United States
Environmental Protection
Agency
Environmental Research
Laboratory
Corvallis, OR 97333
EPA/600/3-89/081
November, 1989
Research and Development
FA Seedling Response to Sulfur, Nitrogen, and
Associated Pollutants
Forest Response Program
Major Program Output #3
V>
United States
Environmental Protection
Agency
United States
Department of Agriculture
Forest Service
-------
EPA/600/3-89/081
November, 1989
SEEDLING RESPONSE TO SULFUR, NITROGEN,
AND ASSOCIATED POLLUTANTS
by
Charles H. Peterson,1 Kim G. Mattson,2 & Robert A. Mickler1
1 NSI Technology Services, Inc.
2 University of Idaho
US EPA Environmental Research Laboratory
Corvallis, OR 97333
Project Officer
Roger Blair
US EPA Environmental Research Laboratory
Corvallis, OR 97333
The Forest Response Program is a cooperative program of the
US Environmental Protection Agency and the USDA Forest Service with support from
the National Council of the Paper Industry for Air and Stream Improvement, Inc.
Environmental Research Laboratory
U.S. Environmental Protection Agency
200 SW 35th Street
Corvallis, OR 97333
LIBRARY
U.S. Environmental Protection Aaencv
Corea,li6isvssrchU8
Corvallis, Oregon 97333
-------
MPO#)
FRP SEEDUNG REPORT
PETERSON ETAL
TABLE OF CONTENTS
ABSTRACT iii
NOTICE 1
EXECUTIVE SUMMARY . 2
INTRODUCTION 5
Forest Response Program . . 5
Synthesis and Integration 5
The Role of Mechanisms as Scientific Questions in the FRP 6
METHODS AND MATERIALS 9
Choice of Experimental Material 9
General Methods 9
Statistical Methods 16
Design and Analysis 16
Relevance 16
Combining Results .. 16
Statistical Power . . 17
Assessment of Data Quality 17
Acid Precipitation and Gaseous Exposure Techniques 18
Growth and Physiology Response Variables 24
Summary 24
RESULTS 30
Red Spruce 30
Visible Injury 30
Growth Changes 33
Mechanism: Foliar Leaching 34
Mechanism: Carbon Allocation \ . .35
Mechanism: Winter Injury 38
Summary 41
Eastern Hardwoods 41
Visible Injury . .41
Growth Changes . .41
Species Sensitivity 42
Summary - .42
Southern Commercial Pines 42
Visible Injury .43
Growth Changes .43
-------
MPO #3 PRP SEEDUNG REPORT PETERSON ET AL
Family Sensitivity SO
Mechanism: Carbon Allocation 50
Summary 55
Western Conifers 55
Visible Injury 55
Growth Changes 56
Species Sensitivity 58
MftrhanUnr Fnliar I aching 59
Summary 59
SUMMARY 60
Visible Injury 60
Growth Effects 60
Carbon Allocation 61
Foliar Leaching 62
Winter Injury 62
CONCLUSIONS AND RECOMMENDATIONS 63
Principal Findings 63
Sulfur Dioxide 63
Simulated Acid Deposition 63
Ozone 63
Pollutant Interactions 64
Summary 64
Recommendations for Future Research 64
Species/Plant Variability 65
Choice of Response Variables 65
Microclimate Characterization 65
Duration of Experiments 65
Statistical Power 66
Repeatability of Experiments 66
Repeated Measures 66
ACKNOWLEDGMENTS 67
LITERATURE CITED 68
ABBREVIATIONS 72
APPENDICES 73
Appendix A: Project Summaries 73
Appendix B: Example of Power Curve 103
ii
-------
MPO #3
FRPSEEDUNG REPORT
PETERSON ETAL
ABSTRACT
In 1986, the National Acid Precipitation Assessment Program (NAPAP) established the Forest
Response Program (FRP) to assess the effects of acidic deposition and associated pollutants on
forests. Seedling exposure studies were initiated to determine acute effects of simulated acid
deposition, ozone, and sulfur dioxide, and to identify hypothesized mechanisms by which these
effects might alter tree condition and hence" result in forest decline. From data available as of
December, 1988, altered post-exposure growth and imbalance in above- and below-ground
responses to sulfur dioxide indicated changes in carbon allocation patterns. Simulated acid
precipitation reduced frost hardiness of red spruce seedlings at pH 3.0 and led to higher rates of
foliar tissue mortality during extreme cold. Loblolly pine showed root and stem growth decreases
at ozone levels 80 ppb and higher. Of western conifers, only ponderosa pine showed consistent
growth decreases due to ozone. Many treatment effects did not show up until the following
growing season. The influences of different exposure methods, exposure durations, and ex-
perimental material are discussed, as are statistical considerations and quality control of treat-
ments. Further results will be presented in a future FRP report and in NAPAP State of
Science/Technology documents.
iii
-------
MPO #3
FRP SEEDUNG REPORT
PETERSON ETAL
1 NOTICE
The information in this document has been funded wholly or in part by the United States
Environmental Protection Agency. It has been subjected to the Agency's peer and administrative
review, and it has been approved for publication as an EPA document.
This document is a preliminary interpretation of the EPA-USFS Forest Response Program's
existing and accumulating data on the response of seedlings to controlled exposures of simulated
acidic deposition, sulfur dioxide, and ozone. Many of the exposure studies had not been
concluded at the time these data were requested. Consequently, some estimates and interpreta-
tions are subject to change as experiments conclude and more data become available.
1
-------
MPO #3
FRP SEEDLING REPORT
PETERSON ETAL
2 EXECUTIVE SUMMARY
2.1 Purpose of the Document
This document, Major Program Output #3, was prepared at the request of the Environmental
Protection Agency (EPA) and the USDA Forest Service as an internal report. Its purpose is to
summarize seedling exposure research conducted in the Forest Response Program (FRP). The
data used in this document were those available from FRP research efforts as of December, 1988,
and are intended to provide input to policy decisions. Forthcoming results from continuing studies
will be integrated with other research efforts in future reports of the FRP and state-of-science
documents for the National Acid Precipitation Assessment Program (NAPAP).
22 Rationale for Seedling Research
In 1986, the FRP was directed by NAPAP to assess the effects of acidic deposition and associated
pollutants on forests. Hypothesized mechanisms were identified by which a pollutant might affect
tree condition, and hence result in forest decline. While recognizing that seedlings have both
advantages and limitations, seedling exposure studies were initiated as the quickest way to
determine acute effects of pollutants and mechanisms by which these effects operate.
23 Assessing Data Quality
The assessment of data quality has not been completed for all projects. When that task is
accomplished, the primary use of the results will be to evaluate the relevance of the data quality
objectives to see if those specifications can be realistically attained. The achievement of an
average target value is relative to the accompanying fluctuations. The purpose of that information
is not to reject data from a given project, but is intended to help the principal investigator monitor
the system and make necessary corrections to keep the system under control. However, infor-
mation such as magnitude and direction of the charted treatment fluctuations will be considered
in a subjective way, when evaluating the biological data from a project. Final data quality analyses
will be contained in a separate data quality document currently in preparation, and updated in
MPO #4.
2.4 Summary of Principal Findings
The main body of this report describes results from individual FRP seedling exposure studies.
The major findings of these studies regarding short-term effects (Le., one exposure season) of
sulfur dioxide, simulated acidic deposition, and ozone on seedlings, as well as possible long-term
implications, are as follows:
Sulfur Dioxide: Two projects examined visible effects of sulfur dioxide, and three examined
growth effects. No visible injury was observed in response to concentrations as high as 66 ppb.
Increased above-ground growth due to sulfur dioxide occurred for Engelmann spruce, white fir,
western redcedar, and Douglas-fir, relative to a control treatment. Douglas-fir, ponderosa pine,
and lodgepole pine showed reductions in root biomass and root/shoot ratios. Compared to a
control, bud elongation was increased by higher sulfur dioxide levels (up to 66 ppb base level) for
ponderosa pine, Douglas-fir, western hemlock, and western redcedar. The altered post-exposure
growth and imbalance in above- and below-ground responses indicate that changes in carbon
2
-------
MPO#J FRP SEEDLING REPORT PETERSON ET/U-
allocation patterns occurred. Under chronic exposure, survival or eventual tree productivity
could potentially be negatively impacted. Sulfur dioxide was not tested with red spruce or
southern pines, and although planned for eastern hardwoods, experimentation problems
precluded a clear study of sulfur effects.
Simulated Add Deposition: The clearest effect of simulated acid deposition was a reduction
of frost hardiness at pH 3.0 and higher rates of foliar tissue mortality during extreme cold for red
spruoe wiling* Most species that were tested at pH levels below 3j0 showed some visible injury.
Across all spedes, there were no conclusive short-term effects of simulated adddcposition by
itself on seedling growth. However, growth of black cheny was decreased by pH 3.0 versus 42.
Furthermore, increased above-ground growth coupled with no apparent effects on below-ground
biomass in western conifers at pH 2.1 compared with pH 5.6 indicates that changes in carbon
allocation patterns occurred.
Ozone: The direct effect of ozone varied from physiological changes in the foliage of red spruce
to suppressed growth of loblolly pine, ponderosa pine, and some hardwood spedes. Eastern
hardwood spedes showed viable injury with ozone of 70 ppb or higher. Yellow-poplar, yellow
birch, sweetgum, red maple, white ash, and black cherry appeared to be the most sensitive spedes
tested. Among western conifers, white fir, subalpirie fir, ponderosa pine,, and western hemlock
also showed visible injury in response to ozone at 70 ppb. Despite considerable differences in
experimental designs and procedures, there was a pattern of root and stem growth decreases at
ozone near 80 ppb or higher for loblolly pine. Atintermediate levels (40 to 80 ppb)results were
more variable; it was not uncommon for growth rate tp be greater at intermediate levels than in
charcoal-filtered air. In the West, only ponderosa pine showed consistent decreases across
several growth variables due to ozone; most other spedes showed increased growth rates at levels
less than 100 ppb. At this point in time, there are no data with which to address the anomaly of
increased seedling growth at these levels (approximately 1.5 times ambient). Amoqg the
hardwoods tested, levels of ozone *70 ppb and higher decreased growth for black cherry, white
oak, red maple, and yellow birch; yellow poplar, white ash, and red oak displayed no response at
the same levels. Cumulative decreases in net photosynthetic rate in response to ozone were found
. for loblolly pine. Red spruce did not exhibit consistent decreases in net photosynthesis. How-
ever, damage to foliar mesophyll cells, decreased photosynthetic pigments, and seasonal changes
in photosynthesis in red spruce in' response to ozone at 40 ppb and higher suggest an increased
potential for winter injury to red spruce.
Pollutant Interactions: There is preliminary evidence of some anion x pH interactions, where
sulfur-based adds' caused greater foliar injury than nitrogen-based adds at the same pH.
Although there are some indications of ozone x add deposition interactions, the inteipretations
of these interactions are incondusive at this time. Any further information that becomes available
will be incorporated in MPO #4.
Spedes Sensitivity: Relative spedes sensitivity was tested only in western conifers and in eastern
hardwoods. Visible injury and growth changes indicate that ponderosa pine was the most
sensitive, and western redcedar the least sensitive, of spedes exposed to expected pollutant
scenarios in the west Results of visible injury to eastern hardwoods from one year of exposure
indicate that black cherry was the most sensitive of spedes exposed to either add predpitation
or ozone.
Long-term Implications: Although there are currently no data on the long-term effects of
multi-year exposures on differential responses in above- versus below-ground biomass
to short-term pollutant exposures indicate long-term problems for seedlings. Under chronic
exposure, survival or eventual tree productivity could be affected.
3
-------
MPO/3
FRP SEEDLING REPORT
PETERSON ETAL
2.5 Recommendations for Future Research
One product of these seedling exposure studies has been the identification of issues that qualify
the above results and that should be addressed in new and continuing studies involving tree
material. Although other issues are likely to arise before the FRP studies are concluded, major
issues identified to date sire:
Species/Plant Variability: A major source of variation in plant response lies in the plant material
itself. Virtually all the analyses demonstrated the large variation in growth that can be attributed
to large differences in initial size (height and/or diameter). Careful selection and randomization
of plants prior to treatment would help to alleviate this nonuniformity. The large variability and
range of responses to pollutant exposures among loblolly pine families show the importance of
reducing that variability if results of different experiments are to be compared.
Choice of Response Variables: The value of studying mechanisms for change in tree conditions
is already apparent and is summarized in the Conclusions and Recommendations of the report.
Currently, there is insufficient information on changes in below-ground biomass. Therefore,
future studies should include measurements on both above-ground and below-ground (i.e., at
least root biomass) changes from the same plant.
Microclimate Characterization: Differences in seedling responses among sites, or among years
at a given site, cannot be correctly interpreted without knowing corresponding spatial and
temporal differences in factors such as light, temperature, and humidity.
Duration of Experiments: Most of the initial studies were designed to evaluate effects over
short periods of exposure (e.g., 12 to 16 weeks). In one study designed to test post-exposure
effects, growth differences due to treatment were reflected in bud elongation (i.e., reduced shoot
growth) in the growing season following the season of treatment. While useful information can
be obtained from short-term exposures, most effects can be better evaluated with multiple-year
exposures. In addition, the variability of results among studies is typically reduced with longer
exposures. Results from ongoing multi-year exposure studies in southern pines and red spruce
will be forthcoming in the next two years.
Statistical Power Power should be considered when planning research and computed at the
conclusion of each project. In the absence of formal significance due to low power, evidence of
treatment effects may still be present in the form of trends or patterns that should not be
overlooked.
Repeatability of Experiments: Any experiment worth doing once should be considered for
replication, regardless of the treatment duration (i.e., single-year versus multi-year exposures) or
the statistical significance of the initial outcome. The ability to replicate an experiment in time
or place is critical to confirmation of results.
4
-------
MPO #3
FRP SEEDLING REPORT
PETERSON ETAL
3 INTRODUCTION
3.1 Forest Response Program
The Forest Response Program (FRP) is a research initiative under Task Group Vof the National
Acid Precipitation Assessment Program (NAPAP). The FRP is responsible for determining the
actual and potential effects of acid deposition and its associated pollutants on trees, forests, and
. forest ecosystems of the United States (Schroeder and Kiester, 1989). The FRP is jointly
administered by the US Environmental Protection Agency (EPA) and the USDA Forest Service.
Operationally, it consists of a National Management Staff, six Research Cooperatives, a Quality
Assurance/Quality Control (QA/QC) Staff, and the Synthesis and Integration (S&I) Project The
six research cooperatives are the Spruce-fir Research Cooperative, Southern Commercial Forest
Research Cooperative, Eastern Hardwoods Research Cooperative, Western Conifers Research
Cpoperative, National Vegetation Survey, and Atmospheric Exposure Cooperative.
The objective of the FRP is to address three broad questions related to environmental policy:
Policy Question #1: Is there significant forest damage in North America caused by acidic
deposition, alone or in combination with other pollutants?
Policy Question #2: By what mechanisms does acidic deposition, alone or in combination with
other pollyj^^s,. contribute to forest damage in North America?
Policy Question #3:; What is the dose-response relationship between acidic deposition, alone or
in combination with other pollutants, and forest damage in North America?
32 Synthesis and Integration
The FRP addresses these policy questions and regulatory needs through many diverse projects.
Therefore, the FRP must ensure that the technical information developed in the six Research
Cooperatives is coordinated across the FRP to understand the effects of pollutants on major
forest species. The Synthesis and Integration Project (S&I) provides the technical focus for the
FRP.
S&I produces the program-wide output documents that provide answers to scientific questions
relevant to the policy questions listed above. S&I synthesizes and integrates results across
individual experiments wherever possible when answering these questions. In order to conduct
a thorough analysis of results across the program, investigators' analyses of their data are made
available to S&I. An additional role for S&I is to review FRP studies and provide suggestions
for further analyses.
The FRP research that addresses the three policy questions is being synthesized in five Major
Program Output documents (MPOs). The MPOs and their expected completion dates are as
follows:
MPO #1: Evaluation of the extent and magnitude of recent changes in forest condition (9/89)
MPO #2: Evaluation of the role of non-air pollution factors in growth reduction and viable
decline (9/89)
MPO #3: Seedling response to sulfur, nitrogen, and associated pollutants (4/89)
MPO #4: Evaluation of the roles of sulfur, nitrogen, and associated pollutants in forest decline
(12/89 and 9/90)
MPO #5: Projection of responses under alternative deposition scenarios (12/89 and 9/90)
5
-------
MPO #3
FRP SEEDUNG REPORT
PETERSON ET AL.
The purpose of this document (MPO #3) is to summarize the seedling exposure research
conducted in the FRP. The seedling studies reported here thus contribute to Policy Question
#2. MPO #4 will also contribute to answering Policy Question #2 by adding to the knowledge
in MPO #3. Policy Question #1 will be addressed in MPO #1 and #2, and Policy Question #3
will be addressed in MPO #5.
