, 1999 ) . However, the biomass reductions were much greater with daytime peak 

### 4 concentrations than with nighttime peak concentrations. Similarly, birch cuttings grown 

### 5 in field chambers that were exposed to O 3 at night only, daytime only, and 24 hours 

### 6 showed similar reductions in biomass in night only and day only treatments. Birch 

### 7 seedling showed greater reductions in grow th in 24

- h exposures than those exposed to O 3 

### 8 at night or day only ( Matyssek et al., 1995 ) . Field mustard ( Brassica rapa ) plants 

### 9Draft

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- 114 September 2011 exposed to O 3 during the day or night showed little significant difference in the amounts 

### 1 of injury or reduced growth response to O 3 treatment, although the stomatal conductance 

### 2 was 70

- 80% lower at night ( Winner et al., 1989 ) . These studies show that effects can be 

### 3 seen with night

- time exposu res to O 3 but when atmospheric conditions are stable at night, 

### 4 it is uncertain how these exposures may affect plants and trees with complex canopies in 

### 5 the field. 

### 6

Seasonal Exposure
Vegetation across the U.S. has widely varying periods of physiological activity during the 

### 7 year due to variability in climate and phenology. In order for a particular plant to be 

### 8 vulnerable to O 3 pollution, it must have foliage and be physiologically active. Annual 

### 9 crops are typically grown for periods of two to three months. In contrast, perennial 

### 10 species may be photosynthetically active longer (up to 12 months each year for some 

### 11 species) depending on the species and where it is grown. In general, the period of 

### 12 maximum physiological activity and thus, potential O 3 uptake for vegetation coincides 

### 13 with some or all of the intra

- annual period defined as the O 3 season, which varies on a 

### 14

state

- by

- state basis (Figure 3

- 19). This is because the high temperature and high light 

### 15 conditions that typically promote the formation of troposph eric O 3 also promote 

### 16 physiological activity in vegetation. There are very limited exceptions to this pattern 

### 17 where O 3 can form in the winter in areas in the western U.S. with intense natural gas 

### 18 exploration ( Pinto, 2009 ) , but this is typically when plants are dormant and there is little 

### 19 chance of O 3 uptake. The selection of any single window of time f or a national standard 

### 20 to consider hourly O 3 concentrations represents a compromise, given the significant 

### 21 variability in growth patterns and lengths of growing season among the wide range of 

### 22 vegetation species that may experience adverse effects associate d with O 3 exposure. 

### 23

Various intra

- annual averaging and accumulation time periods have been considered for 

### 24 the protection of vegetation. The 2010 proposal for secondary O 3 standard (75 FR 2938, 

### 25 p. 3003) proposed to use the maximum consecutive 3

- month period within the O 3 season. 

### 26 The U.S. Forest Service and federal land managers have used a 24

- h W126 accumulated 

### 27 for 6 months from April through September ( 2000 ) . However, some monitors in the U.S. 

### 28 are operational for as little as four months and would not have enough data for a 6

- month 

### 29 seasonal window. The exposure period in the vast majority of O 3 exposure studies 

### 30 conducted in the U.S. has been much shorter than 6 months. Most of the crop studies 

### 31 done through NCLAN had exposure s less than three months with an average of 77 days. 

### 32 Open

- top chamber studies of tree seedlings, compiled by the EPA, had an average 

### 33 exposure of just over three months or 99 days. In more recent FACE experiments, 

### 34 SoyFACE exposed soybeans for an average of approximately 120 days per year and the 

### 35 Aspen FACE experiment exposed trees to an average of approximately 145 days per year 

### 36Draft

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- 115 September 2011 of elevated O 3 , which included the entire growing season at those particular sites. Despite 

### 1 the possibility that plants may be expo sed to ambient O 3 longer than 3 months in some 

### 2 locations, there is a lack of exposure experiments conducted for longer than 3 months. 

### 3

A

B

Figure 9

- 13 Maximum 3

- month, 12

- h W126 plotted against maximum 6

- month, 12

- h W126. Data are from the AQS and CASTNET monitors for the years 2008 and 2009. (A) W126, 3 month versus 6 month, 2008 (Pearson correlation = 0.99); (B) W126, 3 month versus 6 month, 2009 (Pearson correlation = 0.99).Highest 6 month W126

0 10 20 30 40 50 60 70 80 90 100 110

Highest 3 month W126 0 10 20 30 40 50 60 70Highest 6 month W126

0 10 20 30 40 50 60 70 80 90 100 110

Highest 3 month W126 0 10 20 30 40 50 60 70Draft

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- 116 September 2011 In an analysis of the 3

- and 6

- month maximum W126 values calculated for over 1,200 

### 1 AQS (Air Quality System) and CASTNET (Clean Air Status and Trend Network) EPA 

### 2 monitoring sites for the years 2008

- 2009, it was found that these 2 accumulation periods 

### 3 resulted in highly correlated metrics (Figure 9

- 13) . The two accumulation periods were 

### 4 centered on the yearly maximum for each monitoring site, and it is possible that this 

### 5 correlation would be weaker if the two periods were not temporally aligned. In the U.S., 

### 6 W126 cumulated over 3 months, and W126 cumula ted over 6 months are proxies of one 

### 7 another, as long as the period in which daily W126 is accumulated corresponds to the 

### 8 seasonal maximum. Therefore, it is expected that either statistic will predict vegetation 

### 9 response equally well. In other words, the s trength of the correlation between maximum 

### 10 3

- month W126 and maximum 6

- month W126 is such that there is no material difference 

### 11 in their predictive value for vegetation response. 

### 12

9.5.4 Ozone Uptake/Dose Modeling for Vegetation

Another approach for improving risk a ssessment of vegetation response to ambient O 3 is 

### 13 based on estimating the O 3 concentration from the atmosphere that enters the leaf (i.e., 

### 14 flux or deposition). Interest has been increasing in recent years, particularly in Europe, in 

### 15 using mathematically tr actable flux models for O 3 assessments at the regional, national, 

### 16 and European scale ( Matyssek et al., 2008 ; Paoletti and Manning, 2007 ; M and M, 2004 ; 

### 17 Emberson et al., 2000b ; Emberson et al., 2000a ) . Some researchers have claimed that 

### 18 using flux models can be used to better predict vegetation responses to O 3 than exposure



### 19 based approaches ( Matyssek et al., 2008 ) . However, other research has suggested that 

### 20 flux models do not predict vegetation respon ses to O 3 better than exposure

- based models, 

### 21 such as AOT40 ( Gonzalez

- Fernandez et al., 2010 ) . While some efforts have been made in 

### 22 the U.S. to calculate O 3 flux into leaves and canopies ( Fares et al., 2010a ; Turnipseed et 

### 23 al., 2009 ; Uddling et al., 2009 ; Bergweiler et al., 2008 ; Hogg et al., 2007 ; Grulke et al., 

### 24 2004 ; Grantz et al., 1997 ; Grantz et al., 1995 ) , little information has been published 

### 25 relating these fluxes to effects on vegetation. The lack of flux data in the U.S. and the 

### 26 lack of understanding of detoxification processes have made this technique less viable for 

### 27 vulnerability and risk assessments in the U.S. 

### 28
Flux calculations are data intensive and must be carefully implemented. Reducing 

### 29 uncertainties in flux estimates for areas with diverse surface or terrain conditions to 

### 30 wi thin ±50% requires “ very careful application of dry deposition models, some model 

### 31 development, and support by experimental observations ” ( Wesely and Hicks, 2000 ) . As 

### 32 an example, the annual average deposition velocity of O 3 among three nearby sites in 

### 33 similar vegetation was found to vary by ±10%, presumably due to terrain ( Brook et al., 

### 34 1997 ) . Moreover, the authors stated that the actual variation was even greater, because 

### 35Draft

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- 117 September 2011 stomatal uptake was unrealistically assumed to be the same among all sites, and flux is 

### 1 strongly influenced by stomatal conductance ( Brook et al., 1997 ; Massman and Grantz, 

### 2 1995 ; Fuentes et al., 1992 ; Reich, 1987 ; Leuning et al., 1979 ) . This uptake

- based 

### 3 approach to quantify the vegetation impact of O 3 requires inclusion of those factors that 

### 4 control the diurnal and seasonal O 3 flux to vegetation (e.g., climate patterns, species 

### 5 and/or vegetation

- typ e factors and site

- specific factors). The models have to distinguish 

### 6 between stomatal and non

- stomatal components of O 3 deposition to adequately estimate 

### 7 actual concentration reaching the target tissue of a plant to elicit a response ( Uddling et 

### 8 al., 2009 ) . Determining this O 3 uptake via canopy and stomatal conductance relies on 

### 9 models to predict flux and ultimately the “ effective ” flux ( Grunhage et al., 2004 ; 

### 10 Massman, 2004 ; Massman et al., 2000 )

. “ Effective flux ” has been defined as the balance 

### 11 between O 3 flux and detoxification processes ( Heath et al., 2009 ; Musselman and 

### 12 Massman, 1999 ; Grunhage and Haenel, 1997 ; Dammgen et al., 1993 ) . The time



### 13 integrated “ effective flux ” is termed “ effective dose. ” The uptake mechanisms and the 

### 14 resistances in this process, including stomatal conductance and biochemical defense 

### 15 mechanisms, are discussed below. The flux

- based index is the goal for the “ Level II ” 

### 16 critical level for assessment of O 3 risk to vegetation a nd ecosystems across Europe 

### 17 ( Ashmore et al., 2004a )

. 