33 The Role of Mechanisms as Scientific Questions in the FRP
In order to investigate Policy Question #2, scientific questions were outlined that hypothesize
specific mechanisms by which a change in tree condition might occur. Change in tree condition
includes visible injury to the foliage, change in growth, or death. Table 1 lists these hypothesized
mechanisms, and Figure 1 illustrates how the mechanisms relate to tree growth. One single
mechanism is unlikely to be the sole cause of a change in forest condition for the following reasons:
(a)Tree growth is an interrelated set of processes. For example, toxicity to roots or mycorrhizae
(S.Q.2.1a and S.Q.2.1b in Table 1) will have an effect upon both water and nutrient uptake. The
effect of foliar leaching (S.Q.2.1c in Table 1) upon forest growth will also depend upon the rate
and amount of uptake of nutrients. Similarly, susceptibility to insects and disease is influenced
by both nutrient status and water status of trees.
(b)Pol' " ' : icross the United States change in time and space. A change in the ratio between
nitrogen aud sulfur compounds, as acid precursors, seems likely and could have a major impact
upon uptake oi" water and nutrients.
Complete measurement of.all mechanisms in mature trees across a wide range of acidic deposi-
tion and pollutant conditions is not technically possible. It is the objective of S&I to identify
conditions under which one mechanism may be dominant, as well as to assess how mechanisms
may operate in combination to effect, for example, a change in growth. This integration must
take the interrelations of the growth process into account. Data on soil processes and mature
trees are being collected as part of the FRP and as a part of related programs such as the Electric
Power Research Institute's Integrated Forest Study (Lindberg and Johnson, 1989). The FRP
seedling research focuses on Scientific Questions 23 and 2.4, and to a lesser extent on Question
2.2.
Figure 1 illustrates an integrating model of tree growth. This model has been adopted because
it demonstrates plausible interactions of the various processes as well as the possible results of
modifications in one or more of these processes. For example, if repairing pollutant-damaged
tissue increases respiration, less energy will be available for root and foliage maintenance and
growth, even if photosynthetic rate remains constant. Reduced root growth can affect photosyn-
thesis via disruptions of water and nutrient fluxes. Reduced foliage area will result in a diminished
capacity to fix carbon and a decrease in energy available for all plant processes. Therefore, it is
apparent that the three principal components of growth, photosynthesis, water uptake, and
nutrient uptake, are interdependent.
6
-------
MPO#l
PRPSEEDUNO REPORT
PETERSON ET AL.
Table 1. Scientific Questions (SQ 2JQ in the Forest Response Program of mechanisms by which
a change in tree condition may occur.
Sulfur and nitrogen compounds and other air pollutants may affect trees
and forests by the mechanisms of:
SQ2.1a. Direct toxicity to roots, mycorrhizae, or soil microbial
populations by mobilized metals in acidified soil water;
SQ2.1b. Nitrogen toxicity to mycorrhizae;
SQ2.1c. Increased leaching of soil nutrients resulting in reduced
nutrient availability.
SQ2.2. Increased leaching of nutrients from foliage.
SQ2.3. Altered photosynthesis, respiration, and carbon allocation
patterns (e.g., morphology) with possible induction of
water or nutrient stress.
PQ2.4. Delayed cold hardening or premature break in dormancy
resulting in increased winter injury.
SQ2.5. Disruption of reproduction or regeneration.
SQ2.6. Alteration of susceptibility to insects and pathogens.
7
-------
FRP SEEDLING REPORT
PCTERSONETAL
SQ.2.3
Photosynthetic
Foliage
SQ.2.4
O- SO.2.2 -O
Carbohydrate
Water flux
Carbohydrates
Nutrient
(e.g., growth,
SQ.2.3
SQ.2.6
SQ.2.3
SQ.2.1 a
SQ.2.1 b
SQ.2.1c
SQ.2.5
Roots
Root death
SQ.2.1 a
storage,
respiration,
reproduction,
maintenance,
repair)
SQ.2.6
Figure 1: Diagram of basic processes and their interactions resulting in tree growth. Processes
are represented by boxes and interactions by arrows. Functional components are
represented by ellipses. The Scientific Questions (Table 1) ask how changes in these
processes may affect growth.
8
-------
MPO #3
FRP SEEDLING REPORT
PETERSON ETAL
4 METHODS AND MATERIALS
4.1 Choice of Experimental Material
Results from seedling studies, such as visible or latent seedling response, are the major source of
information currently available to address Scientific Questions 12,23, and 2.4 posed in Table
L Information on seedlings is important because seedlings are representative of mature trees in
many physiological processes, and seedling establishment is necessary for future forests. In this
report, the term seedling refers to trees small enough (e.g, height generally less than 1 meter) to
be housed in standard open-top chambers.
The consensus within the FRP is that while seedlings do have limitations, the use of wiling* is
a cost-effective way to obtain information through exposure studies, particularly given the short
time frame of the program (three years). In comparison with larger trees, seedlings are easier to
manipulate and present fewer sampling problems in experimental designs, particularly those that
require growth chamber fumigation systems. The size of seedling plant material allows
mechanisms to be measured over a range of experimental treatments often infeasible with mature
trees.
42 General Methods
Twenty-four studies, currently in various stages of completion, have been funded by the FRP to
quantify seedling responses to simulated acid deposition, sulfur dioxide, and ozone. Twelve
conifer species, twelve hardwood species, and 100 commercially important families of loblolly
pine are being tested. This section describes the general methods employed across the studies.
It should be noted that methods among studies varied and may have affected the particular results
observed. An overview of the experimental approaches used by the four research cooperatives
is given in Tables 2,3,4, and 5.
Seedlings were grown from germinated seeds obtained from known seed sources. These sources
were: 1) specific regions of forest occurrence for spruce fir; 2) tree nurseries for the eastern
hardwoods; 3) commercial and research seed orchards of loblolly pine; and 4) regions of forest
occurrence and forest-tree nurseries for western conifers. Most wtlingc were planted in
individual containers. Rooting media were typically composed of commercial mixtures (e-g.,
peat, vermiculite, perlite), although soil representing a forested site was sometimes used. Ages
of seedlings at time of treatments ranged from 12 weeks to 4 years; the majority were 2 years or
younger at the beginning of the studies. Seedlings were grown under non-stressed conditions
with adequate nutrients, water, and light In some cases, seedlings were screened prior to
treatments and seedlings of atypical growth form were rejected.
To apply the treatments, seedlings were placed in chambers that provided for delivery of
simulated precipitation and allowed some modification of the air space around the seedlings.
Two types of chambers were used, and choice of chamber type involved a trade-off between
experimental control and replication of realistic conditions. Completely enclosed chambers,
referred to as continuously-stirred tank reactors (CSTRs) in greenhouses or laboratories allowed
for higher precision in the application of gaseous treatments. Open-top chambers located
outdoors were used in cases where some exclusion of ambient air, but exposure to sunlight,
humidity, and normal air temperatures was desired. The outdoor chambers mayor may not have
had rainfall exclusion devices, depending upon experimental objectives.
9
-------
MPO #3
FRP SEEDUNO REPORT
PETtRSON ETAL
Table 2. Overview of Spruce-fir seedling exposure research by project number with principal
investigator (* indicates regime used by each project).
.... - - 1
\
Study
Description
SF22
FERET
SF32
GREENWOOD
SF06
JACOBSON
SF07
JENSEN
SF31
KOHUT
SF10
Mclaughlin
SF13
SEILER
SF27
THORNTON
SF14
UNSWORTH
SF16
WEINSTEIN
1988
1989
1988
1987
1987
1988
ozone
only
nnc?d
rain .
Controlled Field
-------
MPO«3
FRF SEEDUNO RETORT
PETERSON ETAL
Table 3. Overview of Eastern Hardwoods seedling exposure research by project number with
principal investigator (' indicates regime used by each project).
Study N.
Description
EH01
DMAS
3 1
JS 8
o o»
EH04
SKELL1
EH06
JENSEN ^
8
o «
11L f! ii J
1 I§| ||| 1|| |S
if i If e l |l if
£ w Controlled FWd
w ControUod Loborotory
*
u Artificial
3 a Uodrfwd Ambient
V s ,
fe g Simulated Ambient
Ambient Profile
Open-Top Chambers
Rain Exclusion
m Air/Rain Exclusion
§j CSTR (Laboratory)
^ , CSTR (Greenhouse)
Growth Chambers
Branch Chambers
Ozone
Acidic Rain
t Acidic Mist
1 Sulfur Dioxide
g Nitrogen Dioxide
S Nitric Acid
Hydrogen Peroxide
Ambient
(sulfate)
* (nitrate)
PUNT
MATERIAL
*
European Beech
Sweetgum
M Hickory spp.
1 Red Oak spp.
& White Oak spp.
Block Oak
Yellow-Poplar
White Ash
Red Maple
Sugar Maple
Bkick Cherry
E. White Pine
Trembling Aspen
Flowering Dogwood
Chestnut Oak
Yellow Birch
¦
11
-------
MPO #3
FRP SEEDUNG REPORT
PETERSON ETAL
Table 4. Overview of Southern Commercial Pine seedling exposure research by project number
with principal investigator (* indicates regime used by each project).
Study
Description
SC14
FLAGLER
SC02
FONG
SC13
JOHNSON
SC06
KRESS
SC15
LOCKABY
SC12
McGregor
SC04
MCLAUGHLIN
SC05
REINERT
SC07
RICHARDSON
^ uj Controlled Field
3 (L
10 Controlled Laboratory
*
0
#
*
*
*
u Artificial
uj Modified Ambient
l/J ^
o ~
5- p. Simulated Ambient
X Uj
UJ ttl
Ambient Profile
*
*
*
*
*
*
*
*
*
Open-Top Chambers
Rain Exclusion
w Air/Rain Exclusion
t CSTR (Laboratory)
<: CSTR (Greenhouse)
Growth Chambers
Branch Chambers
*
*
~
*
*
*
~
*
*
*
*
~
*
*
Ozone
Acidic Rain
Acidic Mist
^ Sulfur Dioxide
_j Nitrogen Dioxide
2 Nitric Acid
Hydrogen Peroxide
Ambient
*
*
~
*
*
*
*
*
*
*
*
* *
*
*
*
*
*
Seedlings
Saplings
^ £ Mature Trees
^ Grafts
*
*
*
*
*
*
* *
*
*
(/i Loblolly Pine
UJ
o Shortleaf Pine
UJ
Slash Pine
*
*
*
*
~
*
* *
*
*
12
-------
MPO#3
PRP SEEDLING REPORT
PETERSON ETAL
Table 5. Overview of Western Conifer seedling exposure research by project number with
principal investigator (* indicates regime usedbyeach project).
Description
WC20
HOURS
WC09
MILLER
WC08
HOGSETT
WC07
TURNER
& u Controlled Field
2 £
" Controlled Laboratory
#
*
u Artificial
g 2 Modified Ambient
. £ § Simulated Ambient
Ambient Profile
*
*
0
Open-Top Chambers
Rain Exclusion
q Air/Rain Exclusion
§ CSTR (Laboratory)
^ CSTR (Greenhouse)
Growth Chambers
Branch Chambers
-
*
*
I
a-
Ozone
Acidic Rain
t Acidic Mist
1 Sulfur Dioxide
-j Nitrogen Dioxide
2 Nitric Add
Hydrogen Peroxide
Ambient
*
~
*
*
Seedlings
^ § Saplings
Mature Trees
«»¦ £ Grafts
*
*
*
*
Subalpine Fir
Ponderosa Pine
Q W. Redcedar
u DouglasFir
Englemann Spruce
W. Hemlock
Lodgepolei Pine
White Fir
*
*
9
*
13
-------
MPO #3
FRP SEEDUNG REPORT
PETERSON ET AL.
The most prevalent treatments included simulated acid precipitation and ozone applied alone or
in combination. One to six levels of acidity were used, ranging from pH 2.1 to S.6. Simulated acid
precipitation typically consisted of a chemical composition that reflected rainfall chemistry of the
study area (sulfur-to-nitrogen ratios were typically 2:1). In this document, terms such as acidity,
acid, or acid deposition refer to the hydrogen ion concentration plus the chemical composition
of the simulated precipitation.
One to six levels of ozone concentration were used, ranging from 0 to 320 ppb. Charcoal filtering
can remove up to 100% of ozone in ambient air. Therefore, CSTRs can attain 0 ppb treatments
while open-top chambers never quite approach 0 ppb due to mixing of filtered air and ambient
air through the open tops. Ozone concentrations in open-top chambers receiving charcoal-fil-
tered air are typically 30% to 50% of ambient concentrations. In addition to acidity and ozone,
one project varied the ratio of sulfur to nitrogen in the precipitation, while others applied
treatments of sulfur dioxide. Finally, several studies tested for interactions of acid precipitation
and/or ozone with winter injury or interactions with water stress. A range of treatments by
pollutants and levels of exposure for a selected number of studies is given in Table 6.
Treatments were applied to the seedlings during periods of active above-ground growth over
intervals varying from ten weeks to seven months. Multiple-year exposures are also being carried
out. However, this document is generally limited to results from a single season of exposures. As
such, some of the conclusions are tentative and may change as more data are obtained.
Simulated precipitation was applied as rain, mist, or fog. Usually precipitation was applied to
both foliage and rooting medium in a pattern reflecting historical trends for a specific region. In
some cases, precipitation was applied only to saturate the foliage; in such cases, the rooting
medium received controlled watering Ozone was applied over regulated time intervals, usually
during daylight hours. Applications varied among studies, but were of two general types. The
first, more simple type was a square-wave regime where a constant concentration of ozone was
applied over a definite time interval during the day. In more sophisticated designs, ozone
applications followed the monitored ambient concentrations for the region, where ozone con-
centrations typically increased to a mid-afternoon peak then decreased until dusk.
Response variables measured on the seedlings are presented as either effects or mechanisms.
Effects represent a change in seedling condition and in this document include visible effects or
growth changes. Mechanisms are the processes by which effects are manifested. The
mechanisms examined include carbon allocation, winter injury, and foliar leaching, reflecting
Scientific Questions 2.2,23, and2.4 posed in Table 1. In this document, carbon allocation is used
as a general term that includes growth, morphology, and general physiology, including photosyn-
thesis and respiration. In the results, growth is discussed separately as an observable effect,
whereas photosynthesis (carbon fixation) and physiological responses are discussed as
mechanisms that may affect growth.
Given the short time frame of the program, effects and mechanisms were examined simultaneous-
ly in the FRP research, as opposed to sequentially. While it was generally accepted that pollutants
at some level affect plants, the goal of the FRP was to quantify these effects on trees while also
identifying mechanisms of the effects, particularly those with relevance for policy.
The actual response variables measured were in some studies quite numerous. Some variables
were measured several times during the treatments, while others were measured only at the
termination of treatments. Visible effects included foliage discoloration (chlorosis, necrosis),
foliage loss (senescence), or whole-tree subjective classification. Growth effects involved some
measure of seedling biomass (linear measures of branches or roots, diameter of stem, or mass of
various components). Carbon allocation involved measures of photosynthetic rates, respiration
rates, tissue damage assessed microscopically, or tissue chemistry (sugars, starch and non-struc-
14
-------
MPO«3 FRPSEEDUNO REPORT PETERSON ETAL
Table,6. Treatment levels used in a cross section of seedling studies of the Forest Response
Program.
PI (Project #)A
Acidity (pB)
Osone (ppb)2
Sulfur dioxide
(ppb)
Jacobson (SF06)
2.5, 3.3, 4.3
2.8, 9.3, 4.2
2.6, 3.4, 4.2
Jensen (SF07)
3.5, 4.0, 4.5
CF, 150(6h), 150<6h)+70(18b)
3.0, 4.2
CF, 50(12h), 100(12h)+50(12h),
ISO (Ufa )-fl00 (12h)
Seller (SF13)
3.0, 4.3, S.6 .
0, 100
¦
<20, 50, 100
Unsworth (SF14)
2.5, 2.7, 3.0,
3.5, 4.0, 5.0
Weinstein (SF16)
CF, A, 2A, 3A, 4A (A-40, 7hm)
Kohut (SF31)
3.1, 4.1, 5.1
CF, A, 0.5A, 1.5A. 2A (A-38, 12tam)
Davis (EB01)
3.0, 4.2
0, 75, 150
Jensen (EB06)
3.0, 3.5, 4.2
CF. 70, 150
0, 20.
Fong (SC02)
CF, 160, 320
McLaughlin (SC04)
4.3
CF, 160, 320
3.3, 4.5, 5.2
CF, A, A+40, A+80, A+160 (A-36, 12hn>)
Reinert (SCOS)
3.3, 4.3, 5.3
0; 80, 160, 240, 320
4.3
0, 80, 160, 240, 320.
Kress (SC06)
3.5, 5.2
CF, A, l.SA, 2.25A, 3A (A-45, 12hm)
Turner (WC07)
2.1, 3.1, 5.6
Hogsett (WC06)
2.1, 3.1, 5.6 .
CF, 1.6B, 1.8B (B-53, 7hm)
CF, B..33B, .67B
(B-66)
Miller (HC09)
2.1, 3.1, 5.6
CF, B, 1.6B, 1.8B (B-53, 7hm)
CF, B..33B, .67B
(B=66)
1 PI - Principal Investigator
Project # - the number assigned to a study within FRP research
cooperatives: SC = Southern Commercial
SF - Spruce-fir
EH - Eastern Harawooas
WC - Western Conifers
2 CF - charcoal-filtered air
A - ambient air at the research site
B - base profile; designed to simulate a potential exposure regime
using a worst-case ambient in the West
h - hours
hm - hour mean
15
-------
MPO#3
FRP SEEDLING REPORT
PETERSON ETAL.
tural carbohydrates, photosynthetic pigments, or enzymes). Winter injury was examined as an
interacting stress; in these cases, seedling responses were measured after treated seedlings were
allowed to over-winter at ambient temperatures or after tissues were exposed to simulated frosts.
Foliar leaching involved some measure of solution chemistry of throughfall or of solutions in which
treated tissues were leached Refer to the project summaries in Appendix A for further details
on measurements taken at each study site.
43 Statistical Methods
All studies were designed to test hypotheses statistically. Building on exposure studies of crops
in the National Crop Loss Assessment Network (NCLAN), the experimental designs were
generally a variation of split-plot or randomized blocks. Most studies also incorporated repeated
measurements (usually five or more intervals) of total plant height and root collar diameter. Data
were analyzed via analysis of variance (ANOVA), analysis of covariance (ANCOVA), or regres-
sion techniques.
Important statistical issues identified by S&I for the seedling exposure experiments are: design
and analysis, relevance, combining results across experiments, and statistical power. These issues
were the subject of several workshops held in 1988 by C.E. Peterson and W.G. Warren with
statisticians and principal investigators associated with FRP seedling studies. In both the
workshops and subsequent contacts, discussions focused on how designs fit objectives (testing
for sensitivity, exposure-response, mechanisms), and whether the model and proposed analyses
would take full advantage of available covariates, time intervals, etc. A short discussion of these
issues follows.
43.1 Design and Analysis
An important function of FRP Work Plans and Quality Assurance Plans is to specify both the
objective and the experimental design with the proposed statistical procedure for the exposure
studies. For instance, are seedling studies intended to screen species or families for sensitivities
to pollutant exposures, help build an exposure-response (E/R) model, or to study mechanisms?
Has an E/R model been selected a priori common to all sites or is the testing of alternative models
an intended use of the data? In the case of ANOVA or ANCOVA, are the statistical models
completely specified (i.e., fixed and random effects identified)? What uncontrollable factors are
not measured or accounted for? Questions such as these must be addressed in order to evaluate
the contribution of each individual project and to identify common objectives among the
experiments when attempting to combine individual results. When considering the possible
geographical inference from combined results, it is also important to determine whether the
experimental sites were chosen purposively or randomly.
432 Relevance
The relevance of seedling experimental material for making inferences requires careful con-
sideration. At a minimum, seedling studies provide information for the evaluation of hypotheses
of changes in seedling condition that are applicable to seedling populations. Ideally, information
might also indicate hypotheses for the study of mature trees. However, direct extrapolation from
seedlings to mature trees is not intended with this research.
433 Combining Results
Although experimental results in forest ecology have not generally been used for regional
assessments, the results from many ongoing and planned studies of forest response to acid
16
-------
MPO #3
FRP SEEDLING REPORT
PETERSON ETAL
deposition are expected to play a significant role in future decision-making. The practice of
combining results from several studies has not been fully addressed in forest ecology with
statistical rigor, although the notion was formally introduced some time ago to researchers
(Fisher, 1932). Combining results from individual studies has the potential to increase the power
of conclusions drawn from the individual experiments. Consequently, an element of regionaliza-
tion can be brought into the evaluation of results.
The methodology for actually combining results of several experiments may take the form of
combining probabilities or test statistics (e^, Fisher, 1932). However, combining results from
the types of exposure studies carried out by the FRP may also involve the use of regression (e^
H eagle, Heck, Rawlings, and Philbcck, 1983; Rawlings, and Cure, 1985; Rawlipgs, Lesser, and
Dassel, 1988).
This challenge is an ongoing concern for the FRP (e.g^ WarTen, 1987). However, it must be
emphasized that many experiments with widely different variations in objectives and designs may
not be comparable, and in this case individual results should probably not be combined. Thus,
the primary goal has been to evaluate the seedling studies as they actually were carried out to
identify potential problems with treatments or experimental procedures that could either cloud
the interpretation of an individual experiment or hinder the combination of results, and to use
this information as feedback to principal investigators.
4.3.4 Statistical Power
Power of :the statistical test, that is, the probability of detecting a consequential change in
condition, should be considered for all controlled exposure studies at both the initiation and
conclusion of the research (e.g^ Rawlings,1986). Power is a function of experimental design,
including sample size and experimental (uncontrolled) error, where sample size refers to both
the number of seedlings used per treatment combination and the number of replicates (commonly
chambers). Each scientist should compute both the confidence limits for differences attributed
to pollutant exposures and the realized power. A simple abbreviated example of a power curve
computed by Project WC08 for ponderosa pine seedlings is included in Appendix B.
4.4 Assessment of Data Quality
The goals for data quality assessment in forestry research are to provide procedures that reduce
random and systematic error in treatment application and response measurement, and to evaluate
data accuracy and precision. To achieve these goals, quantification of the inherent variability
associated with both the treatment application and response variable measurement is necessary.
In this section, the variability inherent in the technology used to apply exposure treatments as
well as the variability of the experimental measures collected are discussed. A detailed summary
of quality control information for individual projects cited in this report is available from the FRP
Program Manager.
In the following discussion, data quality is represented graphically on control charts and frequen-
cy histograms. Control charts indicate when a measurement process has exceeded predeter-
mined statistical limits of variability. Limits on control charts represent the upper and lower
bounds for these estimates. Data quality objectives (DQOs) for upper and lower control limits
have been set by FRP investigators as their best estimate of the bounds within which the process
is in statistical control Warning limits were set at half the data quality objective(see Figure 2 for
an example). Data for acidic precipitation and gaseous exposures at the research sites have been
plotted as fluctuations about target concentrations. The objectives were to identify variation
about the target as well as the source of that variation, and to evaluate the appropriateness of the
17
-------
MPO #3
FRP SEEDLING REPORT
PETERSON ETAL
data quality objectives. A consistent trend outside of the warning limits indicates that the process
is not in control and an adjustment to the process is typically required.
Frequency histograms are also used to summarize large number of observations over time to
identify trends in the data. This technique can be used to show the frequency distribution about
a target, such as pH and ozone concentration, or to plot the coefficient of variation of analytical
data.
4.4.1 Acid Precipitation and Gaseous Exposure Techniques
The difficulties inherent in conducting controlled exposures with mature trees have focused
exposure-response research on seedlings. Growth chambers, continuously-stirred tank reactors
(CSTRs), and open-top chambers represent seedling exposure technologies ranging from highly
controlled but artificial environments to less controlled yet more ambient environments. All three
techniques require that ambient conditions be modified to apply experimental treatments.
Growth chambers permit maximum environmental control with some trade-offs; the small size
of the chambers dictates a limited number of seedlings, and the cost necessitates a limited number
of chambers, allowing little or no replication. Lack of replication may prevent the use of standard
statistical analyses to define treatment effects; thus, it may be only possible to infer biol6gical
trends. Growth chambers are intended to control environmental parameters such as light,
temperate d humidity. However, laminar air flow within the chambers and the associated
pollutant delivery systems may not be able to attain the same degree of treatment control for
gases, as illustrated in Figure 2.
The lack of treatment control in growth chambers maybe due to chamber design that mimics the
air flow over a tree canopy and that may cause improper gas mixing in the air stream. The
chambers also have significant horizontal and vertical gas concentration gradients above and
within the tree canopy. Gas concentration gradients in growth chambers were eliminated in
CSTRs by the use of a mixing impeller to improve the internal mixing of gases. Tighter control
of the gas treatments can be achieved, but it is often at the cost of control of environmental
variables. There are usually no environmental control systems associated with CSTRs. In the
greenhouse, the CSTR environment is affected by the seasonal ambient conditions (Figure 3).
CSTRs located in laboratories combine the tight control of the gas delivery system with better
environmental controls. The artificial light, temperature, and humidity control of the laboratory
does differ significantly from the greenhouse environment.
Although CSTRs are designed for thorough mixing of gases throughout the chamber, the systems
for delivering the gas to the chamber are unique to individual sites. Research projects using
CSTRs had varied amounts of manual control over the gas delivery systems. Adjustments to the
manual system that changes the gas concentrations within the CSTR are based on operator
judgement. The manual system can achieve uniform control of the treatments that meet the data
quality objective (Figure 4). However, the variation inherent in a manually controlled system is
evident in Figure 5. Equipment malfunctions and operator error are additional sources of
treatment variability. These two factors may affect the treatment results (Figure 6).
Open-top chambers are an alternative to the laboratory or greenhouse controlled environments.
Although significant differences exist between the chamber and ambient environments, this
technology is currently the best for controlled exposure of large numbers of trees in the field. Air
temperature increases of 2 to 3°C, light reduction of 10%, and both higher and lower relative
humidity have been observed in chambers when compared to ambient conditions. Modifications
to the original open-top design have had a large impact on the variability of environmental
conditions and treatment applications. The additions of a constricting baffle and rain exclusion
covers to the top of the chamber have minimized ingression of ambient air into the top of the
18
-------
MP0#3
FRP SEEDUNO REPORT
PETERSON ETAL
OZONE EXPOSURE TREATMENT
320 ppb
380
360 +
Ozone Cone.
Out of Control Limit
¦ Warning Limit
340"
300-
280" -
240"
220"
200"
180-
160"
120"
01/03
01/02
liir
01/04
01/05
Date
Figure 2: Ozone treatment in growth chamber with laminar air flow.
19
-------
MPO#3
FRP SEEOUNG REPORT
PETERSON ETAL
Mean Daily Humidity
% Humidity
100
90"
60
60
50"
40
30
10
12
e
9
10
5
e
7
2
0
4
1
Week #
Mean Daily Temperature
Degrees C
Aa~
-A k-
111111
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Ml 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1
Week #
Figure 3: CSTR temperature and relative humidity during 12 weeks of exposure in a greenhouse.
20
-------
MPO #3
PRP SEEOUNO REPORT
PETERSON ETAL.
OZONE EXPOSURE TREATMENT
320 ppb
Ozone Cone.
380
370
360
350
340
330i
320
310
300
290
280
270
260
0 12 3 4
Day #
Figure 4: Accuracy of treatment applications in three CSTRs.
21
-------
MPO #3
FRP SEEDLING REPORT
PETERSON ETAL
OZONE EXPOSURE TREATMENT
320 ppb
Ozone Cone.
Day #
OZONE EXPOSURE FREQUENCY
320 ppb
400
300 -
200
100
Frequency
180 200 220 2*0 260 250 300 320 340 360 380
Ozone Cone, (ppb)
Figure 5: Control chart showing variability of treatment application during one week and a
frequency histogram summarizing the precision of treatment application over 12
weeks.
22
-------
MPO #3
FRF SEEDUNO RETORT
PETERSON ET AL.
OZONE EXPOSURE TREATMENT
320 ppb
1200
Ozone Cone.
1000--
B00-"
000 - ¦
400"
09/02
00/03
09/04
I i i ir-
09/05
Figure 6: Example of equipment failure during treatment application.
23
-------
MP0#3
FRP SEEDLING REPORT
PETERSON ETAL
chamber and can improve filtering efficiency from 50% to 80%. However, the improved
efficiency is attained at the cost of increases in variability of environmental measurements from
ambient.
The large number of open-top chambers at individual research sites requires computer controls
for treatment application. Time sharing of air quality monitors and custom computer software
programs have significant impacts on the accuracy and precision of treatment application.
Although the technology is capable of applying fairly uniform treatments over time, environmen-
tal factors, such as wind speed and direction, can affect the treatments (Figure 7). Analysis of
quality control data indicated significant variability among sites using the same exposure
methodologies. The variability was site dependent with most attributed to the exposure technol-
ogy used at the site. The study type, exposure regime, facility, and pollutant also contributed to
this variability, although these variance components have not been estimated.
Acidic precipitation treatments, rain or mist, are used at most sites. Treatments are formulated
in tank batches and distributed to individual chamber application systems. Batch mixing allows
for high precision in treatments being applied to large numbers of plots, but operator error can
account for fluctuation around target concentrations (Figure 8). Realistic DQOs should be
technology-specific and derived from previously collected experimental data. Those DQOs
established in the Quality Assurance Methods Manuals were a best estimate of precision and, in
many cases, were not based on experimental data. Most DQOs were attained by almost all sites
for acidic precipitation, but not for gaseous pollutants.
4.42 Growth and Physiology Response Variables
To estimate accuracy and precision for growth and physiology measurements, it is best to separate
variables measured by traditional analytical techniques from those requiring more subjective
interpretation. The dry weight of a tissue or its nitrogen content can be determined by analytical
techniques that follow a standard operating procedure. Using National Bureau of Standard
weights and tissue samples, an estimate of accuracy and precision can be determined and the
investigator can follow the DQO criteria for accepting or rejecting data. For variables such as
height and rate of photosynthesis these criteria are not as easy to apply.
Natural variability in plant growth makes height and diameter determinations subjective.
Analysis of repeated measurement data indicated that the precision of diameter measurements
generally fell outside the DQO. Height was measured more precisely (Figure 9). Data from sites
with saplings and mature trees showed the opposite trend.
The rate of photosynthesis is an example of an instantaneous measurement of a dynamic system
that does not permit repeated measurements for precision estimates. When interpreting data it
is important to be aware of the inherent variability of these measurements and determine whether
the data should be viewed as relative or absolute values.
4.43 Summary
Traditionally, variation in the response variables has received some consideration when account-
ing for experimental error, while the inherent variability associated with treatment application
has been ignored or assumed to be zero. For research cited in this report, variability in treatment
application could be a major source of experimental error. Factors contributing to experimental
error include:
System malfunctions: The bimodal distribution of exposure in Figure 10 suggests that a system
failure occurred on numerous occasions during the experiment. Sometimes treatment concentra-
tions can vary enough to actually overlap other treatment levels. As shown in Figure 2, in one case
24
-------
MPO #3
FRP SEEOUNO REPORT
PETERSON ETAL
OZONE EXPOSURE TREATMENT
NON-FILTERED X 2.25
Ratio Actual/Target
1.0--
1.0 -
1.4 --
1.2 --
0.6 - -
o.e --
0.4 --
0.2 --
10/28
10/26
10/26
10/27
10/25
10/23
10/24
Date
OZONE EXPOSURE TREATMENT
NON-FILTERED X 2.25
Ratio Actual/Target
1.8 - -
1.6 -¦
1.4 -¦
1.2 - -
0.8 - ¦
0.6-
0.4 --
0.2 --
6/20
6/23
6/24
Date
Figure 7: Comparison of two exposure weeks in open-top chambers with differing amounts of
ambient air ingression.
25
-------
MPO #3
FRP SEEOUNG REPORT
PETERSON ETAL
Acid Precipitation Treatment
pH 5.0
6.6
6--
5.5--
4.5--
4 --
3.5
8
9
10
3
6
7
2
5
11
4
1
Week #
Acid Precipitation Treatment
pH 5.3
PH
5.7
5.6 --
5.5 ""
5.4 ---
5.3
5.2 ---¦
5.1 --
4.9
8
9
10
12
2
3
6
7
4
5
11
Week #
Figure 8: Variability between two sites in the accuracy of acidic precipitation treatments.
26
-------
MPO #3
FHP SEEOUNO REPORT
PETERSON ET AL
% Deviation From Mean Ht
80
BO
40
20
0
-6 -4 .-3 -2 -1 0 '1 2 3 4 6
% Deviation from Mean Height
% Deviation From Mean Diam
Frequency
26
20
16
10
6
0
-10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 10
% Deviation from Mean Diameter
Figure 9. Variability in height and diameter variables from repeated measurement data.
Frequency
27
-------
MPO#J
FRP SEEDUNG REPORT
PETERSON ETAL.
OZONE EXPOSURE FREQUENCY
3.0 x Ambient Treatment
Frequency
1600 -
1400 -
1200 -
1000 -
800 -
600 -
400 -
O 0.25 0.6 0.75 1 1.26 16 1.75 2 2.25 2.5
Ratio (Actual/Target)
Figure 10: A bimodal histogram indicating a system failure in ozone treatment application.
200
o
28
-------
MPO#3
FRP SEEDLING REPORT
PETERSON ETAL
ozone concentrations in a'chamber targeted for 320 ppb fell below 240 and 160 ppb (other levels
of ozone in the experiment). As another example, the large deviations from the ozone treatment
target seen in Figure 6 signaled system defects that may have adversely affected data quality.
Measurement error As illustrated in Figure 9,the variation in remeasurement of typical
response variables such as height and diameter can affect data quality.
Shifts in environmental parameters: Figure 3 shows how environmental factors such as
temperature and humidity can vary inside CSTRs throughout an experiment, which in turn can
affect seedling response to treatments. These environmental parameters, as well as tree culture
(seed grown or bare root nursery stock), soils, and growing medium (container or field) can
contribute to variation in response.
The assessment of data quality has not been completed for all projects. When that task is
accomplished, the primary use of the results will be to evaluate the relevance of the data quality
objectives to see if those specifications can be realistically attained. The achievement of an
average target value is relative to the accompanying fluctuations. The purpose of that information
is not to reject data from a given project, but is intended to help the principal investigator monitor
the system and make necessary corrections to keep the system under control However, infor-
mation such as magnitude and direction of the charted treatment fluctuations will be considered
in a subjective way, when evaluating the biological data from a project. Final data quality analyses
will be contained -joseparate data quality document currently in preparation, and updated in
MPO #4.