### 18
An important cons ideration in both O 3 exposure and uptake is how the O 3 concentration 

### 19 at the top of low vegetation such as, crops and tree seedlings may be lower than the 

### 20 height at which the measurement is taken. Ambient monitor inlets in the U.S. are 

### 21 typically at heights of 3 to 5 meters. During daytime hours, the vertical O 3 gradient can 

### 22 be relatively small because turbulent mixing maintains the downward flux of O 3 . For 

### 23 example, Horvath et al. ( 1995 ) calculated a 7% decrease in O 3 goi ng from a height of 4 

### 24 meters down to 0.5 meters above the surface during unstable (or turbulent) conditions in 

### 25 a study over low vegetation in Hungary [see section AX3.3.2. of the 2006 O 3 AQ CD 

### 26 ( U.S. EPA, 2006b ) ]. There have been several s tudies indicating decreased O 3 

### 27 concentrations under tree canopies ( Kolb et al., 1997 ; Samuelson and Kelly, 1997 ; Joss 

### 28 and Graber, 1996 ; Fredericksen et al., 1995 ; Lorenzini and Nali, 1995 ; Enders, 1992 ; 

### 29 Fontan et al., 1992 ; Neufeld et al., 1992 ) . In contrast, for forests, measured data may 

### 30 underestimate O 3 concentration at the top of the canopy. The difference between 

### 31

me asurement height and canopy height is a function of several factors, the intensity of 

### 32 turbulent mixing in the surface layer and other meteorological factors, canopy height and 

### 33 total deposition to the canopy. Some researchers have used deposition models to estimate 

### 34 O 3 concentration at canopy

- top height based on concentrations at measurement height 

### 35 ( Emberson et al., 2000a )

. However, deposition models usually require meteorological 

### 36 data inputs that are not always available or well characterized across large spatial scales. 

### 37Draft

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- 118 September 2011 Soil moisture is a critical factor in controlling O 3 uptake through its effect on plant water 

### 1 status and stomatal conductance. In an attempt to relate uptake, soil moisture, and 

### 2 ambient air quality to identify areas of potential risk, available O 3 monitoring data for 

### 3 1983 to 1990 were used along with literature

- based seedling exposure

- respon se data from 

### 4 regions within the southern Appalachian Mountains that might have experienced O 3 

### 5 exposures sufficient to inhibit growth ( Lefohn et al., 1997 ) . In a small number of areas 

### 6 within the region, O 3 exposures and soil moisture availability were sufficient to possibly 

### 7 cause growth reductions in some O 3 sensitive species (e.g., black cherry). The 

### 8 conclusions were limited, however, because of the uncertainty in interpolating O 3 

### 9 exposures in many of the areas and because the hydrologic index used might not reflect 

### 10 actual water stress. 

### 11

The non

- stomatal co mponent of plant defenses are the most difficult to quantify, but 

### 12 some studies are available ( Heath et al., 2009 ; Barnes et al., 2002 ; Plochl et al., 2000 ; 

### 13 Chen et al., 1998 ; Massman and Grantz, 1995 ) . Massman et al. ( 2000 ) developed a 

### 14 conceptual model of a dose

- based index to determine how plant injury response to O 3 

### 15 relates to the traditional exposure

- based parameters. The index used t ime

- varying



### 16 weighted fluxes to account for the fact that flux was not necessarily correlated with plant 

### 17 injury or damage. The model applied only to plant foliar injury and suggested that 

### 18 application of flux

- based models for determining plant damage (yield or biomass) would 

### 19 require a better understanding and quantification of the relationship between injury and 

### 20 damage. 

### 21

9.5.5 Summary

Exposure indices are metrics that quantify exposure as it relates to measured plant 

### 22 damage (i.e., reduced growth). They are summary m easures of monitored ambient O 3 

### 23 concentrations over time intended to provide a consistent metric for reviewing and 

### 24 comparing exposure

- response effects obtained from various studies. No new information 

### 25 is available since 2006 that alters the basic conclusio ns put forth in the 2006 and 1996 O 3 

### 26 AQCDs. These AQCDs focused on the research used to develop various exposure indices 

### 27 to help quantify effects on growth and yield in crops, perennials, and trees (primarily 

### 28 seedlings). The performance of indices was comp ared through regression analyses of 

### 29 earlier studies designed to support the estimation of predictive O 3 exposure

- response 

### 30 models for growth and/or yield of crops and tree (seedling) species. 

### 31
Another approach for improving risk assessment of vegetation resp onse to ambient O 3 is 

### 32 based on determining the O 3 concentration from the atmosphere that enters the leaf (i.e., 

### 33 flux or deposition). Interest has been increasing in recent years, particularly in Europe, in 

### 34Draft

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- 119 September 2011 using mathematically tractable flux models for O 3 assessments at the regional, national, 

### 1 and European scale ( Matyssek et al., 2008 ; Paoletti and Manning, 2007 ; M and M, 2004 ; 

### 2 Emberson et al., 2000b ; Emberson et al., 2000a ) . While some efforts h ave been made in 

### 3 the U.S. to calculate O 3 flux into leaves and canopies ( Turnipseed et al., 2009 ; Uddling et 

### 4 al., 2009 ; Bergweiler et al., 2008 ; Hogg et al., 2007 ; Grulke et al ., 2004 ; Grantz et al., 

### 5 1997 ; Grantz et al., 1995 ) , little information has been published relating these fluxes to 

### 6 effects on vegetation. There is also concern that not all O 3 stomatal uptake results in a 

### 7 yield reduction, which depends to some degree on the amount of internal detoxification 

### 8 occurring with each particular species. Those species having high amounts of 

### 9 detoxification potential may, in fact, show little relationship between O 3 stomatal uptake 

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

### 11 lack of understanding of detoxification processes have made this technique less viable for 

### 12 vulnerability and risk assessments in the U.S. 

### 13

The main conclusions from the 1996 and 2006 O 3 AQCDs regarding indices based o n 

### 14 ambient exposure are still valid. These key conclusions can be restated as follows: 

### 15

 O 3 effects in plants are cumulative; 

### 16

 higher O 3 concentrations appear to be more important than lower 

### 17 concentrations in eliciting a response; 

### 18

 plant sensitivity to O 3 varie s with time of day and plant development stage; 

### 19 and 

### 20

 exposure indices that cumulate hourly O 3 concentrations and preferentially 

### 21 weight the higher concentrations have better statistical fits to growth/yield 

### 22 response data than do the mean and peak indices. 

### 23
Various weighting functions have been used, including threshold

- weighted (e.g., 

### 24 SUM06) and continuous sigmoid

- weighted (e.g., W126) functions. Based on statistical 

### 25 goodness

- of

- fit tests, these cumulative, concentration

- weighted indices could not be 

### 26 differe ntiated from one another using data from previous exposure studies. Additional 

### 27 statistical forms for O 3 exposure indices have been discussed in Lee et al. ( 1988b ) . The 

### 28 majority of studies published since the 2006 O 3 AQCD do not change earlier 

### 29 conclusions, including the importance of peak concentrations, and the duration and 

### 30 occurrence of O 3 exposures in altering plant growth and yield. 

### 31

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

### 32 cumulate and differentially weight the higher hourly average concentrations and also 

### 33 include the mid

- level values continue to offer the most defensible approach for use in 

### 34 developing respo nse functions and comparing studies, as well as for defining future 

### 35 indices for vegetation protection. 

### 36Draft

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- 120 September 2011 

## 9.6 Ozone Exposure

- Plant Response Relationships

9.6.1 Introduction

The adequate characterization of the effects of O 3 on plants for the purpose of setting air 

### 1 qual ity standards is contingent not only on the choice of the index used (i.e. SUM06, 

### 2 W126) to summarize O 3 concentrations (Section 9.5), but also on quantifying the 

### 3 response of the plant variables of interest at specific values of the selected index. The 

### 4
many factors that determine the response of plants to O 3 exposure have been discussed in 

### 5 previous sections. They include species, genotype and other genetic characteristics 

### 6 (Section 9.3), biochemical and physiological status (Section 9.3), previous and current 

### 7 exposure to other stressors (Section 9.4.8), and characteristics of the exposure itself 

### 8 (Section 9.5). Establishing a secondary air quality standard requires the capability to 

### 9 generalize those observations, in order to obtain predictions that are reliable enough 

### 10 under a broad variety of conditions, taking into account these factors. This section 

### 11 reviews results that have related specific quantitative observations of O 3 exposure with 

### 12 quantitative observations of plant responses, and the predictions of respo nses that have 

### 13 been derived from those observations through empirical models. 

### 14
For four decades, exposure to O 3 at ambient concentrations found in many areas of the 

### 15 U.S. has been known to cause detrimental effects in plants ( U.S. EPA, 2006b
, 1996b
, 

### 16 1984
, 1978a ) . Results publishe d after the 2006 O 3 AQCD continue to support this 

### 17 finding, and the following sections deal with the quantitative characterizations of the 

### 18 relationship, and what new insights may have appeared since 2006. Detrimental effects 

### 19 on plants include visible injury , decreases in the rate of photosynthesis, reduced growth, 

### 20 and reduced yield of marketable plant parts. Most published exposure

- response data have 

### 21 reported O 3 effects on the yield of crops and the growth of tree seedlings, and those two 

### 22 variables have been the focus of the characterization of ecological impacts of O 3 for the 

### 23 purpose of setting secondary air quality standards. In order to support quantitative 

### 24

mo deling of exposure

- response relationships, data should preferably include more than 

### 25 three levels of exposure, and some control of potential confounding or interacting factors 

### 26 should be present in order to model the relationship with sufficient accuracy. Le tting 

### 27 potential confounders, such as other stressors, vary freely when generating O 3 exposure



### 28 response data might improve the ‘ realism ’ of the data, but it also greatly increases the 

### 29 amount of data necessary to extract a clear quantitative description of t he relationship. 

### 30 Conversely however, experimental settings should not be so exhaustively restrictive as to 

### 31 make generalization outside of them problematic. During the last four decades, many of 

### 32 the studies of the effects of O 3 on growth and yield of plants have not included enough 

### 33 levels of O 3 to parameterize more than the simplest linear model. The majority of these 

### 34Draft

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- 121 September 2011 studies have only contrasted two levels, ambient and elevated, or sometimes three by 