29
-------
MPO #3
FRP SEEDLfNG REPORT
PETERSON ET AL
5 RESULTS
Results from most of the 1988 seedling experiments will not be available until later in 1989.
Further analyses by the Principal Investigators are in progress, as well as review of those analyses
by S&I and summaries of data quality by QA. Thus, this section contains preliminary results from
two-thirds of the projects' 1986, 1987, and 1988 experiments; the amount of detail presented
reflects the data summaries and interpretations provided by the investigators. Additional results
will be incorporated with other FRP research in MPO #4 (due in 9/89).
Results for individual studies are presented by region (red spruce, eastern hardwoods, southern
commercial pine, and western conifers). For each region, effects (visible injdry and growth
changes) are presented, followed by a discussion of mechanisms (foliar leaching, carbon alloca-
tion, and winter injury). Species or family sensitivities within the region are discussed where
appropriate, and results within each region are summarized.
5.1 Red Spruce
Sixteen reports from seven projects comprise the seedling research on red spruce available for
summary. Most studies involved exposures of seedlings to controlled levels of acid precipitation
and ozone; l-v. . j; studies are continuing as multiple-year exposures. One project [SF13] tested
Fraser fir, a species fbat co-exists with red spruce in the Southern Appalachians; all other projects
used red spruce.
5.1.1 Visible Injury
Leith et al. [SF14] observed from 20% to 65% needle damage (browning) per seedling in red
spruce after the tenth week of twice-weekly applications of the three most acidic mists (pH 2.5,
2.7, and 3.0), as shown in Figure 11. Mists of lower acidity (pH 3.5,4.0, and 5.0) showed less than
5% damage. Primarily current-year needles were affected, particularly those on the upper
surfaces and distal ends of lateral branches. There was considerable seedling-to-seedling varia-
tion; some seedlings showed no damage. In addition to the acid effect, visible damage was linearly
related to accumulated nitrogen or sulfur exposures (Figure 12). Extensive needle loss occurred
about 20 weeks after the first signs of discoloration. Two of the acid treatments (pH 2.5 and 3.0)
also induced earlier bud flushing the following spring. There was no evidence of bud mortality
due to acid mists.
Jacobson et al. [SP06(a)J performed six separate experiments during 1985 through 1987. The
response of red spruce seedlings to total acidity and the ratio of sulfuric to nitric acids was
examined in three field experiments. In three greenhouse experiments, the effects of acidity,
anion ratio, and drying of precipitation on the foliage were evaluated. In the 1985 and 1987 field
experiments, severity of injury increased in mists of pH 2.5 and 2.6 when compared with pH 3.5
or higher (p = 0.0001). At these low acidities, severity of injury (measured as percent of needles
injured) also appeared to be a function of anion ratio; sulfate acids produced greater injury than
nitrate acids (17% versus 2%;p <0.0002). The pH effect was still evident the following spring
after the seedlings overwintered without additional treatments, but the anion ratio effect was not.
The 1985 and 1987 results were not repeated in 1986; there were no effects of acidity, anion ratio,
or their combination, and severity of injury was never greater than 1%.
In Jacobson et al.'s [SP06(a)J greenhouse experiments, foliar injury of seedlings exposed to acid
mists of pH 3.0 was approximately double that observed at pH 4.0 (p - 0.001). Needle injury at
pH 3.0 averaged 33% for nitrate acids and 46% for sulfate acids. Sulfuric acids were associated
30
-------
MPO«
FRP SEEDUNO REPORT
PETERSON ETAL
bO
C
*o
0)
a>
W ce
u
o
v
ex o
b£)
CO "O
(C
o
15
(0
a;
S
¦o
a>
d
Final mist
application
70
60
50
40
JH 30
20
10
0 -
6 14 28
Oct
pH
1987
19 7
Jan Feb
1988
10
Mar
Scoring dates
Figure 11: Visible foliar damage to red spruce seedlings exposed to twice-weekly acidic mists.
Treatments began July 24; no treatment showed any damage prior to October 6.
Symbols represent means; there was considerable variation across seedlings in
amount of damage (from Leith et al. [SF14])
31
-------
MPO #3
FRP SEEDUNG REPORT
PETERSON ETAL
c
_o
Q.
-------
MPO #3
FRP SEEDUNO REPORT
PETERSON ET AL.
with 20% to 100% greater injury than nitrate acids at both pH levels (p = 0.10). In other trials,
intermittent mists were as potentially harmful as continuous mists even though intermittent mists
deposited less total acids on the foliage. Winds between mists enhanced foliar injury, especially
with high sulfate mists. Responses of current-year needles to acidic mist depended somewhat on
seed source.
Patton et aL [SPOT] exposed red spruce to ozone (50,100,150 ppb) and acid precipita-
tion (pH 3.0 and 42) for six months. They examined foliar injury (chlorosis and necrosis) under
water-stressed and well-watered conditions (one versus three waterings per week). No dear
effects of ozone were apparept in any treatment combination. No effects of acidity were observed
under well-watered conditions; generally, more than 90% of these seedlings were classified as
0% foliar injured. Water-stressed «»*Hlingc showed more foliar injury than well-watered see-
dlings, and pH 3.0 treatments appeared to ameliorate the injury somewhat compared to pH 42.
Averaged across pH, from 22% to 47% of the water-stressed seedlings were classified as 1% to
25% foliar injured. Patton et aL suggested that increased nitrogen in the pH 3.0 treatments may
have reduced the foliar injury due to water stress.
To summarize, visible injury to foliage of red spruce seedlings began to occur in these studies at
about pH 3.0. Injury increased with increasing acidity, with increasing acid sulfur content versus
nitrogen content, and with increasing opportunity for acid precipitation to dry on the foliage.
Only one study examined foliar injury due to ozone; no damage was observed.
5.12 Growth Changes
Laurence et al. [SF31] examined the effects of acid (pH 3.1,4.1, and 5.1), ozone (0.5,1.0,15, and
2.0 times ambient concentrations at Ithaca, NY; ambient = 38 ppb, 12-hr average), and their
combination on growth (height, roots, needles, and stems) of red spruce seedlings. During three
months of exposure in 1987, few effects were found (alpha = 0.10). In some cases, interactions
between main effects and time of harvests were observed. However, the response curves were
relatively flat, and Laurence et aL believed such interactions were of little importance. The data
showed marginal linear increases in stem dry mass (14%) and root dry mass (16%) with increasing
ozone levels when averaged across the pH treatments (p = 0.14 and 0.11, respectively).
As part of Laurence et aL's study, a second year of exposures of the same seedlings during 1988
is reported by Kohut et aL [SF31]. Interactions of ozone and add were observed (p < 0.10). The
interactions varied; they generally consisted of a decline in dry mass of needles and stems with
increasing ozone in the pH 5.1 treatment, little or no effect of ozone in the pH 4.1 treatment, and
an increase in dry with increasing ozone in the pH 3.1 treatment An exception to this pattern
was that the combination of pH 3.1 and L5 times ambient ozone resulted in a large depression
in dry mass.
Patton and Jensen [SFD7] observed few effects of simulated add rain (pH 3.5, 4.0, and 4 J) or
ozone (charcoal-filtered air, alone or with additions of ozone up to 150 ppb) on growth of red
spruce seedlings after six months of exposure in 1987. Effects of pH and ozone were detected
on mass of current-year stems only at an intermediate harvest (p.S.0.04 ). The authors reported
no interactions. Patton et aL [SF07] followed up on these results with a two-year study of the
effects of simulated add rain and ozone on growth. In the first year (1988), they observed many
effects of add preapitation on growth. For instance, when data were summed across ozone and
water-stress treatments, pH 3.0 reduced several components of growth when compared with pH
42 (e.g., stem length, needle weight, root weigfrt). These decreases averaged 33% (p typically
.£.0.05). There were few effects of ozone reported (exceptions were roots, p - 0.10, and stem
diameter, p = 0.03), and those that did occur are difficult to interpret. Treatment means for
roots exposed to ozone were not reported. The two higher ozone exposures (100 and 150 ppb),
33
-------
MPO #3
FRP SEEDLING RETORT
PETERSON ETAL
averaged across the pH treatments, resulted in 11% to 18% greater stem diameters compared
with the lower ozone treatment (50 ppb); however, increases were greater in the 100 ppb than in
the 150 ppb exposures. There were some pH x water-stress interactions (p< 0.008). At pH 4.2,
seedlings that were watered three times per week grew more than those watered once per week,
but the reverse was true at pH 3.0. The reason for this effect was unclear.
Alscher et al. [SF16] and Cumming et al. [SF16] observed no effect of ozone (up to 4 times ambient
concentrations at Ithaca, NY; ambient = 40 ppb, 7-hr average) on growth of red spruce seedlings
after either seven months or after 16 weeks of exposure.
Jacobson et aL [SF06(a)] observed various effects of acid mists (pH 25 to 4.5) on red spruce
seedling growth following exposures ranging from six weeks to five months.' In a six-week
greenhouse trial, compared with no mist, acid mists at pH 2.6 had no effect on stem diameter,
reduced shoot lengths by 24% (p = 0.004), and reduced shoot volumes by 31% (p = 0.03). In
another greenhouse trial, pH 3.0 and 4.0 mists led to small increases (<. 7%) on shoot length
compared with no mist (p = 0.08). Shoot length was also affected by anion type; compared with
no ihist, sulfate reduced shoot length while nitrate appeared to increase shoot length (p
-------
MP0#J
FRP SEEOUNG REPORT
PETERSON CTAL.
in the micronutrient boron and the element sodium were also measured. Patton et aL suggested
membrane damage as a mechanism for increased leaching of magnesium and manganese but they
do not discuss the absence of increases in calcium and potassium. Throughfall concentrations of
iron and manganese were higher with precipitation of pH 3.0, while highest levels of potassium
and boron occurred with pH 42.
5.1.4 ^Mechanism: Carbon Allocation
Laurence et aL [SF31] report no effects of acid (pH 3.1,4.1, and 5.1) or ozone (0.5,1, L5, and 2
times ambient concentrations), alone or in combination, on photosynthesis of red spruce see-
dlings following three-month exposures in 1987 (alpha « 0X15). However, photosynthesis rate
decreased by 11% in the two highest ozone levels compared with the two lowest ozone levels. In
1988, Kohut et aL [SF31] observed that rates of photosynthesis in the same seedlings increased
17% at pH 3.1 precipitation treatments when compared with pH 5.1 (p^.0.01) and that ozone
had no effect on photosynthesis rates. The higher rates of photosynthesis at higher acidity were
not reflected in greater growth, however. A third year of exposures is planned.
Patton and Jensen [SFD7] found few effects of simulated acid rain (pH 3.5, 4.0, and 4.5) or ozone
(charcoal-filtered air, either alone or with additions of ozone up to 150 ppb) on carbon allocation
in red spruce seedlings following six-month exposures. Twelve parameters of nonstructural
carbohydrates in needles and roots were examined. Ozone was associated with increases in levels
of starch in the current-year needles when measured during mid-treatment (p = 0.024), but the
effect disappeared with continued treatments (p 0.980).
Thornton et al. [SF27] observed some enhancement of photosynthesis in commercially grown red
spruce seedlings following either exclusion of clouds but not ambient ozone or exclusion of both
clouds aiid ozone on top of Whitetop Mountain, VA (Figure 13).. Native stock seedlings did not
appear to respond in a similar way. Statistical analyses have not yet been performed.
Fincher et aL [SF16] observed effects of ozone (charcoal-filtered air and 1,2, and 3 times ambient
concentrations at Ithaca, NY) on mesophyll cells of red spruce seedlings, following seven-month
exposures during the summer and fall of 1987. In November and December, 1987, after the onset
of frosts, mesophyll cell damage was 2.0 to 4.8 times greater in the ozone treatments compared
with charcoal-filtered air as shown in Figure 14 (p = 0.017). High cell-to-cell variation in damage
was observed, and no visible signs of foliar damage were evident at the time of data collection.
There was no evidence that photosynthetic rates, chlorophyll content, or carotenoid contents
were different among ozone treatments. However, by-May following overwintering with no
additional ozone treatments, seedlings exposed to ozone did show 5% to 50% reductions of
photosynthetic pigments (chlorophyll a, chlorophyll b, and carotenoids) when compared with
charcoal-filtered air. The observations of visual damage in spring did not correlate with the prior
mesophyll damage (see Fincher et aL in Section 5.1.5).
Cumming et al. [SF16], in combination with the work of Fincher et aL [SF16], reported several
trends due to ozone in red spruce seedlings during exposures in 1986. Compared with charcoal-
filtered air, ozone at 2 times ambient resulted in 12% decreases in total chlorophyll concentrations
(p j£.0.05), 20% increases in the rate of photosynthesis (expressed on per mg chlorophyll basis,
p <0.05). but no changes in the rate of respiration or electron transport (alpha «= 0.05). The
effects of ozone appeared to be a function of season; while there were no differences in
photosynthesis in July, seedlings exposed to charcoal-filtered air showed decreased photosyn-
thesis in October, while those exposed to all levels of ozone did not (p^.0.05). Photosynthesis
normally decreases in the fall as frost hardening; occurs. Therefore, ozone could have a negative
effect on seedlings by musing photosynthesis to be maintained at pre-hardening levels. A
35
-------
2
3
8
o
o
tn
,
S n
D O
B
cr _j
&> ^
Photosynthetic rate, current year's growth
(u mol CC^* g dw
z-\
o
o
ro
o
o
S 1)
Oi
o
o
A
o
o
<
©
(/)
W
v
or
(0%
2 o
in
D lO
»o
3
shade c!oth
ambient chamber
cloud removal chamber
(ambient air with clouds removal)
0jj+ cloud removal chamber
(charcoalfiltered air with clouds removed)
shade cloth
ambient chamber
c
2
O
fflaartffaa remov,,)
mmmmMm
0^+ cloud removal chamber
(charcoal-filtered air with clouds removed)
-------
MPO #3
FRP SEEDUNO REPORT
PETERSON ETAL
X
U
_C
o
o>
n
E
a
Q
0.8-
0.7-
0.6-
0.5-
0.4-
0.3-
0.2-
0.1 -
0.0--
Mesophyll Injury in early winter
r
2
T
3
Q Nov and Dec
Dec.
-i
4
Ozone Level
Figure 14: Mesophyll cell damage in red spruce seedlings observed in December following
exposure to ozone for seven months (from Fincher et al [SF16]).
37
-------
MPO #3
FRP SEEDUNG REPORT
PETERSON ETAL.
reduction in densely-staining material was also noted in microscopic examinations of tissues from
ozone treatments; this is suggestive of reduction in the rate of frost hardening.
Tseng et al. [SF13] measured no effects of ozone (< 20, 50,100 ppb) on transpiration or needle
conductance of Fraser fir seedlings (alpha = 0.05). Photosynthesis rates decreased in all
treatments during the experiment's duration, possibly due to needle aging or handling. The
decrease in photosynthesis was 30% to 100% greater in the highest ozone treatment at the fifth
week than in the lower treatments (p^.0.05). Although the highest ozone treatment was as-
sociated with a nearly 100% greater decrease in photosynthetic rates at the tenth week, differen-
ces were not significant, implying a great amount of variability in the results. There were no
significant interactions of ozone with moisture stress.
Overall, there appears to have been little effect of acidity on physiological processes associated
with carbon allocation. Ozone may have caused alterations in seasonal patterns of photosynthesis
(Cumming et al. [SF16]), damage to mesophyll cells (Fmcher et al. [SF16]), and depressions of
photosynthetic pigments (Fmcher et al. [SF16], Cumming et al. [SF16]). However, there is no
evidence that ozone caused changes in rates of photosynthesis (Kohut et al. [SF31], Thornton et
al. [SF27], Fmcher et al. [SF16], Tseng et al. [SF27]).
5.1.5 Mechanism: Winter Injury
Fincher et al. [SF16] assessed the effects of ozone expressed as winter injury. They classified
seedlings for visible injury in spring after summer and fall exposures to ozone as high as three
times ambient at Ithaca, NY (ambient = 40 ppb, 7-hr average) followed by exposure to winter
conditions. They found no effect of ozone on either total seedling condition or the number of
shoots with brown needles among 85 seedlings examined (p - 0.93 and 0.87, respectively). Half
of the seedlings did show some browning, and among these seedlings there was an effect of ozone
(p = 0.005). However, the effect was not consistent; compared with charcoal-filtered air,
intermediate ozone levels were associated with increased browning while the highest ozone level
was associated with reduced browning. It should be noted that the same pattern was observed
with mesophyll damage (see Figure 14).
Murray et al. [SF14] found increases in the rate of foliar leaching from red spruce seedlings at
successively colder exposures below freezing (Figure 15). Analysis of variance indicated two
different leaching rates (p <_0.05). The lower leaching rate was produced by shoots exposed to
higher temperatures. These shoots were regarded as still living. The higher leaching rate was
produced by shoots exposed to lower temperatures. These shoots were regarded as dead. To
separate live from dead tissue, the authors derived a critical leaching rate of 0.4% h"1 (expressed
as a percent of that amount leached when the shoots were autoclaved).