### 1 adding carbon filtration in OTC studies, with little or n o consideration of quantitatively 

### 2 relating specific values of exposure to specific values of growth or yield. This is not to 

### 3 say that studies that did not include more than two or three levels of O 3 exposure, or 

### 4 studies that were conducted in uncontrolled environments, do not provide exposure



### 5 response information that is highly relevant to reviewing air quality standards. In fact, 

### 6 they can be essential in verifying the agreement between predictions obtained through the 

### 7 empirical models derived from experime nts such as NCLAN, and observations. The 

### 8 consensus of model predictions and observations from a variety of studies conducted in 

### 9 other locations, at other times, and using different exposure methods, greatly increases 

### 10 confidence in the reliability of both. Furthermore, if they are considered in the aggregate, 

### 11 studies with few levels of exposure or high unaccounted variability can provide 

### 12 additional independent estimates of decrements in plant growth and yield, at least within 

### 13 a few broad categories of exposu re. 

### 14
Extensive exposure

- response information on a wide variety of plant species has been 

### 15 produced by two long

- term projects that were designed with the explicit aim of obtaining 

### 16 quantitative characterizations of the response of such an assortment of crop p lants and 

### 17 tree seedlings to O 3 under North American conditions: the NCLAN project for crops, and 

### 18 the EPA National Health and Environmental Effects Research Laboratory, Western 

### 19 Ecology Division tree seedling project (NHEERL/WED). The NCLAN project was 

### 20 initi ated by the EPA in 1980 primarily to improve estimates of yield loss under field 

### 21 conditions and to estimate the magnitude of crop losses caused by O 3 throughout the U.S. 

### 22 ( Heck et al., 1991 ; Heck et al., 1982 ) . The cultural conditions used in the NCLAN studies 

### 23 approximated typical agronomic practices, and the primary objectives were: (1) to define 

### 24 relationships between yields of major agricultural crops and O 3 exposure as required to 

### 25 provide data necessary for econo mic assessments and development of O 3 NAAQS; (2) to 

### 26 assess the national economic consequences resulting from O 3 exposure of major 

### 27 agricultural crops; and (3) to advance understanding of cause

- and

- effect relationships that 

### 28 determine crop responses to pollut ant exposures. 

### 29
NCLAN experiments yielded 54 exposure

- response curves for 12 crop species, some of 

### 30 which were represented by multiple cultivars at several of 6 locations throughout the U.S. 

### 31 The NHEERL/WED project was initiated by EPA in 1988 with the same o bjectives for 

### 32 tree species, and yielded 49 exposure

- responses curves for multiple genotypes of 11 tree 

### 33 species grown for up to three years in Oregon, Michigan, and the Great Smoky 

### 34 Mountains National Park. Both projects used OTCs to expose plants to three t o five 

### 35 levels of O 3 . Eight of the 54 crop datasets were from plants grown under a combination 

### 36 of O 3 exposure and experimental drought conditions. Figure 9

- 14 through 9

- 17 

### 37 summarize some of the NCLAN and NHEERL/WED results. 

### 38Draft

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- 122 September 2011 It should be noted that data from FACE experiments might also be used for modeling 

### 1
exposure

- response. They only use two levels of O 3 (ambient concentration at the site and 

### 2 a multiple of it), but given that the value of both levels of exposure changes every year, 

### 3 and that they are typicall y run for many consecutive years, aggregating data over time 

### 4 produces twice as many levels of O 3 as there are years. As described in Section 9.2.4, 

### 5 FACE experiments seek to impose fewer constraints on the growth environment then 

### 6 OTCs. As a consequence, FAC E studies have to contend with larger variability, 

### 7 especially year

- to

- year variability, but the difference in experimental conditions between 

### 8 the two methodologies makes comparisons between their results especially useful. 

### 9
Growth and yield of at least one crop (soybean) has been investigated in yearly 

### 10 experiments since 2001 at a FACE facility in Illinois ( University of Illinois, 2010 ; 

### 11 Morgan et al., 2006 ) , however almost all analyses of SoyFACE published so far have 

### 12 been based on subsets of one or two years, and have only contrasted ambient versus 

### 13 elevated O 3 as categorical variables. They have not modeled the response of growth and 

### 14 yield to O 3 exposure continuously over the range of exposure values that have occurred 

### 15 over time. The only exception is a study by Betzelberger et al. ( 2010 ) , who used a linear 

### 16 regression model on data poo led over 2 years. Likewise, trees of three species (trembling 

### 17 aspen, paper birch, and sugar maple) were grown between 1998 and 2009 in a FACE 

### 18 experiment located in Rhinelander, Wisconsin ( Pregitzer et al., 2008 ; Dickson et al., 

### 19 2000 ) . The Aspen FACE experiment has provided extensive data on responses of trees 

### 20 beyond the seedling stage under long

- term exposure, and also on ecosystem

- level 

### 21 responses (Section 9.4), but the only attempt to use those data in a continuous model of 

### 22 the response of tree growth to O 3 exposur e ( Percy et al., 2007 ) suffered severe 

### 23 methodological problems, some of which are discussed in Section 9.6.3. Finally, one 

### 24 experiment was able to exploit a naturally occurring gradient of O 3 concentrations to fit a 

### 25 linear regression model to the growth of cottonwood ( Gregg et al., 2006
, 2003 ) . Factors 

### 26 such as genotype, soil type and soil moisture were under experimental control, and the 

### 27 authors were able to partition out the effects of potential confounders such as 

### 28 temperature, atmospheric N deposition, and ambient CO 2 . 

### 29
A serious difficulty in assessing results of exposure

- response research is the multiplicity 

### 30 of O 3 metrics that have been used in reporting. As described in Section 9.5, metrics that 

### 31 entail either weighting or thresholding of hourly values cannot be converted into one 

### 32 another, or into unweighted metrics such as hourly average. When co mputing O 3 

### 33 exposure using weighted or thresholded metrics, the computation of each metric has to 

### 34 start with the original hourly data. Comparisons of exposure

- response models can only be 

### 35 made between studies that used the same metric, and the value of expos ure at which a 

### 36 given plant response is expected using one metric of exposure cannot be exactly 

### 37 converted to another metric. Determining the exposure value at which an effect would be 

### 38Draft

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- 123 September 2011 observed in a different metric can only be accomplished by first computin g the 

### 1 experimental exposures in this metric from the hourly data, then estimating (fitting) 

### 2 model coefficients again. This problem is irremediable, although useful comparisons 

### 3 might be made using categorical exposures such as ‘ current ambient exposure ’ or ‘ 2050 

### 4 projected exposure ’ , which can serve as a common reference for quantitative values 

### 5 expressed in various metrics. Studies that contained growth or yield exposure

- response 

### 6 data at few levels of exposure, and/or using metrics other than W126 are summari zed in 

### 7 Tables 9

- 18 and 9

- 19. 

### 8

9.6.2 Estimates of Crop Yield Loss a nd Tree Seedling Biomass Loss in the 1996 and 2006 Ozone AQCDs

The 1996 and 2006 O 3 AQCDs relied extensively on analyses of NCLAN and 

### 9 NHEERL/WED by Lee et al. ( 1994 ; 1989
, 1988b
, 1987 ) , Hogsett et al. ( 1997 ) , Lee and 

### 10 Hogsett ( 1999 ) , Heck et al. ( 1984 ) , Rawlings and Cure ( 1985 ) , Lesser et al. ( 1990 ) , and 

### 11 Gumpertz and Rawlings ( 1992 ) . Those analyses concluded that a three

- parameter 

### 12 Weibull model

– 

### 13

β



### η 

# α        

−



# = 126 W



# e Y

Equation 9

- 2
is the most appropriate model for the response of absolute yield and growth to O 3 

### 14 exposure, because of the interpretability of its parameters, its flexibility (given the small 

### 15 number of parameters), and its tractability for estimation. In addition, removing the 

### 16 intercept α results in a model of relati ve yield (yield relative to [yield at exposure=0]) 

### 17 without any further reparameterization. Formulating the model in terms of relative yield 

### 18 or relative yield loss (yield loss=[1

− relative yield]) is essential in comparing exposure



### 19 response across species, genotypes, or experiments for which absolute values of the 

### 20 response may vary greatly. In the 1996 and 2006 O 3 AQCDs, the two

- parameter model 

### 21 of relative yield was used in deriving common models for multiple species, multiple 

### 22 genotypes within species, and multiple locations. 

### 23
Given the disparate species, genotypes, and locations that were included in the NCLAN 

### 24 and NHEERL/WED projects, and in the absence of plausible distributional assumptions 

### 25 with respect to those variables, a three step process using robust methods was used to 

### 26 obtain parameter estimates that could be generalized. The models that were derived for 

### 27 each species or group of species were referred to as median composite functions. In the 

### 28 first step, the three parameters of the Weibull model were c omputed for absolute yield or 

### 29Draft

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- 124 September 2011 biomass data from each NCLAN and NHEERL/WED experiment (54 crop datasets and 

### 1 49 tree seedling datasets), using nonlinear regression. When data were only available for 

### 2 three levels of exposure because of experimental problems, the shape parameter β was 

### 3 constrained to 1, reducing the model to an exponential decay model. In the second step, α 

### 4 was dropped, and predicted values of relative yield or biomass were then computed for 

### 5
12

- hr W126 exposures between 0 and 60 ppm

- h. At each o f these W126 exposure values, 

### 6 the 25th, 50th, and 75th percentiles of the response were identified among the predicted 

### 7 curves of relative response. For example, for the 34 NCLAN studies of 12 crop species 

### 8 grown under non

- droughted conditions for a complete cropping cycle (Figure 9

- 14), the 3 

### 9 quartiles of the response were identified at every integer value of W126 between 0 and 

### 10 60. The third step fitted a two

- parameter Weibull model to those percentiles, yielding the 

### 11 median composite function for the relativ e yield or biomass response to O 3 exposure for 

### 12 each grouping of interest (e.g., all crops, all trees, all datasets for one species), as well as 

### 13 composite functions for the other quartiles. In the 1996 and 2006 O 3 AQCDs this 

### 14 modeling of crop yield loss and tree seedling biomass loss was conducted using the 

### 15 SUM06 metric for exposure. This section updates those results by using the 12

- hr W126 

### 16 as proposed in 2007 (72 FR 37818 ) and 2010 (75 FR 2938, p. 3003). Figures 9

- 14 

### 17 through 9

- 17 present quantiles of predicted relative yield or biomass loss at seven values 

### 18 of the 12

- h W126 for some representative groupings of NCLAN and NHEERL/WED 

### 19 results. Tables 9

- 10 through 9

- 12 give the 90

- day 12

- h W126 O 3 exposure values at 

### 20 whic h 10 and 20% yield or biomass losses are predicted in 50 and 75% of crop or tree 

### 21 species using the composite functions. 

### 22Draft

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- 125 September 2011 Source of Weibull parameters: Lee and Hogsett ( 1996 )

. Quantiles of the predicted relative yield loss at 7 values of 12

- hour 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 cy cle.