Fowler et al. [SF14], building on Murray et al.'s work [SF14], also found that foliar tissues of red
spruce seedlings exposed to increasingly colder temperatures resulted in increased foliar leaching
rates. Leaching rate corresponded with visual damage, and a temperature that resulted in a
leaching rate corresponding to more than 50% needle death was established. The temperature
that produced this critical leaching rate was designated as an LT50. LTsos decreased over all
treatments with time as the seedlings developed frost hardiness during the fall months. However,
lower pH mists applied over a 22-week interval elevated the LT50 by as much as 12°C or,
alternatively, delayed frost hardening by two to four weeks (Figure 16).
These studies indicate that acidity increased potential for winter injury, particularly during the
frost hardening periods. The mechanism of action appears to have been cellular disruptions
caused by freezing resulting in excess foliar leaching.
38
-------
MPO#) FRPSEEDUNOREPORT PETERSON ET AL.
0.8
0.6
0.4
0.2
-196 C°
(liquid nitrogen)
-12°C
40
80
120
160
200
Time
(h)
Figure 15: Increases in conductivity over time (leaching rate) of red spruce seedling foliage
exposed to varying temperatures (from Murray et aL [SF14]).
39
-------
MPO #3
FRP SEEDUNG REPORT
PETERSON ET AL
LTS0 <° c>
-10
-20
-30
-40
-50
Key
~
~
pH
2.5
2.7
21 Sept. 5 Oct. 19 Oct. 2 Nov. 16 Nov. 30 Nov.
Date
Figure 16: Elevated LTsos (lethal temperature affecting 50% of the shoots tested) of red spruce
seedling foliage following exposure to acid mist beginning July 24 (from Fowler et al.
[SF14]).
40
-------
MPO #3
FRF SEEOUNO REPORT
PETERSON ET AL
5.1.6 Summary
While there is some evidence that acidity and ozone are deleterious to red spruce seedlings, the
results are inconclusive at this time. The variations in seedling response across studies, and in
some cases within studies, leads to the conclusion that there were sources of uncontrolled
variation in some of the studies.
Controlled exposures of generally one-season duration did not decisively demonstrate growth
depressions due to acidity and ozone. Visible injury due to add did begin to occur at pH levels
about 3.0 and lower. Acid and ozone may induce foliar leaching. There is evidence that ozone
may damage foliar mesophyil cells and thus disrupt photosynthesis. However, consistent depres-
sions in photosynthesis due to ozone were not evident across the studies. The most conclusive
evidence for potential damage to fed spruce is the reduction of frost hardiness due to acid
precipitation.
5.2 Eastern Hardwoods
Two reports from two projects are reported here [EH01, EH06]. Twelve species of hardwoods
were examined for visible injury and growth changes due to add deposition and ozone. Alth'ough
sulfur dioxide exposures were planned, experimentation problems preduded a dear study.
5.2.1 Visible Injury
Jensen and Dochinger [EH06] measured stipple injury and defoliation in response to 16-week
exposures to all combinations of add predpitation (pH 3.0,3.5, and 4.2), ozone (0,70, and 150
ppb), and sulfur dioxide (0 and 20 ppb). There were no effects of sulfur dioxide or addity on
stipple injury or defoliation of any of 10 hardwood spedes tested. No stipple injury due to ozone
was observed on white oak, shagbark hickory, American beech, sugar maple, and European
beech. The five remaining species (yellow-poplar, yellow birch, sweetgum, red maple, and white
ash) showed an increase in stipple injury with increasing ozone. The five spedes also showed
defoliation in response to ozone; yellow birch was the most sensitive, losing 30% of the total leaves.
Davis and Skelly [EH01] observed no consistent effects of 12 weekly applications of add
predpitation (pH 3.0 compared to 42) on foliar response variables of hardwood seedlings, either
alone or in combination with ozone. However, ozone (75 or 150 ppb, 2 days/week) was associated
with several types of foliar injury. Adaxial stipple was observed on the older leaves, and most
predominantly on black cherry, followed by sweetgum, yellow-poplar, white ash, red maple and
yellow birch. No stippling occurred on leaves of either red oak or white oak. Chlorosis occurred
in some degree on all spedes, but was most common on red maple, red oak, and white oak.
Necrosis was most common on black cherry and yellow-poplar, while fleck was seen on white ash.
522 Growth Changes
Jensen and Dochinger [EH06] measured height, leaf dry weight, leaf-area, and new-growth dry
weight at the end of 16-week exposures to the combinations of add predpitation, ozone, and
sulfur dioxide described above. Because of experimentation problems, the effect of sulfur dioxide
on all spedes was incondusive. No significant growth changes were observed in response to
addity, ozone, or sulfur dioxide for white oak, shagbark hickory, American beech, and European
beech. For the remaining five spedes, (yellow birch, sweetgum, sugar maple, red maple, and
white ash) varying pH had a greater effect on the growth variables than did ozone. However,
depending upon the spedes and variable considered, the response to addity was sometimes
41
-------
MPO#3
FRP SEEDLING REPORT
PETERSON ETAL
inhibitory and sometimes stimulatory. White ash showed decreases of 6% to 20% in the four
growth measures. Red maple, ycLlow-poplar, sweetgum, and yellow birch showed increases in
most growth measures, typically ranging from 3% to 20%, in response to increased acidity.
Increased ozone was associated with decreases in some growth measures for red maple, yellow-
poplar, white ash, and yellow birch.
Davis and Skelly [EH01] report that, of eight species tested, only black cherry seedlings exhibited
a consistent decrease in growth variables in response to increased acidity (pH 3.0 versus pH 42).
Ozone (75 and ISO ppb, 2 days/week) was associated with suppressed growth measures for black
cherry, red maple, yellow birch, and possibly white oak seedlings compared with charcoal-filtered
air. Ozone appeared to have no effect on growth measures for yellow-poplar, white ash, or red
oak seedlings. Growth of sweetgum seedlings appeared to have been stimulated by ozone. Ozone
and acidity did not appear to have interactive effects on growth of hardwood seedlings.
523 Species Sensitivity
Jensen and Dochinger [EH06] found white oak, shagbark hickory, American beech, and
European beech to be tolerant to acid precipitation, ozone, and sulfur dioxide. Although sugar
maple had decreased new-growth dry weight with increasing acidity, it exhibited no foliar injury.
The species showing foliar injury and/or growth responses to acidity or ozone included the
remainder: white ash, yellow birch, sweetgum, red maple, yellow-pop1' r' sugar maple.
Davis and Skelly [EH01] found black cherry to be the most sensitive species to acidity based on
growth reductions and the most sensitive to ozone based upon stipple response.
5.2.4 Summary
Neither acid precipitation (pH 3.0 versus 4.2) nor sulfur dioxide was associated with any visible
injury on hardwood seedlings. Ozone (75 and 150 ppb) was associated with several types of foliar
injury on hardwood seedlings: defoliation, adaxial stipple, chlorosis, necrosis, and fleck. The
response appeared to be species-specific; black cherry was most sensitive to ozone injury.
Growth responses were varied in response to acid precipitation; as acidity increased, white ash
and black cherry showed decreases in growth measures, while red maple, yellow-poplar, sweet-
gum, and yellow birch showed increases in growth measures. Increasing ozone was associated
with decreased growth for black cherry, red maple, yellow-poplar, white ash, and yellow birch.
53 Southern Commercial Pines
Reports from five projects are discussed here, based on four study sites: SC05 at North Carolina
State University (NCSU), SC04 at Oak Ridge National Laboratory (ORNL), SC06 at Duke Forest
(DUKE), and SC02 at Texas A & M University (TAMU). An additional study (SC07) used plant
material treated under project SC06. Descriptions of these projects are in Appendix A. Ap-
proximate ambient ozone levels observed at the field sites (see Table 6) were 43 ppb and 47 ppb
for DUKE (1987 and 1988, respectively) and 26 ppb for ORNL in 1986.
Most of the research on southern commercial pines has focused on the response of loblolly pine
seedlings, particularly the relative responses of 100 commercially important families (in this
report, all family references are to open-pollinated plant material). Evaluation of responses by
different families was based on quantitative measurement of stem height, rootcollar diameter,
above-ground biomass, and net photosynthetic efficiency, with biomass as the major growth
variable.
42
-------
MPO«3
FRP SEEDLING REPORT
PETERSON ET AL.
In addition to results from the individual studies, Shafer et aL used a subset of data from the study
sites (up to eight common families ofloblolly pine) to examine both inter- and intra-site continuity
of growth results. For each family, relative response models were computed so both the observed
and predicted values were adjusted to predicted values -control treatment levels. One of the
first questions asked was whether bio mass responses of eight famil"*-* to ozone were consistent
among the three sites with indoor facilities (CSTRs and growth chambers at SC04, SOQ5, and
SC02) in a way that permitted pooling of data. It was determined thatthc responses of die eight
families were too heterogeneous, to pool, lading to the conclusion that exposure/response
relationships on a family basis differed among sites. Examples of the results are given in Figure
17 for relative biomass changes, indicating no dose patterns of family response among the sites,
except for one that the authors acknowledge could have occurred by chance alone (Family C).
Given the heterogeneity of responses from the eight common families, many contrasts reported
by Shafer et aL in the following discussions are qualitative, and thus no probability levels are
specified.
53.1 Visible Injury
Unlike the other regions, the motivating factor for studying effects of air pollutants in southern
pines is a suspected growth decline, without visible injury observed in the field. Therefore, any
visible injury reported is discussed with other effects.
532 Growth Changes
Kress et aL [SC06] noted reductions in four growth variables of three families exposed to ozone
in open-top chambers when compared with charcoal-filtered (CF) air (61 ppm-hrs) after the first
year of exposure (from March to December, 1987). Averaged over the three families, seedlings
exposed to non-filtered air (132 ppm-hrs, considered ambient) showed reductions from 1% to
9% in height, diameter, foliage biomass, and stem biomass. Ensure to 193 ppm-hrs (L5 times
ambient, 12 hr/day) resulted in increases of 3% to 20% in these four measures. Seedlings exposed,
to 288 pipm-hrs (225 times ambient), exhibited growth reductions ranging from 9% to 37%. In
all cases, the larger changes in growthwere seen for biomass!. Shafer et aL [SC06] produced a
linear regression for the foliage biomass data, but the regression severely underestimated, the
biomass of the 193 ppm-hrs treatment; a reduction of 95% was predicted, while the data showed
a 6% increase. After the first exposure season, Schoeneberger (pers. comrtL, SC06) found
changes in Up root biomasses of +5% to -21% (0% average) and -19% to -27% (-26% average)
for respective treatments of L5 and 3 times ambient when compared with CF.
At the end of the second year of exposure (1988), Kress et aL [SC06] found that, compared with
CF (60 ppm-hrs), non-filtered air (136 ppm-hrs)led to reductions in 1988 growth measures
ranging from 14% to 32%, with total above-ground biomass and total stem biomass showing larger
changes than diameter and height' increments. Furthermore, all growth variables exhibited
reductions'ranging from 30% to 80% at 298 and 396 ppm-hrs (2^5 and 3 times ambient),
respectively. However, at 205 ppm-hrs (1.5 times ambient) growth stimulations of 1% to 6%
occurred compared with CF air, as well as some increases compared with 136 ppm-hrs (p = 0.05
for stem and diameter increment;/? = 0.10 for total treebiomass). This finding suggests a possible
charcoal-filtered versus non-filtered air protocol problem (SC06 03/15/89 quarterly report).
In comparison to CF air, Mclaughlin et aL [SO04] found that for 33 of the 44 families ofloblolly
pine seedling? tested at ORNL, 12-week Exposures to. ambient levels vof ozone (non-filtered air)
suppressed height and diameter growth (Figure 18). They also report that height growth of
Kedlings was stimulated by moderate levels of ozone (ambient + 40 ppb) while diameter growth
of the same seedlings was suppressed (Figure 19). At higher levels of ozone (ambient + 160 ppb)
43
-------
MPO #3 PRP SEEDUNG REPORT PETERSON ET Al-
Family C
NCSU
ORNL
TAMU
0 20 40 60 60 100
Cumulative Ozone Dose (ppm x h)
ra
E
o
E
o
>
ra
a>
EC
Family E
120 -
NCSU
"a ORNL
TAMU
0 20 40 60 B0 100
Cumulative Ozone Dose (ppm x h)
140
Family G
120
¦o-
100
NCSU
ORNL
TAMU
0
20
40
80
100
60
140
Family H
120
80
o NCSU
-a- ORNL
TAMU
40
0
20
40
60
80
1 00
Cumulative Ozone Dose (ppm x h) Cumulative Ozone Dose (ppm x h)
Figure 17: Relative total above-ground biomass (1986 data) of four open-pollinated families of
Pinus taeda after exposure to ozone in controlled-environment facilities at NCSU,
ORNL, or TAMU (from Shafer et al. [SC04, SC05, SCOT]).
44
-------
MPO«J
FRP SEEDLING REPORT
PETERSON ETAL
ORM.-OWQ 862-6268
GROWTH RATIO (Ambient/Charcoal Filtered)-!
§
0
0.4
0 3
0.2
0.1
0
-0.1
-0.2
-0.3
-0.4
-0.5
-0.6
0.15
0.1
0.05
0
0 05
-0 1
-0 15
-0.2
-0.25
HEIGHT
<
(E
¦x -O 05 -
III
I ll I l-ll I
I!.,,,-!... I III" ¦ I
DIAMETER
111 131 151 17| 19|2l|24|26|28l30|32|34|36|38|40|42|44|46U8|50|52i54!
12 14 16 18 20 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55
FAMILY NUMBER
Figure 18: A comparison of 44 loblolly pine families for the ratio (minus 1.0) of their height and
diameter growth in ambient ozone to that in charcoal filtered chambers (Figure 3.8
in McLaughlin et aL [SC04]).
45
-------
MPO #3
FRP SEEOUNG REPORT
PETERSON ETAL
OZONE EFFECTS BY FAMILY
(Ambient +40 vs Charcoal Filtered)
I
Q
UJ
CO
LL)
tj
LL
<
o
0
cr
<
1
o
\
o
+
I-
z
UJ
n
£
<
CO
o
"n3
cc
£
o
5
o.a
0.7
0.8
0.5
0.4
0.3
0.2
0.1
0
-0.1
-0.2
-0.3
11
&
HOOMT Q«OWTH
n
F
gj
0
10
2i-
27
32
37
42
47
62
0.04
0.04
0.12
11 18 21 27 32 37 42 47
FAMILY NUMBER
Figure 19: A comparison of 44 loblolly pine families for the ratio (minus 1.0) of their height and
diameter growth in ambient + 40 ppb to that in charcoal-filtered chambers (Figure
3.8 in McLaughlin et al. [SC04]).
46
-------
MPO#3
FRP SEEDLING REPORT
PETERSON ETAL
both measures of growth were suppressed. Finally, compared to pH 52, height growth was
stimulated at pH 45 and depressed at pH 33 (Figure 20).
Reinert et aL [SC05] report that for both the 1986 and 1987 single-season (11-week) exposures
of three loblolly families to either acidjty or acidity and ozone, an acidity x ozone interaction was
found for diameter growth of one family. Ozone alone suppressed both height growth and
diameter growth in both 1986 and 1987 (p <0.05). The data means are graphically depicted in
Figure 2L Shafer et aL [SC05] report that, in general, family responses at NCSU in 1986 were
replicated in 1987 at the same facility. Diameter growth for 12 families was suppressed by ozone
in 1986, and 11 of these 12 showed similar suppressions in 1987. In 1987, Shafer et aL [SC05] also
examined repeatability of family responses to an ozone x run acidity interaction found at NCSU
in 1986. The general nature of the 1986 response curves was repeated; diameter growth was
suppressed by ozone in the three families tested in both years. However, the effect of acidity
varied by family. There was no effect for two families, and an altered response of the third due
to a treatment interaction; in the absence of acidity, low levels of ozone produced a positive
response and high levels of ozone produced a negative response. However, with increased acidity,
the stimulatory effect of ozone disappeared.
Comparability of indoor and field results was examined by Shafer et aL using data from three
common families. One comparison was of responses to a combination of ozone and acid
precipitation for three families of loblolly pine in greenhouse conditions at NCSU [SC05] versus
responses from an ORNL [SC04] field experiment. Biomass was unaffected by either ozone or
acidity at ORNL, and although low levels of ozone at NCSU produced a positive response and
high levels of ozone produced a negative response in the absence of acidity (Le., a possible
treatment interaction), the stimulatory effect of ozone disappeared with increased acidity.
Overall, both types of experiments showed a pattern of negative biomass responses to ozone. At
ORNL, only two of eight families tested showed the same linear ozone response surface when
comparing field results (at pH 4.5 or 5.2) iand indoor results (at pH 43) at the same site. For the
NCSU-indoor and DUKE-field comparison of three families, the ozone exposure/response
models were quadratic and acidity-dependent at NCSU, but linear and unaffected by acidity at
DUKE.
Available analyses by Shafer et al. from studies in open-top chambers show differences among
families in sensitivity to acid rain and ozone [SC04, SC06]. Responses to ozone were variable,
with apparent stimulation of height but not diameter growth at the lowest ozone levels (no ozone
in CSTRs and CF in field), and reduction in height and diameter with increasing ozone concentra-
tions. Near-ambient levels of acidity in simulated acid rain stimulated height growth at below-
ambient ozone levels. Overall sensitivity of seedling growth to ozone appears to be greater under
field conditions than in associated laboratory studies. This greater sensitivity may be due to
greater growth in the field or to higher cumulative ozone exposures due to ambient levels in the
field environment. This difference might also be due to other factors such as variability in age of
seedling material, exposure length,'soils, or fertility regimes.
In general, decreased diameter growth with increased ozone was observed at ORNL [SC04] for
the majority of 44 families of potted seedlings tested. Kress et aL [SC06] noted the same effects
for three open-pollinated families, with reductions at approximately ambient exposures. Al-
though,both facilities noted apparent growth stimulations at moderate ozone levels compared
with non-filtered air, the reason for the apparent stimulation is unclear.
47
-------
FRP SEEDLING REPORT PETERSON ET AL
ORNL-DWG 83Z-11536
ACID RAIN COMPARISON
o
£
DC
o
cc
0
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0. 1
0
-0. 1
-0.2
-0.3
0.3
] \ 1 I I | I M I ) \ i M | I I I I j I I 1 I | I [ { I j 1
pH 4.5 VS 5.2
iMll^JliiilJlll llulllil^l
i ''' ¦"' i' ' i ''' 11' i 111 i i'' 11' 111 ' i i ' i i '' 11111 11 i i
<
a:
$
o
DC
0
pH 3.3 VS 5.2
i
2 7 12 17 23 28 33 38 43 48 53
FAMILY NUMBER
Figure 20: A comparison of 53 loblolly pine families for the ratio (minus 1.0) of their height growth
at nominal pH 33 or pH 4.5 to that at pH 5.2. A value of 0 indicates equal growth
rates, and a value of +0.20 indicates 20% better growth in the lower pH rain (Figure
3.13 in McLaughlin et al. [SC04]).
48
-------
MPO#3
PRP SEEDUNO REPORT
PETERSON ETAL
Height (cm)
8
Diameter (mm)
4 "
2 -
80 160 240
Ozone Concentration (ppb)
15
1.5
0.5
320
80 160 240
Ozone Concentration (ppb)
320
Figure 21: Final heights and diameters averaged across three loblolly pine families after 12 weeks
of exposure to ozone.' Two separate experiments from 1986 and 1987 are presented
(from Table 4 in Reinert et aL [SC05]).
49
-------
MPO #3
FRP SEEDUNC REPORT
PCTERSONCTAL
533 Family Sensitivity
McLaughlin et al. [SC04] examined variation of eight loblolly pine families in sensitivity to ozone
in CSTRs and of more than 44 families to combinations of ozone and acid precipitation in
open-top chambers. Ratings of sensitivity were based on changes in average height and diameter
of seedlings after 12-week exposures. Responses to acid rain and ozone varied by family. Slightly
more Piedmont families were responsive to acid rain treatments in both diameter growth and
height growth than Coastal families (Table 7). In response to ozone, Piedmont families are also
reported to show a higher percentage of changes in height growth compared to families of Coastal
source (Table 7). Although these are important observations, given that height/diameter family
responses were not always in the same direction (i.e., some positive and some negative), these
tables require further stratification according to a known direction of response.
Reinert et aL [SC05] report on the sensitivity of 12 loblolly families exposed to ozone (ranging
from 0 to 320 ppb) in two experiments conducted in 1986 and in 1987. Although the magnitude
of growth changes and some rankings changed with year of exposure, the most ozone-sensitive
(HS 5-56) and least ozone-sensitive (HS 1-68) families retained relative rankings. The com-
parison of those two families with the average of all 12 is given in Figure 22.
53.4 Mechanism: Carbon Allocation
Kress et [rc?61 observed decreases in total and retained needles, and accelerated needle
abscission of early cohorts of foliage on loblolly pine seedlings as levels of ozone exposure were
increased from CF to 3 times ambient (Figure 23). This result suggests that accumulation of foliar
injury can eventually cause premature abscission.
Richardson and Sasek [SC07] measured photosynthesis rates using an infrared gas analyzer. They
found that net photosynthesis rates of loblolly pine seedlings were not affected by acid rain
treatments (pH 3.5 versus 5.2). Furthermore, there were no interactive effects of acidity with
ozone treatment or acidity with family. Net photosynthesis rates of loblolly pine seedlings were
negatively affected, however, by cumulative ozone exposure^. Compared to charcoal-filtered air,
the highest ozone level (3 times ambient) was associated with 80% reductions in net photosyn-
thetic rates after four-month exposures (Figure 24). It appears that the product of concentration
and length of exposure was a good predictor of net photosynthetic response because low
exposures over long periods had similar effects to high exposures over shorter periods. Thus,
long-term chronic exposures to ozone appeared to have cumulative effects that resulted in
reduced net photosynthetic capacity. Results further suggest that ozone decreased concentra-
tions of chlorophyll a, chlorophyll b, and carotenoids (Richardson, pers. comm.).
McLaughlin et al. [SC04] report that more acidic treatments (i.e., pH 33 and 4.5 versus pH 52)
were associated with higher photosynthesis rates (52% at 12 weeks) in loblolly pine seedlings. A
fertilization effect of increasing nitrogen when increasing acidity was speculated to be a cause for
the observed increases. Compared to charcoal-filtered air (14 ppb ozone), 25% reductions in
photosynthetic rates were found after 12 weeks of 167-ppb ozone treatments in the Geld studies
at ORNL. In contrast, the laboratory studies at ORNL showed no clear trends with respect to
ozone treatments; ozone was associated with both increases and decreases in photosynthetic rates
over time. These contrasting results between field and laboratory studies raise concern over the
validity of extrapolating freely from lab studies to the field for studies on photosynthesis.
These two studies (SC04 and SCO7) yielded conflicting results with respect to acid effects on
photosynthesis of loblolly pine seedlings. The enhancement of photosynthesis at lower acidity
(pH 33) observed by McLaughlin et al. [SC04] at ORNL suggests that acid treatments may
stimulate growth. However, it should be noted that McLaughlin et al. [SC04] actually observed
growth suppression at pH 33 (Figure 20). Both studies showed negative response to ozone.
50
-------
MPO«3
FRP SEEDUNO REPORT
PETERSON ETAL
Table 7. A summary of growth responses to acid rain and ozone for common loblolly pine families
tested in field chambers at ORNL (from Tables 3.9 and 3.10 in McLaughlin et al.
[SC04]).
Mean growth .response expressed as a
percent of growth in pH 5.2 rain
pH 4 .3
PH 3.3
Seed
gourea
Piedmont
Coastal
Common
Huaber of
fawillei
25
28
9
Haloh£
132%
120
120
Diameter
106%
106
100
Height
79%
81
77
Mmnatar"
99%
97-
92
Percent of families
with statistically
significant
responses to rain
pH levels
fp <-0.101
Height
48%
39
33
Dlaaater
40%
32
11
Seed
Bourea
Piedmont
coastal
Common
Huaber of
faalliea
25
28
.9
Mean growth response expressed as a
percent of growth in CP air
AmManfr
Ainhlont- + ISO mb
Height
76%
78
Piaaeter
97%
95
Hniqht
78%
88
93
Planeter
93%
92
92
Percent of families
with statistically
significant
responses to
ocone levels
n.ioi
Haloht
64%
39
11
Diameter
28%
32
11
Common denotes families that are also being tested at NC State and Texas A&M.
51
-------
FRP SEEDLING REPORT
PETERSON ETAL
SEEDLING HEIGHT
o
cc
I-
z
o
o
LL
o
se
140
120
100
80
60
40
20
stem Diameter
-A HS 1-68
HS 5-56
- 12HSAVG
_1_
80 160 240 3 HO
OZONE CONCENTRATION (PPB)
O
tr
o
o
LL
o
a?
HS 1-68
HS 5-56
12 HS AVG
80 160 240 320
OZONE CONCENTRATION (PPB)
SECONDARY NEEDLE FRESH WT
STEM FRESH WT
C
.
¦
""a
-
a HS 1-68
.
¦-a HS 5-56
-
12 HS AVG
» i
i , i
80 160 240 320
OZONE CONCENTRATION (PPB)
O
oc
t-
z
o
o
u.
o
a?
-it HS 1-68
Q HS 5-56
- 12 HS AVG
80 160 240 320
OZONE CONCENTRATION (PPB)
Figure 22: Average responses of loblolly pine seedlings to ozone from two separate experiments
(1986 and 1987) using the same families; heavy (middle) trend in each graph repre-
sents average of 12 families. HS 1-68 and HS 5-56 were the least and most sensitive
families, respectively (from Figure 1 in Reinert et al. [SC05]).
52
-------
MPO»> PRP SEEDUNO REPORT PETERSON ETAL
150
125
100
75
50
25
0
Flush
Ytar
I
I RETAINED
SENESCEt)
V
i >
ET
d
1234612346
1987 1988
60
1234612346
1987 1988
136
12 34612346
1987 1988
1234612346
1987 1988
205
OZONE DOSE
298
ppm^hour
1234612346
1987 1988
396
Figure 23: Needle response of loblolly pine seedlings by flush number for 1987 and 1988 as a
function of ozone. Seedlings had also been exposed to similar treatments in 1987.
Data for the 1987 needles reflect the status at the beginning of the 1988 exposures
(from Kress, pcrs. comm.).
53
-------
vpo#3
FRP SEEDUNG REPORT
PETERSON ETAL
w
w
G>
sz
4->
c
v>
o
H'
o
£
Q.
CD
>
jO
a)
DC
O June
A August
n October
100 200 300
Cumulative Ozone Dose (ppm hrs)
400
Figure 24: Photosynthesis rates relative to the charcoal-filtered (CF) treatment versus cumulative
ozone dose determined at three different measurement dates during 1987. Photosyn-
thesis is reported at each sampling date. Within a sampling date, the five data points
represent (from left to right) CF, NF, 1.5, 2.25, and 3.0 x ambient ozone treatments
(from Richardson and Sasek [SC07]).
54
-------
MPO#J
FRPSEEDUNO REPORT
PETERSON ETAL
ORNL [SC04] showed a positive response to acidity. DUKE [SO07] showed no response from
the first year of acid treatments (1987). However, by the end.of the second season of treatments,
the seedlings in the pH 33 treatment showed greater diameter increments than those in the pH
S3 treatment (p = 0.05).
In both studies, field results indicated that the highest ozone levels were associated with substan-
tial reductions in photosynthetic rates of loblolly pine seedlings. However, short-term lab studies
failed to show ozone effects. Shafer et aL [SC07] found that reductions in growth were also
associated with the highest ozone levels at Richardson and Sasek's [SC07] site and with ambient
levels at the Mclaughlin [SC04] site (see Section 532).
S3S Summary
Despite considerable differences in experimental designs and procedures, and regardless of the
presence or absence of significant effects, most experiments showed patterns of either biomass
or diameter growth suppressions in response to ozone treatments. For the most part, effects of
acidity, effects of ozone x acidity, and shapes of exposure/response models for determining these
effects differ from site to site. In general, there was no clear pattern of a direct acidity effect on
either growth or photosynthesis of loblolly pine from these early short-term results.
Foliar injury from ozone, followed by premature abscission, was observed at the DUKE field site
[SC07] for all ozone levels above charcoal-filtered air. Although net photosynthetic rates of
loblolly seedlings were not affected by acidity, they were negatively affected by cumulative ozone
exposures. The fact that height and diameter growth'changes under ozone were notalways in
the same direction and were sometimes associated with reduced root biomass indicates that
patterns of carbon allocation can change. Thus, there is the potential for long-term chronic
exposures to ozone to reduce photosynthetic capacity, whidi, if coupled with reduced root
biomass, might ultimately affect the growth and survival of seedlings.
With the exception of some preliminary information from the second year at DUKE, results on
Southern pines in this report were limited to studies of one exposure season conducted in 1986
and 1987. However, several exposure studies were established in 1988 that, in addition to the
ongoing work at the DUKE site, are designed to cany the same seedlings through multi-year
exposures.
5.4 Western Conifers
Results from three projects are reported here [WC07, WC08, and WC09]. The latter two projects
are determining the sensitivity of western conifer species to three deposition exposure scenarios
likely to occur in the western United States: 1) gaseous sulfur dioxide during fall and winter, 2)
acid fog during the fall and winter; and 3) acid fog during the fall and winter preceded by summer
ozone exposures. In addition to immediate effects, both projects measure changes in seedling
condition during the growing season following conclusion of exposure treatments (Le., post-ex-
posure effects).
5.4.1 Visible Iqjuiy
Milleret aL [WC09] report that Douglas-fir and Engelmann spruce did not exhibit any injury
from ozone following exposures from 53 ppb to 71 ppb oyer three months. However, white fir,
subalpine fir, and ponderosa pine showed increased injury with increased ozone levds. Visible
injury on needles exposed to acid fog (pH 2.1 and 3.1 versus pH 5.6) was evident on ponderosa
pine, Douglas-fir, subalpine fir, and white fir, but not Engelmann spruce.
55
-------
MPO #3
FR? SEEDLING REPORT
PETERSON ETAL
Turner et al. [WC07] observed foliar injury (necrosis) associated with acid fog alone on western
hemlock (with pH 2.1 and 3.1) and western red cedar (with pH 2.1) when compared with control
fog (pH 5.6). No effects were observed in Douglas-fir or ponderosa pine. Ponderosa pine and
western hemlock were the only species that showed typical chJorotic mottle and banding during
the ozone exposure period. Douglas-fir and western redcedar showed no injury with ozone. In
a repetition of the exposure scenario in the second year, ponderosa pine appeared again to have
the greatest susceptibility to seasonal ozone and acidic fog treatments. Western redcedar
appeared least susceptible to this same regime.
5.42 Growth Changes
Miller et al. [WC09] found that, after two months of exposure to 66 and 42 ppb of sulfur dioxide,
height growth of Engelmann Spruce and white fir was increased relative to a base treatment of
21 ppb (p < 0.006 andp < 0.03, respectively). There were no effects of sulfur dioxide treatment
on heights of subalpine fir, ponderosa pine, or Douglas-fir or on diameter increment for any
species.
Preliminary results from ozone exposures in 1987/88 indicated that medium to high ozone
concentrations (67 and 71 ppb) stimulated height growth of Douglas-fir and white fir. ¦ The
height-growth stimulation from ozone continued during the acid fog treatment. With the pH 2.1
acid fog, subalpine fir also responded with increases in height growth, and diameter increases
were noted for Douglas-fir, Engelrnaan spruce, ponderosa pine, and subalpine fir. Analysis of
harvest data is in progress.
Hogsett and Tingey [WCQ8] examined seedling growth after one year of exposure scenarios.
While all species exhibited growth over time in all measured variables, certain growth variables
were altered with one or both exposure scenarios (p < 0.10). A repeat of the exposure scenarios
in the second year (1987-1988) substantiated the previous year's pattern of species susceptibility
to each of the exposure scenarios (Table 8). Results of Hogsett and Tinge/s [WC08] repeated
experiments are summarized below.
Under the ozone and acid fog treatment, there was a marginal reduction of diameter growth in
Douglas-fir (p = 0.08) and ponderosa pine (p = 0.08). Diameter growth of ponderosa pine was
progressively reduced toward the conclusion of the exposure period, resulting in a reduction the
following spring. Diameter was reduced 7% and 11% from controls with the two elevated ozone
regimes (67 and 71 ppb, respectively). In 1987/1988, western hemlock, lodgepole pine, and
western redcedar did not show any changes in diameter or height growth rates over the course
of the ozone exposures, nor did differences occur in final height measures taken at the conclusion
of the second growth season. Following exposures to acid fog without ozone, final harvest showed
9% and 48% increases in stem diameter at pH 3.1 and 2.1, respectively, for Douglas-fir (p =
0.08), and 8% and 35% increases at pH 3.1 and 2.1, respectively, for ponderosa pine (p = 0.07).
Stem height of ponderosa pine was increased 7% at pH 3.1 and 19% at pH 2.1 compared with
the control fog of pH 5.6. Stem height of Douglas-fir was not affected.
None of the five species showed significant changes in height or diameter in response to sulfur
dioxide (alpha = 0.10), although there were relatively large increases (10% to 27%) in height
and diameter of western redcedar and Douglas-fir relative to the controls. The largest increase
occurred at the lowest exposure level.
Bud elongation of ponderosa pine appears to have been decreased by acidic fog, depending on
the level of ozone. The pH 3.1 treatment with base ozone had no effect on bud elongation, while
there was a marked decrease due to pH 2.1 at the same ozone level. Both pH 2.1 and 3.1 caused
a similar decrease in the rate of bud elongation with the highest ozone treatment (71 ppb, 7-hr
average; 30-day sum = 34ppm). Although there were some strong differences in bud elongation
56
-------
MPO #3 FRPSEEDUNC REPORT PETERSON BTAL.
Table 8. Summary of growth effects on western conifers exposed to three different exposure
scenarios over one year (1987-88, from Table 7 in Hogsett and Tingey [WC08]).