### Figure 9

- 14 Quantiles of predicted relative yield loss for 34 NCLAN crop experiments. 10 th Pctile 25 th Pctile 50 th Pctile 75 th Pctile 90 th Pctile

0 10 20 30 40 50 60 70 80 90 100

0 10 20 30 40 50 60Percent Yield Loss (%)

12 hr W126 (ppm

- hr) 34 crop datasetsDraft

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- 126 September 2011 Source of Weibull parameters: Lee and Hogsett ( 1996 )

. 

### Figure 9

- 15 Quantile s of predicted relative yield loss for 4 crop species in NCLAN experiments. Quantiles 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 f or the full duration of 1 cropping cycle. The number of studies available for each species is indicated on each plot. 25 th Pctile 50 th Pctile 75 th Pctile

0 10 20 30 40 50 60 70 80 90 100

0 10 20 30 40 50 60 7 Wheat datasets

25 th Pctile 50 th Pctile 75 th Pctile

0 10 20 30 40 50 60 70 80 90 100

0 10 20 30 40 50 60Percent Yield Loss (%) 11 Soybean datasets

50th Pctile

0 10 20 30 40 50 60 70 80 90 100

0 10 20 30 40 50 60
12 hr W126 (ppm

- hr) 2 Corn datasets

25 th Pctile 50 th Pctile 75 th Pctile

0 10 20 30 40 50 60 70 80 90 100

0 10 20 30 40 50 60Percent Yield Loss (%)

12 hr W126 (ppm

- hr 5 Cotton datasetsDraft

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- 127 September 2011 Source of Weibull parameters: Lee and Hogsett ( 1996 )

. 

### Figure 9

- 16 Quantiles of predicted relative biomass loss for 49 tree species in NHEERL/WED experiments. Quantiles of the predicted relative above

- ground biomass loss at 7 values of 12

- h W126 for 49 Weibull curves estimated using nonlinear regression o n 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. 10 th Pctile 25 th Pctile 50 th Pctile 75 th Pctile 90 th Pctile

0 10 20 30 40 50 60 70 80 90 100

0 10 20 30 40 50 60Percent Biomass Loss (%)

90 day 12 hr W126 (ppm

- hr) 49 tree seedlings datasetsDraft

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- 128 September 2011 Source of Weibull parameters: Lee and Hogsett ( 1996 )

. 

### Figure 9

- 17 Quantiles of predicted relative biomass loss for 4 tree species in NHEERL/WED experiments. Quantiles of the predicted relative above

- ground biomass loss at 7 exposure values of 12

- h W126 for Weibull curves estimated usin g nonlinear regression 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. 25 th Pctile 50 th Pctile 75 th Pctile

0 10 20 30 40 50 60 70 80 90 100

0 10 20 30 40 50 60Percent Biomass Loss (%) 14 Aspen datasets

25 th Pctile 50 th Pctile 75 th Pctile

0 10 20 30 40 50 60 70 80 90 100

0 10 20 30 40 50 60 11 Ponderosa pine datasets

50 th Pctile 0 10 20 30 40 50 60 70 80 90 100

0 10 20 30 40 50 60Percent Biomass Loss (%)

90 day 12 hr W126 (ppm

- hr) 7 Douglas fir datasets

50 th Pctile

0 10 20 30 40 50 60 70 80 90 100

0 10 20 30 40 50 60
90 day 12 hr W126 (ppm

- hr) 3 Tulip poplar datasetsDraft

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- 129 September 2011 

### Table 9

- 9 Ozone exposures at which 10 and 20% yield 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 standar dized 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 34 curves Relative yield=exp(

- (W126/104.82)**1.424) 22 37 Model for the 75th Percentile of 34 curves Relative yield=exp(

- (W126/78.12)**1.415) 16 27

Source of parameters for the 34 curves: Lee and Hogsett ( 1996 )



### Table 9

- 1 0 Ozone exposures at which 10 and 20% yield lo ss is predicted for 50 and 75% o f 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

Source of parameters for the 16 curves: Lee and Hogsett ( 1996 )Draft

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- 130 September 2011 

### Table 9

- 1 1 Ozone 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 (ppm

- h) 90 day 12 h W126 for 20% yield loss (ppm

- h)

Model for the 50th Percentile of 49 curves
Relative yield=exp(

- (W126/131.57)**1.242) 21 39

Model for the 75th Percentile of 49 curves

Relative yield=exp(

- (W126/65.49)**1.500) 15 24

Source of parameters for the 49 curves: Lee and Hogsett ( 1996 )



### 9.6.3 Validation of 1996 and 2006 Ozone AQCD Models and Methodology Using the 90 day 12

- h W126 and Current FACE Data

Since the completion of the NCLAN and NHEERL/WED projects, almost no studies 

### 1 have been published that could provide a basis for estimates of exposure

- response that 

### 2 can be compared to those of the 1996 and 2006 O 3 AQCDs. Most experiments, 

### 3 regardless of exposure methodology, include only two levels of exposure. In addition, 

### 4 very few studies have included measurements of exposure using the W126 metric, or the 

### 5 hourly O 3 concentration data that would allow computing ex posure using the W126. Two 

### 6 FACE projects, however, were conducted over multiple years, and by adding to the 

### 7 number of exposure levels over time, may support independent model estimation and 

### 8 prediction using the same model and the same robust process as sum marized in Section 

### 9 9.6.2. Hourly O 3 data were available from both FACE projects. 

### 10
The SoyFACE project is situated near Champaign, IL, and comprises 32 octagonal rings 

### 11 (20m

- diameter), 4 of which in a given year are exposed to ambient conditions, and 4 of 

### 12 wh ich are exposed to elevated O 3 as a fixed proportion of the instantaneous ambient 

### 13 concentration ( Betzelberger et al., 2010 ; University of Illinois, 2010 ; Morgan et al., 2006 ; 

### 14 Morgan et al., 2004 ) . Since 2002, yield data have been collected for up to 8 genotypes of 

### 15 soybean grown in subplots within each ring. The Aspen FACE project is situated in 

### 16 Rhinelander, WI, and comprises 12 rings (30m

- diamet er), 3 of which are exposed to 

### 17 ambient conditions, and 3 of which are exposed to O 3 as a fixed proportion of the 

### 18 instantaneous ambient concentration ( Pregitzer et al., 2008 ; Karnosky et al., 2005 ; 

### 19 Dickson et al., 2000 ) . In the summer of 1997, half the area of each ring wa s planted with 

### 20 small (five to seven leaf sized) clonally propagated plants of five genotypes of trembling 

### 21Draft

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- 131 September 2011 aspen, which were left to grow in those environments until 2009. Biomass data are 

### 1 currently available for the years 1997

- 2005 ( King et al., 2005 ) . Ozone exposure in these 

### 2 two FACE projects can be viewed as a categorical variable with two levels: ambient, and 

### 3 elevated. However, this overlooks the facts that yearly ambient and elevated exposure 

### 4 b oth vary with every year, and that the proportionality between them also changes. This 

### 5 change has two sources: first, the dispensing of O 3 into the elevated exposure rings varies 

### 6 from the proportionality set point to some extent, and for SoyFACE, the set p oint 

### 7 changed between years. Second, the proportionality does not propagate predictably from 

### 8 the hourly data to the yearly value when using threshold or concentration

- weighted 

### 9 cumulative metrics (such as AOT40, SUM06 or W126). Hourly average elevated 

### 10
exposu res that are, for example, a constant 1.5 times greater than ambient do not result in 

### 11 AOT40, SUM06 or W126 values that are some constant multiple of the ambient values of 

### 12 those indices. The greater the fraction of elevated hourly values that are above the 

### 13 threshold or heavily weighted, compared to the fraction of hourly ambient values that are, 

### 14 the greater the difference between ambient and elevated yearly exposure, as measured 

### 15 using weighted cumulative indices. When elevated exposure is a multiple of ambie nt 

### 16 hourly intervals, the number of hours for which elevated exposure meets the threshold for 

### 17 inclusion can vary widely, even though the hourly mean for the year retains the 

### 18 proportionality. As a consequence, the number of exposure levels in multi

- year 

### 19
expe riments is twice the number of years. In the case of SoyFACE for the period between 

### 20 2002 and 2008, ambient exposure in the highest year was approximately equal to elevated 

### 21 exposure in the lowest year, with 14 levels of O 3 exposure evenly distributed from l owest 

### 22 to highest. The particular conditions of the Aspen FACE experiment resulted in 12 

### 23 exposure levels between 1998 and 2003, but they were not as evenly distributed between 

### 24 minimum and maximum over the 6

- year period. 