OZONE + ACID FOG
ACID FOG
ONLY
SULFUR
DIOXIDE
SPECIES
OZONE
ACID FOG
OZONE
x
ACID FOG
Fonderosa
pine
Douglas -
fir
Western
hemlock
Western
redcedar
Final
Harvest1-2
sdw, rdw,
2nw, 3nw,
r/s, r/n,
dia
r/s, r/n,
3NW
rdw
Lodgepole rdw, lnw,
pine 2nw
Final
Harvest.
2dw, 3DV,
r/s, r/n
2nw, 3NW,
BUD
SDW, NDW,
r/s, r/n
r/s, r/n
SDW, 3NW,
r/s, r/n
Final
Harvest
3NW, BDD
rdw, 3NW,
r/s, r/n
rdw, r/s,
r/n, bud
SDW, NDW
2nw, r/n
Final
Harvest
3NW, r/s,
HT, DIA
SDW, 2nw,
3NW, r/n,
r/s, DIA
NA
NA
NA
Final
Harvest
r/n
3NW, r/s,
r/n, BUD
ndw
sdw, NDW,
R/S
rdw, 2nw
Listing a variable indicates a difference between at least one of
the treatment exposures and the control exposure (p < 0.10).
Variables listed as lower case indicate that reductions were
observed; variables listed in UPPER CASE indicate that increases
were observed.
bud -» spring bud elongation rate or the final length of apical
bud after spring elongation
dia - stem diameter
ht - stem height
ndw - needle dry weight
rdw = root dry weight t
r/n - root/needle dry weight ratio
r/s - root/shoot dry weight ratio
sdw - stem dry weight
xnw - needle dry weight by age class (x=l,2 or 3)
NA - not applicable (i.e., species not included in treatment)
57
-------
MPO #3
FRP SEEDLING REPORT
PETERSON ETAL.
at final harvest, neither ozone nor acid fog effects could be detected in any species due mainly to
large coefficients of variation. The largest response to any of the treatments was a 37% increase
in bud elongation of western hemlock in the pH 2.1 acidic fog treatment. The largest change in
bud elongation in response to ozone was a 10% increase for western hemlock; no effects of the
acid fog without ozone treatment were detected for either ponderosa pine or Douglas-fir.
Douglas-fir showed respective increases in mean bud elongation of 12% and 18% at the two
highest sulfur dioxide treatments (42 and 66 ppb, 60-day average; p = 0.03). Growth rate of
western redcedar increased by more than 40% for all sulfur dioxide treatments (p = 0.001). With
increasing sulfur dioxide, there were moderate decreases (11% to 13%) in bud elongation for
Douglas-fir and lodgepole pine, and small decreases ( < 10%) for ponderosa pine and western
hemlock.
Compared with the other species, ponderosa pine showed the greatest vegetative biomass
response to the ozone/acidic fog treatment. All tissue parts (stem, roots, and needles) were
reduced. Stem dry weight was reduced by 8% to 11% with elevated ozone (p = 0.004). Root
dry weight was reduced 5% to 26% with increasing ozone levels (p = 0.001). Second-year needles
had 10% to 32% less biomass than controls, whereas third-year needles were increased in biomass
above controls with increasing ozone.
The acidic fog effect was similar to that of ozone. Second-year needles biomass was reduced
approximately 12% compared with the pH 5.6 control, while third-year needles increased 9% to
26%. Both root/shoot and root/needle ratios were reduced 10% to 24% by both ozone and acidic
fog in the combined exposure scenarios. Douglas-fir, western hemlock, and lodgepole pine
showed intermediate responses to this exposure scenario, while western redcedar appeared to
be the least responsive species and ponderosa pine the most responsive. Both Douglas-fir and
ponderosa pine showed increased biomass in response to the acidic-fog-only treatment. Stem
dry weight of Douglas-fir increased 7% to 30% at pH 3.1 and pH 2.1, respectively. The
cun-ent-year needles increased 50% to 117% above controls at the lower pH treatments.
Root/needle and root/shoot ratios were reduced up to 27% in acid fog treatments. Ponderosa
pine exhibited similar, but less dramatic, increases in growth variables. For example, needle dry
weight increased 20% to 57% with exposure. Above-ground biomass appeared to increase in
both species in response to the acidic fog treatments with no effect on below-ground biomass.
Most of the changes in response to sulfur dioxide were increases in above-ground biomass,
although there were reductions in root biomass and root/shoot ratios for Douglas-fir, ponderosa
pine and lodgepole pine. Western hemlock showed a marginal (8%) increase in needle dry weight
at the second highest treatment (42 ppb, 60-day average). There were minor reductions in root
dry weight of Douglas-fir (7%) as a result of exposure to sulfur dioxide. There is slight evidence
that stem dry weight of western redcedar was reduced by 11% to 13% with the three sulfur dioxide
treatments (p = 0.10).
Turner et al. [WC07] found highest foliar and root tissue dry weights with the pH 3.1 fog treatment
for ponderosa pine, red cedar, and Douglas-fir. These increases were not significant, however
(alpha = 0.10). Root dry weight of western hemlock was reduced at the higher acid fogs (pH 2.1
and 3.1 versus 5.6).
S.43 Species Sensitivity
Preliminary observations of foliar injury suggest that western hemlock and western redcedar were
more sensitive to low pH fogs than were Douglas-fir and ponderosa pine (Turner et al. [WC07]
and Hogsett and Tingey [WC08]). Western redcedar exhibited significant increases in many
growth variables as a result of ozone/acid fog exposures, including the bud elongation the
58
-------
MPO#J
PRPSEEDUNO REPORT
PETERSON ETAL
following spring. Both western hemlock and western redcedar appeared to be more responsive
to acid fog at both pH 2.1 and 3.1 than any other species.
SAA Mechanism: Foliar liwrhlng
Turner et aL [WC07]grew Douglas-fir in solution-cultures whereby nutrient depletion in the
solution estimated seedling uptake. They found that foliar leaching of potassium, calcium, and
magnesium was greater with twice-weekly fogs of pH 3.1 than for pH 5.6. However, the rate of
foliar cation leaching during applications of four-hour fogs at pH 3.1 were relatively small
compared with the daily uptake rates for these seedlings. Furthermore, under conditions of low
nutrient availability where growth was suppressed, low-pH fogs were not associated with reduc-
tions in foliar nutrient concentrations. Epicuticular waxes were not affected by either pH
treatment
54i Summary
The evaluation of responses of western conifer species to ozone, acid fog, and sulfur dioxide is
made with one season of treatment, rather than multi-year exposures. Confidence in the
conclusions is increased by one study's replication of treatments in a second year on a separate
population of seedlings [WC07]. Overall, the same responses were observed in the second year.
Visible injury attributable to ozone exposure was found on ponderosa pine, white fir, subalpine
fir, and western hemlock. Only western hemlock" and;western redcedar showed visible injury after
exposure to acidic fog, although western redcedar appeared less susceptible to the ozone and
acidic fog seasonal exposure.
Only Douglas-fir and ponderosa pine were exposed to add-fog-only, and Douglas-fir appeared
to be most susceptible to this treatment Both species showed increases in above-ground biomass
with no apparent change in below-ground biomass. This imbalance in biomass and the relative
strengths of the source/sink functions of these tissues could cause problems in seedling estab-
lishment.';
It is important to note that most growth effects due to treatment, especially changes in biomass,
did not occur during the exposure period, but were observed following the spring bud elongation
period. These changes in spring growth after exposure, whether increases or decreases, may
indicate treatment effects on carbohydrate allocation patterns or on strengths of the carbohydrate
sources for initiating spring growth. These conclusions are drawn from one year of treatment
and further study is necessary to determine if multiple seasons of exposure alter the responses as
the seedlings integrate their environment.
59
-------
MPO #3
FRP SEEDLING REPORT
PETERSON ETAL
6 SUMMARY
In the previous section, results within each region were discussed by the general categories of
response (i.e., visible injury and growth effects, and the mechanisms of foliar leaching, carbon
allocation, and winter injury). The results pertaining to a specific response are now discussed
across regions in an attempt to draw general conclusions regarding effects of acid deposition,
ozone, and other pollutants on seedlings.
6.1 Visible Injury
All nine studies that examined visible injury to seedlings reported some effect of exposures to
either acid precipitation or ozone. No visible injury was associated with sulfur dioxide in the two
studies that examined these effects (Jensen and Dochinger [EH06], Hogsett and Tingey [WC08]).
Exposure to acid precipitation resulted in visible injury to most seedlings at or below pH 3.0.
Only one of the six studies that examined acid effects did not observe some visible injury to some
of the species (Davis and Skelly [EH01]). However, that study did not expose seedlings to acid
below pH 3.0. Six studies reported visible injury associated with acid below pH 3.0 (Leith et al.
[SF14], Jacobson and Lassoie [SF06], Jensen and Dochinger [EH06], Hogsett and Tingey [WC08],
Miller et al. [WC09], and Turner et al. [WC07]). Only Engelmann spruce (Miller et al. [WC09]),
Douglas-fir, and ponderosa pine (Turner et al. [WC07], Hogsett and Tingey [WC08]) showed no
visible injury when exposed to acid at pH less than 3.0. It should be noted that Douglas-fir and
ponderosa pine did show visible injury when exposed to acid less than 3.0 in Miller et al.'s [WC09]
study.
Seedlings exhibited injury at ambient ozone concentrations (Kress et al. [SC06]) or slightly above
ambient levels (Miller et al. [WC09], and Davis and Skelly [EH01]). All six studies that examined
visible injury from ozone observed other effects (Fincher et al. [SF16], Jensen and Dochinger
[EH06], Davis and Skelly [EH01], Kress et al. [SC06], Hogsett and Tingey [WC08], and Miller et
al. [WC09]). Injury was observed at about 70 ppb (Miller et al. [WC09], and Davis and Skelly
[EH01]). In addition, Kress et al. [SC06] showed a trend of increasing injury with increasing
ozone concentrations. It should be noted that elevated ozone was associated with both increases
and decreases in needle browning in red spruce (Fincher et al. (SF16J).
These short-term studies generally failed to show correlations of visible injury with changes in
plant growth. Perhaps the seedlings have a capacity to recover from injury. Alternatively, growth
responses might not be detected in the first season of measurement. The multiple-year studies
in progress will help to determine whether visible injury will translate into growth effects.
62 Growth Effects
The effects of acidic precipitation on growth responses were highly variable among the seven
studies that addressed this question. Results depended on species and pH level. Growth of most
hardwood species was stimulated at pH levels of 3.0 and 3.5 versus 4.2 (Jensen and Dochinger
[EH06], Davis and Skelly [EH01]). Growth of loblolly pine was stimulated at pH 4.5 versus 5.2
but depressed at 3.2 (McLaughlin [SC04]). Growth of red spruce was suppressed at pH levels
below 3.0 when compared to 42 (Jacobson et al. [SF06(a)]). Growth of most species of western
conifers was stimulated at pH levels of 2.1 (Miller et al. [WC09]). Other potential sources of
variation included method of exposure; results from Jacobson et al. [SF06(a)J suggested that
60
-------
MPO*3
FRP SEEDUNO REPORT
PETERSON ET AL
intermittent mists may have been more deleterious than continuous mists and sulfate acids may
have been more harmful than nitrate acids.
Ozone effects on growth were most evident in the results for loblolly pine. Growth of roots and
stems of loblolly pine appeared to be consistently suppressed at high concentrations of ozone
(greater than 100 ppb); -At intermediate lewis (near 70 ppb), suppression and sometimes
enhancement of growth occurred, depending on family. Neither red spruce nor the eastern
hardwoods showed consistent growth; effects due to ozone. Results for the western conifers
indicated that some stimulation of growth occurred with medium to high levels of ozone (less
than 100 ppb), except for ponderosa pine where growth suppression occurred.
Sulfur dioxide was associated with enhanced' height growth of Engelmann spruce, white Gr,
Douglas-fir, and western redcedar in the western conifers (Miller et aL [WC09], Hogsett and
Tingey'[WC08]). Douglas-fir, ponderosa pine, and lodgepole pine showed reductions in root
biomass and root/shoot ratios (Hogsett and Tingey [WC08]). Compared to a control, bud
elongation was increased by higher .sulfur dioxide levels (up to 66 ppb base level) for ponderosa
pine, Douglas-fir, western hemlock, and western redcedar (Hogsett and Tingey [WG08]). . No
consistent effects were observed in the eastern hardwoods. Sulfur dioxide was not examined in
the other regions.
An important finding in western conifers was that some effects, particularly growth changes, were
not apparent until the spring after pollutant exposures were conducted. This result illustrates
that seedling'studies should be routinely carried over through the winter to the spring growing
season following exposure.
63 Carbon Allocation
Most of the information on the effects of acid and ozone on carbon allocation is from research
on red spruce and loblolly pine. Acid precipitation did not appear to suppress rates of photosyn-
thesis based on the five studies reviewed (Laurence'et al. [SF31], Patton and Jensen [SF07],
Thornton et aL [SF27], McLaughlin et aL [SC04], and Richardson and Saselc {SC07]). Three
studies found no effect of acid on photosynthesis, Thornton et aL's [SF27] study suggested
suppression of photosynthesis of red spruce seedlings with cloud exclusion, while McLaughlin et
aL [SC04] found enhanced rates of photosynthesis of loblolly pine seedlings at pH 3.3 and 4.5
(possible due to nitrogen fertilization). It should hie noted that none of the photosynthesis studies
employed exposures below pH 3.1. Thus, it is not appropriate to contrast the results of photosyn-
thesis studies (e&, lack of effect above pH 3.1) with those of growth and visible injury (e.g.,
suppression below pH 3.0).
Although there was variability among studies, ozone at high levels (2 to 4 times ambient) appeared
to disrupt photosynthesis. These concentrations reduced levels of photosynthetic pigments, and
increased mesophyU cell disruptions in red spruce seedlings (Fincheret aL [SF16J). The effects
oh photosynthesis of red spruce seedlings are yet undetermined; three studies found no effects
(Fincher et aL [SF16], Patton and Jensen [SFD7], and Laurence et aL [SF31]), one study reported
both enhancement and suppression depending on season (Cumming et aL [SF16]), and one study
reported suppression associated with, ambient ozone versus ozone removal (Thornton et aL
[SF27]). The highest levels of ozone were associated with reductions in photosynthesis of loblolly
pine seedlings (Richardson and Sasek [SG07]); furthermore, the effects of ozone on photosyn-
thesis appeared to be cumulative (Le., low concentrations for long periods also caused reduc-
tions).
61
-------
MPO #3
FRP SEEDUNG REPORT
PETERSON ETAL
6.4 Foliar Leaching
Although acid fog treatments increased foliar leaching of potassium, calcium, and magnesium
from Douglas-fir seedlings, the amounts leached were small compared to uptake (Turner et al.
[WC07]). Leaching of nutrients from the foliage, as a process induced by acidity, did not appear
to be a problem.
6.5 Winter Injury
Acid rain reduced the ability of red spruce seedlings to withstand simulated overnight frosts
during the winter hardening period. The degree of reduction in frost hardiness was proportional
to acidity; compared to pH 5.0, mists of pH 2-5 raised the LTjos by as much as 12°C, or,
alternatively, delayed frost hardening by two to four weeks. While there was no overall effect of
ozone on visible injury to red spruce seedlings following overwintering, a subset of seedlings did
demonstrate some potential for visible injury.
62
-------
MPom
ntPSEEOUNO REPORT
PETERSON ETAL
7 CONCLUSIONS AND RECOMMENDATIONS
7.1 Principal Findings
Thus far, the objective has been to summarize results from individual seedling exposure studies.
The contribution of these results toward understanding potential effects of sulfur dioxide,
simulated acidic pretipitation, and ozone on seedling populations is now discussed.
7iJ Sulfur Dioxide
Two projects examined visible effects of sulfur dioxide, and three examined growth effects. No
visible injury was observed in response to concentrations as high as 66 ppb. Increased above-
ground growth due to sulfur dioxide occurred for Engelmann spruce, white fir, western redcedar,
and Douglas-fir; relative to a control treatment Douglas-fir, ponderosa pine, and lodgepole pine
showed reductions in root biomass and root/shoot ratios. Compared to a control, bud elongation
was increased by higher sulfur dioxide Revels (up to 66 ppb base level) for ponderosa pine,
Douglas-fir, western hemlock, and western redcedar. The altered post-exposure growth and
imbalance in above- and below-ground responses; indicate that changes in caibon allocation
patterns occurred. Under chronic exposure, survival or eventual tree productivity could poten-
tially be negatively impacted. No effects of sulfur dioxide were seen for eastern hardwoods.
Sulfur dioxide was not tested with red spruce or southern pines.
712 Simulated Add Deposition
The clearest effect of simulated acid deposition was a reduction of frost hardiness in red spruce
seedlings at pH 3.0 and higher rates of foliar tissue mortality during extreme cold. Most species
that were tested at pH levels below 3.0 showed some,visible injury. Across all species, there were
no conclusive short-term effects of simulated acid deposition by. itself on seedling growth.
However, growth of black cherry was decreased by pH 3.0 versus AX Furthermore, increased
above-ground growth coupled with no apparent effects on below-ground biomass in western
conifers at pH 2.1 compared with pH 5.6 indicated that changes in carbon allocation patterns
occurred.