### 25
There are necessary differences in th e modeling of exposure

- response in annual plants 

### 26 such as soybean, and in perennial plants such as aspen trees, when exposure takes place 

### 27 over multiple years. In annual plants, responses recorded at the end of the life cycle, i.e., 

### 28 yearly, are analyzed in r elationship to that year ’ s exposure. Yield of soybeans is affected 

### 29 by exposure during the year the crop was growing, and a new crop is planted every year. 

### 30 Thus an exposure

- response relationship can be modeled from yearly responses matched 

### 31 to yearly exposur es, with those exposure

- response data points having been generated in 

### 32 separate years. For perennial organisms, which are not harvested yearly and continue to 

### 33 grow from year to year, such pairing of exposure and response cannot be done without 

### 34 accounting fo r time. Not only does the size of the organism at the beginning of each year 

### 35 of exposure increase, but size is also dependent on the exposure from previous years. 

### 36 Therefore the relationship of response and exposure must be analyzed either one year at a 

### 37 tim e, or by standardizing the response as a yearly increment relative to size at the 

### 38 beginning of each year. Furthermore, the relevant measurement of exposure is 

### 39Draft

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- 132 September 2011 cumulative, or cumulative yearly average exposure, starting in the year exposure was 

### 1 initiated, u p to the end of the year of interest. When analyzing the growth of trees over 

### 2 several years, it would be evidently incorrect to pair the exposure level in every discrete 

### 3 year with absolute size of the trees that year, and posit a direct relationship betwee n them, 

### 4 without taking increasing age into consideration. In the Aspen FACE experiment, for 

### 5 example, one could not establish an exposure

- response relationship by matching 

### 6
12 yearly exposures and 12 yearly tree sizes, while disregarding age as if size did n ot also 

### 7 depend on it. This is the basis of the 2007 study of Aspen FACE data by Percy et al. 

### 8 ( 2007 ) , which compares the size of trees of various ages as if they were all the same age, 

### 9 and was therefore not informative. 

### 10

9.6.3.1 Comparison of NCLAN

- Based Prediction and SoyFACE Data

For this ISA, EPA conducted a comparison between yield of soybean as predicted by the 

### 11 composite function three

- step process (Section 9.6.2) using NCLAN data, and 

### 12 observations of yield in SoyFACE. The median composite function for relative yield was 

### 13 derived for the 11 NCLAN soybean Weibull functions for non

- droughted studies, and 

### 14 comparisons between the predictions of the median composite and SoyFACE 

### 15 observations were conducted as follows. 

### 16
For the years 2007 and 2008, SoyFACE yield data were available for 7 and 6 genotypes, 

### 17 respectively. The EPA used those data to compare the relative change in yield observed 

### 18 in SoyFACE in a given year between ambient O 3 and elevated O 3 , versus the relative 

### 19 change in yield predicted by the NCLAN

- based median composite functi on between 

### 20 those same two values of O 3 exposure. The two parameter median composite function for 

### 21 relative yield of soybean based on NCLAN data was used to predict yield response at the 

### 22 two values of exposure observed in SoyFACE in each year, and the change between yield 

### 23 under ambient and elevated was compared to the change observed in SoyFACE for the 

### 24 relevant year (Table 9

- 1 2 ). This approach results in a direct comparison of predicted 

### 25 versus observed change in yield. Because the value of relative response b etween any two 

### 26 values of O 3 exposure is independent of the intercept α, this comparison does not require 

### 27 prediction of the absolute values of the responses. 

### 28
Since comparisons of absolute values might be of interest, the predictive functions were 

### 29 also scale d to the observed data: SoyFACE data were used to compute an intercept α 

### 30 while the shape and scale parameters (β and η) were held at their value in the NCLAN 

### 31 predictive model. This method gives a comparison of prediction and observation that 

### 32 takes all the observed information into account to provide the best possible estimate of 

### 33Draft

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- 133 September 2011 the intercept, and thus the be st possible scaling (Table 9

- 13 and Figure 9

- 18). For the 

### 1 comparison of NCLAN and SoyFACE, this validation was possible for 2007 and 2008, 

### 2 where data f or 7 and 6 soybean genotypes, respectively, were available. The median 

### 3 composite function for relative yield was derived for the 11 NCLAN soybean Weibull 

### 4 functions for nondroughted studies, and the values of median yield under ambient 

### 5 exposure at SoyFACE i n 2007 and 2008 were used to obtain an estimate of the intercept 

### 6 α for the NCLAN median function in each of the two years. 

### 7

Table 9

- 1 2 presents the results of ambient/elevated relative yield comparisons between 

### 8 the NCLAN

- derived predictions and SoyFACE obse rvations. Table 9

- 1 3 and figure 9

- 18 

### 9 present the results of comparisons between NCLAN

- derived predictions and SoyFACE 

### 10 observations of yield, with the predictive function scaled to provide absolute yield values. 

### 11

Table 9

- 1 2 Comparison between change in yiel d observed in the SoyFACE experiment between elevated and ambient ozone, and change 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) observed at SoyFACE Yie ld in Elevated O 3 Relative to Ambient O 3 (%)
Ambient Elevated Predicted by NCLAN Observed at SoyFACE 2007 4.39 46.23 75 76 2008 3.23 28.79 85 88



### Table 9

- 1 3 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 absolute yield model with intercept scaled to SoyFACE data)

Year 90

- day 12

- h W126 (ppm

- h) observed at SoyFACE Yield predicted by NCLAN (g/m 2 ) Yield observed at SoyFACE (g/m 2 )
Ambient Elevated Ambient Elevated Ambient Elevated 2007 4.39 46.23 309.2 230.6 305.2 230.6 2008 3.23 28.79 350.3 298.2 344.8 304.4Draft

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- 134 September 2011 Source of data: Betzelberger et al. ( 2010 ) ; Morgan et a l. ( 2006 ) ; Lee and Hogsett ( 1996 )

. Note: Black dots are the median of 7 or 6 soybean genotypes in SoyFACE (2007, 2008); bars are IQR for genotypes; dashed line is median composite model for 11 studie s in NCLAN.


### Figure 9

- 18 Comparison of yield observed in SoyFACE experiment in a given year with yield predicted by the median composite function based on NCLAN.

Finally, a composite function for the 25th, 50th, and 75th percentiles was developed from 

### 1 SoyFACE annual yield data, and compared to the NCLAN

- based function. The process 

### 2 described in Section 9.6.2 was applied to SoyFACE data for individual genotypes, 

### 3 aggregated over the years during which each was grown; one genotype from 2003 to 

### 4 2007, and six genotypes in 2007 and 2008. First, the three parameter Weibull model 

### 5 described in Section 9.6.2 was estimated using nonlinear regression on exposure

- yield 

### 6 data for each genotype separately, over the years for which data were available, totaling 

### 7

seven curv es. The 25th, 50th, and 75th percentiles of the predicted values for the two 

### 8 parameter relative yield curves were then identified at every integer of W126 between 0 

### 9 and 60, and a two

- parameter Weibull model estimated by regression for the three 

### 10 quartiles. The comparison between these composite functions for the quartiles of relative 

### 11 yield loss in SoyFACE and the corresponding composite functions for NCLAN is 

### 12 presented in Figure 9

- 19. 

### 13 0 50 100 150 200 250 300 350 400

0 10 20 30 40 50 60 70Yield (g/m2)

90 day 12 hr W126 (ppm

- hr) 2007, 7 genotypes

0 50 100 150 200 250 300 350 400

0 10 20 30 40 50 60 70Yield (g/m2)

90 day 12 hr W126 (ppm

- hr) 2008, 6 genotypesDraft

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- 135 September 2011 Source of data: Betzelberger et al. ( 2010 ) ;Morgan et al. ( 2006 ) ; Lee and Hogsett ( 1996 )

. 

### Figure 9

- 19 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.

As seen in Tables 9

- 13 and 9

- 14, and in Figure 9

- 18, the agreement between predictions 

### 1 based on NCLAN data and SoyFACE observations was notably close in single

- year 

### 2 comparisons. Together with the very high agreement between median composite models 

### 3 for NCLAN and SoyFACE (Figure 9

- 19), it provides very strong mutual confirmation of 

### 4 those two projects ’ results with respect to the response of yield of soybeans to O 3 

### 5 exposure. It is readily apparent from these results that the methodology described in 

### 6 Section 9.6.2 for obtaining predictions of yield or yield loss from NCLAN data is 

### 7 strongly validated by SoyFACE results. As described in Section 9.2, the exposure 

### 8 technologies used in the two projects were in sharp contrast, specifically with respect to 

### 9 the balance each achieved between control of potential interacting factors or confounders, 

### 10 and fidelity to natural conditions. The comparisons that EPA conducted the refore 

### 11 demonstrate that the methodology used in developing the composite functions is resistant 

### 12 to the influence of nuisance variables and that predictions are reliable. They may also 

### 13 suggest that the aspects in which the two exposure technologies differ h ave less influence 

### 14
on exposure

- response than initially supposed. These results are also in agreement with 

### 15 comparative studies reviewed in 9.2.6. 

### 16 FACE 75 th Pctile 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 90 100

0 10 20 30 40 50 60 70Percent Yield Loss (%)

90 day 12 hr W126 (ppm

- hr)Draft

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- 136 September 2011 

### 9.6.3.2 Comparison of NHEERL/WED

- Based Prediction of Tree Biomass Response and Aspen FACE Data

EPA also conducted two comparisons between prediction of above

- ground biomass loss 

### 1 based on NHEERL/WED results and observations from Aspen FACE. The median 

### 2 composite function was developed from NHEERL/WED data for 11 studies that used 

### 3 wild

- type seedlings of aspen as well as four clonally propagated genotypes. All plants 

### 4 were grown in OTCs for one growing season before being destructively harvested. Aspen 

### 5 FACE data were from clonally propagated trees of five genotypes grown from 1998 to 

### 6 2003, with above

- ground biomass calculated u sing allometric equations derived from 

### 7 data for trees harvested destructively in 2000 and 2002 ( King et al., 2005 )

. 

### 8
The two parameter median composite function for relative biomass was used to pr edict 

### 9 biomass response under the observed elevated exposure, relative to its value under 

### 10 observed ambient exposure, for each separate year of Aspen FACE. EPA first compared 

### 11 Aspen FACE observations of the change in biomass between ambient and elevated 

### 12
expos ure with the corresponding prediction at the same values of exposure. Comparisons 

### 13 between observed and predicted absolute biomass values were then conducted for each 

### 14 year by scaling the predictive function to yearly Aspen FACE data as described for 

### 15 soybean data in Section 9.6.3.1. In all cases, yearly 90 day 12

- hour W126 values for 

### 16 Aspen FACE were computed as the cumulative average from the year of planting up to 

### 17 the year of interest. A comparison of composite functions between NHEERL/WED and 

### 18 Aspen FACE, si milar to the one performed for NCLAN and SoyFACE, was not possible: 

### 19 as discussed in the introduction to Section 9.6, the pairing of 12 exposure values from 

### 20 separate years and 12 values of biomass cannot be the basis for a model of exposure



### 21 response, becaus e the trees continued growing for the six

- year period of exposure. 

### 22 Because the same trees were used for the entire duration, and continued to grow, data 

### 23 could not be a ggregated over years. Table 9

- 14 presents the results of ambient/elevated 

### 24 relative biomas s comparisons between the NHEERL/WED

- derived predictions and 

### 25 Aspen FACE observations. Table 9

- 1 5 and Figure 9

- 20 present the results of the 

### 26 comparison between NHEERL/WED

- derived predictions and Aspen FACE observations 

### 27 for absolute biomass, using Aspen FACE data to scale the NHEERL/WED

- derived 

### 28 composite function. 

### 29Draft

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- 137 September 2011 

### Table 9

- 1 4 Comparison between change in above

- ground biomass elevated and ambient ozone in Aspen FACE experiment in 6 year, and change predicted at the same values of ozone by the median composite f unction for NHEERL/WED (two

- parameter relative biomass model)

Table 9

- 15 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 with intercept scaled to Aspen FACE data)

Year 90 day 12

- h W126 (ppm

- h) Cumulative Average observed at Aspen FACE Biomass Predicted by NHEERL/WED (g/m 2 ) Biomass Observed at Aspen FACE (g/m 2 )

Ambient Elevated Ambient Elevated Ambient Elevated 1998 3.19 30.08 276.0 203.2 274.7 204.9 1999 2.61 33.85 958.7 668.3 955.3 673.3 2000 2.43 30.16 1382.4 1022.8 1400.3 998.6 2001 2.55 31.00 1607.0 1173.7 1620.7 1154.9 2002 2.51 30.27 2079.0 1532.1 2125.9 1468.41 2003 2.86 29.12 2640.1 1981.2 2695.2 1907.8 Year 90

- day 12

- h W126 (ppm

- h) Cumulative Average observed at Aspen FACE Above

- Ground Biomass in Elevated O 3 relative To Ambient O 3 (%)
Ambient Elevated Predicted by NHEERL/WED Observed at Aspen FACE 1998 3.19 30.08 74 75 1999 2.61 33.85 70 70 2000 2.43 30.16 74 71 2001 2.55 31.00 73 71 2002 2.51 30.27 74 69 2003 2.86 29.12 75 71Draft

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- 138 September 2011 Source of data: King et al. ( 2005 ) , Lee and Hogsett ( 1996 )

. Note: Black dots are aspen biomass/m 2 for 3 FACE rings filled with an assemblage of 5 clonal genotypes of aspen at Aspen FACE; bars are SE for 3 rings; dashed line is median composite model for 4 c lonal genotypes and wild

- type seedlings in 11 NHEERL/WED 1

- year OTC studies.


### Figure 9

- 20 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.

As in the comparisons between NCLAN and SoyFACE, the agreement between 

### 1 predictions based on NHEERL/WED data and Aspen FACE observations was very close. 

### 2 The results of the two projects strongly reinforce each other with respect to the response 

### 3 of aspen biomass to O 3 exposure. The methodology used for obtaining the median 

### 4 composite function is shown to be capable of deriving a predictive model despite 

### 5 potential confounders, and despite the added measurement error that is expected from 

### 6 calculating biomass using allo metric equations. In addition, the function based on 

### 7

one year of growth was shown to be applicable to subsequent years. 

### 8
The results of experiments that used different exposure methodologies, different 

### 9 genotypes, locations, and durations converged to the same values of response to O 3 

### 10 exposure for each of two very dissimilar plant species, and predictions based on the 

### 11 earlier experiments were validated by the data from current ones. However, in these 

### 12 comparisons, the process used in establishing predictive functions involved aggregating 

### 13 data over variables such as time, locations, and genotypes, and the use of a robust statistic 

### 14 1998 1999 2000 2001 2002 2003

0 500 1000 1500 2000 2500 3000

0 10 20 30 40 50 60 70Biomass (g/m2)

90 day 12 hr W126 (yearly cumulative average, ppm

- hr)Draft

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- 139 September 2011 (quartiles) for that aggregation. The validating data, from SoyFACE and Aspen FACE, 

### 1 were in turn aggregated over the same variables . The accuracy of predictions is not 

### 2 expected to be conserved for individual values of those variables over which aggregation 

### 3 occurred. For example, the predicted values for soybean, based on data for five 

### 4 genotypes, are not expected to be valid for each g enotype separately. As shown in the 

### 5 validation, however, aggregation that occurred over different values of the same variable 

### 6 did not affect accuracy: composite functions based on one set of genotypes were 

### 7 predictive for another set, as long as medians wer e used for both sets. A study of 

### 8 cottonwood ( Populus deltoides ) conducted using a naturally occurring gradient of O 3 

### 9
exposure ( Gregg et al., 2006
, 2003 ) may provide an illustration of the response of an 

### 10 individual species whose response is far from the median response for an aggregation of 

### 11 species. 

### 12

9.6.3.3 Exposure

- Response in a Gr adient Study

Gregg et al. ( 2003 ) grew saplings of one clonally propagated genotype of cottonwood 

### 13 ( Populus deltoides ) in seven locations within New York City and in the surrounding 

### 14 region between July and September in 1992, 1993 and 1994, and harvested them 72 days 

### 15 after planting. Owing to regional gradients of atmospheric O 3 concentration, the 

### 16 experiment yielded eight levels of exposure (Figure 9

- 21), and the authors were able to 

### 17 rule out environmental variables other than O 3 to account fo r the large differences in 

### 18 biomass observed after one season of growth. The deficit in growth increased 

### 19 substantially faster with increasing O 3 exposure than has been observed in aspen, another 

### 20 species of the same genus ( Populus tremuloides , Section 9.6.3. 2). Using a three 

### 21 parameter Weibull model (Figure 9

- 21), the biomass of cottonwood at a W126 exposure 

### 22 of 15 ppm

- h, relative to biomass at 5 ppm

- h, is estimated to be 0.18 (18% of growth at 

### 23
5 ppm

- h). The relative biomass of trembling aspen within the same 5

- 15 ppm

- h range of 

### 24 exposure is estimated to be 0.92, using the median composite model for aspen whose 

### 25 very close agreement with Aspen FACE data was shown in Section 9.6.3.2. Using a 

### 26 median composite function for all deciduous trees in the NHEERL/WED projec t (6 

### 27 species in 21 studies) also gives predictions that are very distant from the cottonwood 

### 28 response observed in this experiment. For all deciduous tree species in NHEERL/WED, 

### 29 biomass at a W126 exposure of 15 ppm

- h, relative to biomass at 5 ppm

- h, was est imated 

### 30 to be 0.87. 

### 31Draft

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- 140 September 2011 Source: Modified with permission from Nature Publishing Group ( Gregg et al., 2003 )

. 

### Figure 9

- 21 Above

- ground biomass for one genotype of cottonwood grown in seven locations for one season in 3 years. Line represents th e three

- parameter Weibull model.

These cottonwood data confirm that, as should be expected, some individual tree species 

### 1 are substantially more sensitive than the median of NHEERL/WED (Figure 9

- 16). As 

### 2 shown in Section 9.6.2, the median models available f or trembling aspen and soybean 

### 3 have verifiable predictive ability for those particular species. This suggests that the 

### 4 corresponding NCLAN

- and NHEERL/WED

- based models for multiple crop and tree 

### 5 species can provide reliable estimates of losses for similar assortments of species. 

### 6 However, their predictive ability would likely be poor for individual species not tested. 

### 7
An alternative hypothesis for the difference between the response of cottonwood in this 

### 8 experiment and deciduous tree species in NHEERL/WED, or the difference between the 

### 9 response of cottonwood and aspen in NHEERL/WED and Aspen FACE, could be the 

### 10 presence of confounding factors in the environments where the experiment was 

### 11 conducted. However, variability in temperature, moisture, soil fertility, and atmospheric 

### 12 deposition of N were all ruled out by Gregg et al. ( 2003 ) as contributing to the observed 

### 13 response to O 3 . In addition, this hypothesis would imply that the unrecognized 

### 14 confounder(s) were either absent from both OTC and FA CE studies, or had the same 

### 15 value in both. This is not impossible, but the hypothesis that cottonwood is very sensitive 

### 16 to O 3 exposure is more parsimonious, and sufficient. 

### 17 0 10 20 30 40 50 60 70 80 90 100

0 10 20 30 40 50 60 70Biomass (g)

72 day 12 hr W126 (ppm

- hr)Draft

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- 141 September 2011 

### 9.6.3.4 Meta

- analyses of growth and yield studies

Since the 2006 O 3 AQCD, five studies have used meta

- analytic methods to integrate 

### 1 results from experimental studies of crops or tree species relevant to the U.S. It is 

### 2 possible to obtain exposure

- response data for growth and yield from those meta

- analyses, 

### 3 but because all of them provided summary measurements of O 3 exposure as hourly 

### 4 averages of various lengths of exposures, comparisons with exposure

- response results 

### 5 where exposure is expressed as W126 are problematic. Table 9

- 1 6 summarizes the 

### 6 characteristics of the five meta

- analyses. They all i ncluded studies conducted in the U.S. 

### 7 and other locations worldwide, and all of them expressed responses as comparative 

### 8 change between levels of exposure to O 3 , with carbon filtered air (CF) among those 

### 9 levels. Using hourly average concentration to summari ze exposure, CF rarely equates 

### 10 absence of O 3 , although it almost always near zero when exposure is summarized as 

### 11 W126, SUM06, or AOT40. 

### 12

Table 9

- 1 6 Meta

- analyses of growth or yield studies published since 2005

Study Number of articles included Years of publication surveyed Crop, species or genera Response Number of O 3 levels Duration of exposure
Ainsworth ( 2008 ) 12 1980

- 2 007 rice Yield 2 unreported Feng et al. ( 2008b ) 53 1980

- 2007 wheat Yield 5 > 10 days Feng and Kobayashi ( 2009 ) All crops together : 81 1980

- 2007 Potato, barley, wheat, rice, bean, soybean Yield 3 > 10 days

Grantz et al. ( 2006 ) 16 1992

- 2004 34 herbaceous dicots 21 herbaceous monocots 5 tree species Relat ive Growth Rate 2 2

- 24 weeks

Wittig et al. ( 2009 ) All responses:263 Articles that included biomass:unreported 1970

- 2006 4 gymnosperm tree genera 11 angiosperm tree genera Total biomass 4 > 7 days

The only effect of O 3 exposure on yield of rice reported in Ainsworth ( 2008 ) was a 

### 13 decrease of 14% with exposure increasing from CF to 62 ppb average concentration. 

### 14 Feng et al. ( 2008b ) were able to separate exposure of wheat into four classes with average 

### 15 concentrations of 42, 69, 97, and 153 ppb, in data where O 3 was the only treatment. Mean 

### 16 responses relative to CF were yield decreases of 17, 25, 49, and 61% respectively. Feng 

### 17 et al. ( 2008b ) observed that wheat yield losses were smaller under conditions of drought, 

### 18 and that Spring wheat and Winter wheat app eared similarly affected. However, mean 

### 19 exposure in studies of Winter wheat was substantially higher than in studies of Spring 

### 20 wheat (86 versus 64 ppb), which suggests that the yield of Spring wheat was in fact more 

### 21 severely affected, since yield was appro ximately the same, even though Spring wheat was 

### 22 exposed to lower concentrations. Exposures of the six crops considered in Feng and 

### 23 Kobayashi ( 2009 ) were classified into two ranges, each compared to CF air. In the lower 

### 24Draft

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- 142 September 2011 range of exposure (41

- 49 ppb), potato studies had the highest average exposure (45 ppb) 

### 1 and wheat and rice the lowest (41 ppb). In the higher range (51

- 75 ppb), wheat studies 

### 2 had the highest average exposure (65 ppb), and potato, barley and rice the lowest (63 

### 3 ppb). In other words, across the studies included, all crops were exposed to very similar 

### 4 levels of O 3 . At approximately 42 ppb, the yield of potato, barley, wheat, rice, bean, and 

### 5 soybean declined by 5.3, 8.9, 9.7, 17.5, 19, and 7.7% respectively, relative to CF air. At 

### 6 approximately 64 ppb O 3 , declines were 11.9, 12.5, 21.1, 37.5, 41.4, and 21.6%. Grantz 

### 7

et al. ( 2006 ) reported Relative Growth Rate (RGR) rather than growth, and did not report 

### 8 O 3 exposure values in a way that would allow calculation of mean exposure for each of 

### 9 the three categories of plants for which RGR changes are reported. All studies used only 

### 10 two levels of exposure, with CF air as the lower one, and most used elevated exposu re in 

### 11 the range of 40 to 70 ppb. Decline in RGR was 8.2% for the 34 herbaceous dicots, 4.5% 

### 12 for the 21 herbaceous monocots, and 17.9% for the 5 tree species. Finally, Wittig et al. 

### 13 ( 2009 ) divided the studies analyzed into three classes of comparisons: CF versus ambient, 

### 14 CF versus elevated, and ambient versus elevated, but reported comparisons between three 

### 15 average levels of exposure besides CF: 40 ppb, 64 ppb, and 97 ppb. Corresponding 

### 16 decreases in total biomass relative to CF were 7, 17, and 17%. 

### 17

The se meta

- analyses provide very strong confirmation of EPA ’ s conclusions from 

### 18 previous O 3 AQCDs: compared to lower levels of ambient O 3 , current levels in many 

### 19 locations are having a substantial detrimental effect on the growth and yield of a wide 

### 20 variety of crops and natural vegetation. They also confirm strongly that decreases in 

### 21 growth and yield continue at exposure levels higher than current ambient levels. 

### 22 However, direct comparisons with the predictions of exposure

- response models that use 

### 23 concentration

- weighted cumulative metrics are difficult. 

### 24

9.6.3.5 Additional exposure

- response data

The studies summarized in Tables 9

- 18 and 9

- 19 contain growth or yield exposure



### 25 response data at too few levels of exposure for exposure

- response models, and/or used 

### 26 metrics ot her than W126. These tables update Tables AX9

- 16 through AX9

- 19 of the 

### 27 2006 O 3 AQCD. 

### 28

9.6.4 Summary

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

### 29 AQCD has modified the assessment of quantitative exposure

- response relationships that 

### 30 was presented in that document. This assessment updates the 2006 exposure

- response 

### 31Draft

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- 143 September 2011 models by computing them using the W126 metric, cumulated over 90 days. Almost all 

### 1 of the experimental research on the effects of O 3 on growth or yield of plants publi shed 

### 2 since 2006 used only two levels of exposure. In addition, hourly O 3 concentration data 

### 3 that would allow calculations of exposure using the W126 metric are generally 

### 4 unavailable. However, two long

- term experiments, one with a crop species (soybean), 

### 5

on e with a tree species (aspen), have produced data that can be used to validate the 

### 6
exposure

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

### 7 derive them. 

### 8
Quantitative characterization of exposure

- response in the 2006 O 3 AQCD was base d on 

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

### 10 Network (NCLAN) and EPA National Health and Environmental Effects Research 

### 11 Laboratory, Western Ecology Division (NHEERL

- WED) projects, using OTCs to expose 

### 12 crops and tre es seedling to O 3 . In recent years, yield and growth results for two of the 

### 13 species that had provided extensive exposure

- response information in those projects have 

### 14 become available from studies that used FACE technology, which is intended to provide 

### 15 condi tions much closer to natural environments ( Pregitzer et al., 2008 ; Morgan et al., 

### 16 2006 ; Morgan et al., 2004 ; Dickson et al., 2000 ) . The robust metho ds that were used 

### 17 previously with exposure measured as SUM06 were applied to the NCLAN and 

### 18
NHEERL

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

- species 

### 19 median models for soybean and aspen from studies involving different genotypes, yea rs, 

### 20 and locations. The resulting models were used to predict the change in yield of soybean 

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

### 22 experiments. Results from these new experiments were exceptionally close to predict ions 

### 23 from the models. The accuracy of model predictions for two widely different plant 

### 24 species provides support for the validity of the corresponding multiple

- species models for 

### 25 crops and trees in the NCLAN and NHEERL

- WED projects. However, variability amo ng 

### 26 species in those projects indicates that the range of sensitivity is likely quite wide. This 

### 27 was confirmed by a recent experiment with cottonwood in a naturally occurring gradient 

### 28 of exposure ( Gregg et al., 2006 ) , which established the occurrence of species with 

### 29 responses substantially more severe under currently existing conditions than are predicted 

### 30 by the median model for multiple species. 

### 31

Results from several meta

- analyses have provided approximate values for responses of 

### 32 yield of soybean, wheat, rice and other crops under broad categories of exposure, relative 

### 33 to charcoal

- filtered air ( Ainsworth, 2008 ; Feng et al., 2008b ; Morgan et al., 2003 )

. 

### 34 Likewise, Feng and Kobayashi ( 2009 ) have summarized yield data for six crop specie s 

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

### 36 263 studies that reported effects on tree biomass. However, these analyses have proved 

### 37Draft

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- 144 September 2011 difficult to compare with exposure

- response models, especially given that exposure was 

### 1 not expressed in the same W126 metric. 

### 2

Table 9

- 1 7 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 (percent 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 al. ( 2009 )

Bean ( Phaseolus vulgaris I. cv Borlotto) OTC; ground

planted Curno, Italy 3 months, 2006 Seasonal 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 al. ( 2009 )

Big Blue Stem ( Andropogon gerardii ) OTC Alabama 4 months, 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 )

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. ( 2009b )
Corn ( Zea mays cv. Chambord) OTC France 33 days AOT40 ppm

- 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% chang e) Leitao et al. ( 2007c )

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 ) Eastern Gamagrass ( Tripsacum dactyloides ) OTC Alabama 4 months, 2003 12

- h avg: CF (14ppb), Ambient (29 ppb), Elevated (71 ppb) (N/A) Final harvest biomass; RVF +68 (+42);

- 17 (

- 12) Lewis et al. ( 2006 )

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 )

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 )

Oilseed Rape ( Brassica napus ) OTC Yangtze Delta, China 39 days Daily avg: 100 ppb, one with diurnal variation and one with constant concentration (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 )

Peanut ( Arachis hypogaea ) OTC Raleigh, NC 3 yr 12

- h avg: CF (22 ppb), Ambient (46 ppb), Elevated (75ppb) (CO 2 : 375 ppm; 548 ppm; 730 ppm) Yield (seed weight, g/m) - 33 (

- 8) Burkey et al. ( 2007 )

Poa pratensis OTC Braunschweig, Germany 2000

- 2002: 4

- 5 wk in the Spring 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 )Draft

– Do Not Cite or Quote 9

- 145 September 2011 Species Facility Location Exposure Duration O 3 Exposure (Additional Treatment) Response Measured percent change from CF (percent change from ambient) Reference

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 yield averaged across 5 field

- sites; Tuber starch content regressed against [O 3 ] report sig.
± slope with increasing [O 3 ] N/A (

- 27 %

- +27%, most comparisons n.s.) Lin ear regression slope =

- 0.0098) Vandermeiren et al. ( 2005 )

Rice ( Oryza sativa ) OTC Raleigh, NC 1997

- 1998, June

September 12

- h mean ppb: CF (27.5), Elevated (74.8) (CO 2) Total biomass; Seed yield - 25(N/A)

- 13 to 20 (N/A) Reid, et al. ( 2008 )

Rice ( Oryza sativa ) 20 Asian cultivars 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 )

Seminatural grass FACE Le Mouret, Switzerland 5 yr Seasonal AOT40: Ambient (0.1

- 7.2 ppm

- h); Eleva ted. (1.8

- 24.1 ppm

- h) (N/A) Relative annual yield N/A (2×faster decrease in yield/yr) Volk et al. ( 2006 )

Soybean OTC; CRA Bari, Italy 2003

- 2005 growing seasons Seasonal AOT40 ppm

- h: CF (0), Ambient (3.4), High (9.0) (Drought) Yield

- 46 (

- 9) Bou Jaude et al. ( 2008 )
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 al. ( 2006 )
Soybean ( Glycine max cv. Essex) Chambers; 21 L Raleigh, NC 2×3 months 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 )
Soybean ( Glycine max cv. Essex) OTCs; 21

- L pots Raleigh, NC 2×3 months 12

- h avg: CF (18); Elevated (72) (CO 2 : 367 & 718) Seed mass per plant

- 34 (N/A) Booker et al. ( 2004a )
Soybean ( Glycine max cv. Tracaja) Chambers; pots Brazil 20 days 12

- h avg: CF & 30 ppb (N/A) Biomass

- 18 (N/A) Bulbovas et al. ( 2007 )
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 )
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 acro ss all experiments N/A (significant negative correlation) N/A (sig negative correlation) Piikki et al. ( 2008a )

Strawberry ( Fragaria x ananassa Duch. Cv Korona & Elsanta) Growth chambers Bonn, Germany 2 months 8

- h avg: CF (0 ppb) & Elevated (78 ppb) (N/A) Fruit yield (weight/plan t) - 16 (N/A) Keutgen et al. ( 2005 )

Sugarbeet ( Beta vulgaris cv. Patriot) OTC Belgium 2003, 2004; 5 months 8

- h avg: Ambient (36 ppb); Elevated (62 ppb) (N/A) Sugar yield N/A (

- 9) De Temmerman et al. ( 2007 ) Sugarcane ( Saccharum spp ) CSTR San Joaquin Valley, CA 2007; 11

- 13 wk. 12

- h avg: CF (4 ppb); Ambient (58); Elevated (147) (N/A) Total biomass (g/plant) - 40 (

- 30) Grantz and Vu ( 2009 )Draft

– Do Not Cite or Quote 9

- 146 September 2011 Species Facility Location Exposure Duration O 3 Exposure (Additional Treatment) Response Measured percent change from CF (percent change from ambient) Reference

Sweet Potato Growth chambers Bonn, Germany 4 wk 8

- h avg: CF (0 ppb), Ambient (<40 ppb) Elevated (255 ppb) (N/A) Tuber weight

- 14 (

- 11.5) Keutgen et al. ( 2008 )

Tomato ( Lycopersicon esculentum ) OTC Valencia, Sp ain 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 )

Trifolium Subterraneum OTC; 2.5

- L pots Madrid, Spain 29 days 12

- h avg: CF (<7.9±6.3); Ambient (34.4±10.8) ; Elevated (56.4±22.3) (N: 5, 15 & 30 kg/ha) Above

- ground biomass - 45 (

- 35) Sanz et al. ( 2005 )

Watermelon ( Citrullus lanatus) OTC Valencia, Spain 2000, 2001. 90 days AOT40: CF (0 ppm

- h) Ambient (5.7 ppm

- h), Elevated (34.1 ppm
- h) (N:0, 14.0 & 29.6 g/pot) total fruit yield (kg) n.s. (54) Calatayud et al. ( 2006 )

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 )

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.



### Table 9

- 1 8 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 Parlier, CA 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. ( 2010b )

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 years CF (459 g/m 2 ), CF+ (457 g/m 2 ), CF2+ (398 g/m 2 ) Pfleeger et al. ( 2010 )

Highbush blackberry ( Rubus argutus ) OTC Auburn, AL 2004, May

- August 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 )

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 )

Red Oak ( Quercus rubrum ) Forest sites Look Rock & Twin Creeks Forests, TN 2001

- 2003, April

- October AOT60: 2001 (11.5), 2002 (24.0), 2003 (11.7) (Observational study with multiple environmental variables) Annual circumference increment (change relative to 2001 i n year 2002;2003) - 42.8%; +1% McLaughlin et al. ( 2007a )Draft

– Do Not Cite or Quote 9

- 147 September 2011 Species Facility Location Exposure Duration O 3 Exposure (Additional Treatment) Response Measured Response Reference

Pine species Forest sites Look Rock Forest , TN 2001

- 2003, April

- October AOT60: 2001 (11.5), 2002 (24.0), 2003 (11.7) (Observational study with multiple environmental variables) Annual circumference increment (change relative to 2001 in year 2002;2003) - 62.5%;

- 2.9% McLaughlin et al. ( 2007a )

Hickory species Forest sites Look Rock Forest, TN 2001

- 2003, April

- October AOT60: 2001 (11.5), 2002 (24.0), 2003 (11.7) (Observational study with multiple environmental variables) Annual circumference increment (change relative to 2001 in year 2002;2003) - 14%; +30% McLaughlin et al. ( 2007a )

Chestnut Oak ( Quercus prinus ) Forest sites Look Rock Forest, TN 2001

- 2003, April

- October AOT60: 2001 (11.5), 2002 (24.0), 2003 (11.7) (Observational study with multiple environmental variables) Annual circumference increment (change relative to 2001 in year 2002;2003) +44%; +55% McLaughlin et al. ( 2007a )

Black Cherry ( Prunus rigida ) Forest sites Twin Creeks Forest, TN 2002

- 2003, April

- October AOT60: 2002 (24.0), 2003 (11.7) (Observational study with multiple environmental variables) Annual circumference increment (change relative to 2003 in year 2002) - 75% McLaughlin et al. ( 2007a )

Shortleaf pine ( Pinus echinata ) Forest sites Twin Creeks Forest, TN 2002

- 2003, April

- October AOT60: 2002 (24.0), 2003 (11.7) (Observational study with multiple environmental variables) Annual circumference increment (change relative to 2003 in year 2002) - 16.8% McLaughlin et al. ( 2007a )

Hemlock ( Tsuga canadensis ) Forest sites Twin Cree ks Forest, TN 2002

- 2003, April

- October AOT60: 2002 (24.0), 2003 (11.7) (Observational study with multiple environmental variables) Annual circumference increment (change relative to 2003 in year 2002) - 21.9% McLaughlin et al. ( 2007a )

Red Maple ( Acer rubrum ) Forest sites Twin Creeks Forest, TN 2002

- 2003, April

- October AOT60: 2002 (24.0), 2003 (11.7) (Observational study with multiple environmental variables) Annual circumference increment (change relative to 2003 in year 2002) - 59.6% McLaughlin et al. ( 2007a )

Yellow Poplar ( Liriodendron tulipifera ) Forest sites Look Rock, Oak Ridge, & Twin Creeks Forest, TN 2002

- 2003, April

- October AOT60: 2002 (24.0), 2003 (11.7) (Observational study with multiple environmental variables) Annual circumference increment (change relative to 2001 in years 2002; 2003) - 45.9%;

- 15.25% McLaughlin et al. ( 2007a )Draft

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- 148 September 2011 Species Facility Location Exposure Duration O 3 Exposure (Additional Treatment) Response Measured Response Reference

Sugar Maple ( Acer saccharum ) Forest sites Twin Creeks Forest, TN 2002

- 2003, April

- October AOT60: 2002 (24.0), 2003 (11.7) (Observational study with multiple environmental variables) Annual circumference increment (change relative to 2003 in year 2002) - 63.8% McLaughlin et al. ( 2007a )

Trembling aspen ( Populus tremuloides ), 5 genotypes Aspen FACE Rhinelander, WI 1998

- 2004, May

- October Cumulative avg 90

- day 12

- h W126. Ambient 3.1 ppm
- h Elevated: 27.2 ppm

- h (Competition with birch, maple) main stem volume after 7 years Ambient: 6.22 dm 3 ; Elevated: 4.73 dm 3 Kubiske et al. ( 2006 )

Hybrid Poplar ( Populus trichocarpa x Populus deltoides ) OTC Seattle, WA 2003, 3 months Daily mean (μg/g): CF(<9), Elevated (85

- 128) Total biomass CF to elevated:

- 12.9% Woo and Hinckley ( 2005 )

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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