7.13 Ozone
The direct effect of ozone varied from physiological changes in,the foliage of red spruce to
suppressed growth of loblolly pine, ponderosa pine, and some hardwood species. Eastern
hardwobd species showed visible injury with ozone of 70 ppb or higher. Yellow-poplar, yellow
birch, sweetgum, red maple, white ash, and black cheriy appeared to be the most sensitive species
tested., Among western conifers, white fir, subalpine fir, ponderosa pine, and western hemlock
also showed visible injury in response to ozone at 70 ppb: Despite considerable differences in
experimental designs and procedures, there was a pattern of root and stem growth decreases at
ozone near 80 ppb or higher for loblolly pine. At intermediate levels (40 to 80 ppb) results were
more variable; it was not uncommon for growth rate to be greater at intermediate levels than in
charcoal-filtered air. In the West, only ponderosa pine showed consistent decreases across
several growth variables due to ozone; most other species showed increased growth rates at levels
less than 100 ppb. At this point b time, there are no data with which to address the anomaly of
increased seedling growth at these levels (approximately L5 times ambient). Among the
hardwoods, there was some association between ozone of 70 ppb and higher and decreased
growth for black cherry, white oak, red maple, and yellow birch; yellow poplar, white ash, and
red oak displayed no response at the same, levels. Cumulative decreases in net photosynthetic
63
-------
MPO#3
FRP SEEDUNG REPORT
PETERSON ETAL
rate in response to ozone were found for loblolly pine. Red spruce did not exhibit consistent
decreases in net photosynthesis. However, damage to foliar mesophyll cells, decreased photosyn-
thetic pigments, and seasonal changes in photosynthesis in red spruce in response to ozone at 40
ppb and higher suggest an increased potential for winter injury to red spruce.
7.1.4 Pollutant Interactions
There is preliminary evidence of some anion x pH interactions, where sulfur-based acids caused
greater foliar injury on red spruce than nitrogen-based acids at the same pH. Nine projects
exposed seedlings to both acidity and ozone, eight of which discussed interactions. Of these, four
reported no interactions of acidity and ozone for stem growth of seedlings. Of the four projects
that did observe some interactions, only two provided discussions adequate to evaluate the nature
of the effect. In a second year of exposures of red spruce seedlings, an interaction was observed;
elevated ozone caused reduced growth at pH 5.1 but caused increased growth at pH 3.1. A similar
interaction in loblolly pine seedlings was found when S3 families were analyzed simultaneously.
However, only 20% of the individual families showed a significant acidity x ozone interaction on
height or diameter growth (p
-------
MPO#3
FRPSEEDUNG REPORT
PETERSON BT AI.
results and that should be addressed in new and continuing studies involving tree material.
Although many other issues will likely surface before these studies are concluded, some of the
major issues at this point are:
7.2.1 Spedes/Plant Variability
A major source of variation in plant response lies in the plant material itself. Virtually all the
analyses demonstrated the large variation in growth that can be attributed to large differences in
initial size (height and/or diameter). Careful selection and randomization of plants prior to
treatment would help to alleviate this nonuniformity. The large variability and range of responses
to pollutant exposures among loblolly pine families show the importance of reducing that
variability if results of different experiments are to be compared.
122 Choice of Response Variables
The value of studying mechanisms for change in tree conditions is already apparent, as shown in
the summary above. Choosing the appropriate response variables to correspond with the
mechanism is essential For instance, early indications from changes in rates of photosynthesis,
below-ground growth changes, and above-ground growth changes in response to short-term
exposures of pollutants point to altered patterns of carbon allocation. Presently, there is
insufficient information from existing studies on changes in below-ground biomass to evaluate
this mechanism. Therefore, future studies need measurements from the same plant on both
below-ground (i.e. at least root biomass) and above-ground changes.
Before the end of NAPAP, exposure/response information for sapling-sized trees and for foliage
response of mature trees will be collected. The most promising way in which seedling studies will
provide insight into the response of mature trees to pollutants over time appears to be through
examination of physiological processes (e.g., carbon allocation). While remaining NAPAP
studies will contribute additional information, all future exposure studies involving seedlings and
larger/older trees must emphasize physiological measurements.
It should also be noted that other sources of variability such as seedling genotype should be
considered as a major source of variation in response. Genotypic variability currently ranges
from well-documented open-pollinated families to commercial nursery stock.
123 Microclimate Characterization
Possible interactions between air pollutants and other environmental factors, such as those in the
chamber microclimate, need to be characterized In addition to allowing assessment of interac-
tions, this information is important when replicating experiments.' Differences in seedling
responses among sites, or among years at a given site, cannot be correctly interpreted without
knowing corresponding spatial and temporal differences in factors such as light, temperature,
and humidity.
12A Duration of Experiments
Most of the initial studies were designed to look at effects over short periods of exposure (tg,
12 to 16 weeks). While useful information can be obtained from short-term exposures, certain
types of information such as growth response and winter injury can be better evaluated with
multiple-year exposures. In addition, the variability of results among studies maybe reduced with
longer-term exposures. Variability in short-term studies is often a result of variation in initial age
or size of plant material. These differences cannot always be solved through use of a covariate
in the statistical analyses. Biologically, the cumulative effects or even lag in effects will remain
65
-------
MPO *3
FRP SHEDUNG REPORT
PETERSON ET AL
unknown unless those changes are monitored on the same plants exposed over two to three
growing seasons. An important finding from experiments on western conifers [WC08] was that
some effects, particularly growth effects, were not apparent until the spring after pollutant
exposures were conducted; growth differences due to treatment were reflected in bud elongation
(Len reduced shoot growth) in the growing season following the season of treatment. There are
currently ongoing multi-year exposure studies in southern pines and red spruce from which results
will be forthcoming in the next two years.
12£ Statistical Power
The adequacy of the sample size to determine effects due to treatments should be calculated at
the conclusion of each project by computing power (i.e., the probability of detecting a consequen-
tial difference). This calculation includes measurements made from destructive sampling as well
as additional sampling and design considerations as a function of measurement intervals or
frequency of repeated measures. Successful planning of future experiments requires specifica-
tion of the magnitude of treatment differences to be detected and knowledge of expected
variation. Therefore, when possible, power should be considered when planning research as well
as at the conclusion of each project. In the absence of formal significance due to low ppwer,
evidence of treatment effects may still be present in the form of trends or patterns, and should
not be overlooked. However, it is important that users of the experimental results have realistic
expectations of the confidence to be placed in treatment effects. Statistical uovver has been
addressed to date for projects studying conifers in the west and loblolly pines in th". southeast
[WC08, WC09, SC02, SC04, SC05, and SC06]. Preliminary calculations suggest that for most
growth variables of interest, these experiments have an 80% probability of detecting treatment
differences of 20% and greater for a significance level of 0.05.
12.6 Repeatability of Experiments
Any experiment worth doing once should be considered for replication, regardless of the
treatment duration (i.e., single-year versus multi-year exposures) or the statistical significance of
the initial outcome. The ability to replicate an experiment in time or place is critical to
confirmation of results. For example, trends in height and diameter response of loblolly pine to
ozone, averaged across three families, were similar between the 1986 exposures and a replication
of the experiment in 1987. Furthermore, the relative responses among the three loblolly pine
families observed in 1986 were reproduced in 1987 for diameter changes, albeit not for height
changes. As another example, in the west, two consecutive single-season exposure scenarios of
acid fog plus ozone followed by sulfur dioxide were conducted on independent samples of
Douglas-fir, western redcedar, ponderosa pine, western hemlock, and lodgepole pine. The
1986-1987 season indicated that the most sensitive species was ponderosa pine and the least
affected species was redcedar. A repeat of the experiment in 1987-1988 confirmed these results.
On the other hand, a pH effect on visible injury of red spruce in 1985 was confirmed from a
repeated experiment in 1987, but not from a repeated experiment in 1986.
12.7 Repeated Measures
Although most studies have repeated measurements over time, very few projects have attempted
any repeated measures analysis in the statistical treatment of the data. There is a strong effort
by some statisticians within the FRP to adapt the existing literature on repeated measures analysis
to use on seedling exposure research. Projects that currently have repeated measurements, yet
have no repeated measures analysis, are not taking full advantage of available information.
66
-------
MPO#3
FRPSEEDUNO REPORT
PETERSON ETAL
8 ACKNOWLEDGMENTS
We would like to thank all the Principal Investigators of the seedling studies for continued
cooperation in response to our requests. We are grateful to W.G. "Bill" Warren for his special
efforts in helping promote and coordinate consensus-building for issues on statistical analysis,
and to the numerous other statisticians involved with each; individual project The continuing
support of the Co-op Direction and National Program Management is also appreciated. Finally,
we would like to thank Jeff Brandt for his assistance in the revisions and Lynn Araaut for help
with editing and production.
-------
MPO #3
FR? SEEDLING REPORT
PETERSON ETAL
9 LITERATURE CITED
Sources are organized first by topic or region. For each region, individual projects funded by the
FRP are identified by their project number and by one or more of the Principal Investigators.
Sources within each project are listed by author. All sources are on file in Corvallis.
GENERAL
Lindberg, S.E. and D.W. Johnson, eds. 1989. Draft of 1988 Annual Report of the Integrated
Forest Study. Report prepared for the Electric Power Research Institute, RP2621.
Schroeder, P. and RA Kiester. 1989. The Forest Response Program: National research on forest
decline and air pollution. J. Forestry. Jan:27-32.
STATISTICAL METHODS
Fisher, RA. 1932. Statistical Methods for Research Workers. Edinburgh: Oliver and Boyd.
Heagle, A.S., W.W. Heck, J.O. Rawlings, and R.B. Philbeck. 1983. Effects of chronic doses of
ozone and sulphur dioxide on injury and yield of soybeans in open-top field chambers- Crop
Sci. 23:1184-1191.
Rawlings, J.O. and W.W. Cure. 1985. The weibull function as a dose-response model to describe
ozone effects on crop yieids. Crop Sci. 25:807-814.
Rawlings, J.O. 1986. Design of experiments for controlled exposure studies. In Proceedings of
Workshop on Controlled Exposure Techniques and Evaluation of Tree Responses to Airborne
Chemicals. Technical Bulletin No. 500. National Council of the Paper Industry for Air and
Stream Improvement, Inc.
Rawlings, J.O., V.M. Lesser, and FLA. Dassel. 1988. Statistical approaches to assessing crop
losses, pp. 389-416. In: Heck, W.W., Taylor, O.C., and Tingey, D.T., eds. Assessment of Crop
Loss from Air Pollutants. Elsevier Applied Science, New York.
Warren, W.G. 1987. On the combining of independent tests of the same hypothesis. Internal
report: Synthesis and Integration Report Number 12. U.S. Environmental Protection Agen-
cy, Environmental Research Laboratory, Corvallis, OR.
SPRUCE-FIR
[SF06] Jacobson and Lassoie
(a) Jacobson, J.S., L.I. Heller, K.E. Yamada, J.F. Osmeloski, andT. Bethard. Submitted. Foliar
injury and growth response of red spruce to sulfate and nitrate acidic mist.
(b) Jacobson, J.S., J.P. Lassoie, J. Osmeloski, and K. Yamada. Foliar accumulation and loss of
macro- and micro-elements in red spruce seedlings after exposure to sulfate and nitrate
acidic mist. Report to the Forest Response Program, U.S. Environmental Protection
Agency, Corvallis, OR, February, 1989.
[SF07] Jensen and Schier
Patton, R. and K. Jensen. Responses of red spruce seedlings to ozone and acid deposition.
Report to the Forest Response Program, U.S. Environmental Protection Agency, Corvallis,
OR, November, 1988.
Patton, R.L., K.F. Jensen, and G-A. Schier. Impact of atmospheric deposition and drought on
foliar leaching and growth of red spruce seedlings. Report to the Forest Response Program,
U.S. Environmental Protection Agency, Corvallis, OR, March, 1989.
68
-------
MPO#3
FRP SEEDUNO REPORT
PETERSON ETAL
ISF13] Seller and Chevone
Seiler, JJ*., E.C. Tseng, B A Chevone, D J. Pagnalli. The impact of acid rain on Fraser fir seedling
growth and physiology. Report to the Forest Response Program, U.S. Environmental
Protection Agency, Corvallis, OR, December, 1988.
Seiler, JJL and BJ. Chevone. Impact of water stress, ozone, and add rain on the growth and
water relations of Fraser fir. Report to.the Forest Response Program, US. Environmental
Protection Agency, Corvallis, OR, December, 1988.
Tseng, E.C, J.R. Seiler, and BX Chevone. 1988. Effects of ozone and water stress on green-
house-grown fraser fir seedling growth and physiology. Environ. Exp. Bet 28^1): 37-4L
[SF14] Unsworth
Leith, ID., M.B. Murray, Li>. Sheppard, J.N. Cape, J.D., Deans, and D. Fowler. Submitted.
Visible injury of red spruce seedlings subjected to simulated acid mist
Murray,MB., J.N. Cape, D. Fowler, and I. Alvarez Asensio. Submitted. Quantification of frost
damage in plant tissues by rates of electrolyte leakage.
Fowler, D., J.N. Cape, JD., Deans, ID. Leith, M.B. Murray, R J. Smith, LJ. Sheppard, and Mil.
Unsworth. Submitted. Effects of acid mist on the frost hardiness of red spruce seedlings.
[SF16] Weinstein
Fmcher, J., J.R. Cumming, R.G. Alscher, and L. Weinstein. In preparation. Effect of long term
ozone exposure on winter hardiness of red spruce seedlings. October, 1988.
Alscher, R.G., R.G. Amundson, G. Rubin, J.R. Cumming, S. Fellows, J. Fincher, and Lii.
Weinstein. In preparation. Seasonal changes in the pigments; carbohydrates, and growth
of red spruce seedlings exposed to ozone. October, 1988.
Cumming, J.R., R.G. Alscher, J. Chabot, and L.H. Weinstein. Effects of ozone on the physiology
of red spruce seedlings. Report to the Forest Response Program, U.S. Environmental
Protection Agency, Corvallis, OR, October, 1988.
[SF27] Thornton
Thornton, F.C., PA. Pier, and C McDufiie. Effects of clouds and ozone on spruce seedlings:
A field chamber study at Whitetop Mountain, Virginia. Report to the Forest Response
Program, U.S. Environmental Protection Agency, Corvallis, OR, October, 1988.
[SF31] Kohut
Kohut, RJ., J.A. Laurence, R.G. Amundson, R.M. Raba, and J J. Melkonian. Effects of ozone
and acidic precipitation on the growth and photosynthesis of red spruce. Report to the
Forest Response Program, U.S. Environmental Protection Agency, Corvallis, OR, January,
1989.
Laurence, JA^ RJ. Kohut, and R.G. Amundson. 1989. Response of red spruce seedlings
ejqx)sed to ozone and simulated acidic precipitation in the field. Arch. Environ. Contam.
Toxicol. 18:285-290.
EASTERN HARDWOODS
[EH01] Davis and Skelly
Davis, D.D. and J.M. Skelly. Relative sensitivity of eight eastern hardwood tree species to ozone
and/or acidic precipitation. Report to the Forest Response Program, U.S. Environmental
Protection Agency, Corvallis, OR, October, 1988.
69
-------
MPO*3
FRP SEEDUNO REPORT
PETERSON CTA1.
[EH06] Jensen and Dochinger
Jensen, K.F. and L.S. Dochinger. Response of eastern hardwood species to ozone sulfur dioxide
and acid precipitation. Presented at the 81st Annual Meeting of APCA, Dallas, TX, June
19-24,1988. Report #88-703.
SOUTHERN COMMERCIAL PINES
[SC04] McLaughlin
McLaughlin, S.B., M.B. Adams, N.T. Edwards, P J. Hanson, PA. Layton, E.G. O'Neill, and WJC
Roy. Comparative sensitivity, mechanisms, and whole plant physiological implications of
responses of loblolly pine genotypes to ozone and acid deposition. ORNI/TM-10777,
Environmental Sciences Division Public. No. 3105, September, 1988.
Shafer, S., S. Spruill, and M. Somerville. Responses of loblolly pine seedlings to ozone and
simulated acidic rain in controlled exposures; results of studies conducted by the Southern
Commercial Forest Research Cooperative, 1986-1987. Report to the Forest Response
Program, U.S. Environmental Protection Agency, Corvallis, OR, January, 1989.
[SC05] Reinert and Wells
Reinert, RA., M.M. Schoeneberger, S.R. Shafer, G. Eason, SJ. Horton, and C. Wells. Reponces
of loblolly pine half-sib families to ozone. Presented at the 81st Annual Meeting of APCA,
Dallas, TX, June 19-24,1988.
Reinert, RA., M.M. Schoeneberger,G. Eason, and S.R. Shafer. In preparation. Responses of
loblolly pine to ozone and simulated acid rain. December, 1988.
Shafer, S., S. Spruill, and M. Somerville. Responses of loblolly pine seedlings to ozone and
simulated acidic rain in controlled exposures; results of studies conducted by the Southern
Commercial Forest Research Cooperative, 1986-1987. Report to the Forest Response
Program, U.S. Environmental Protection Agency, Corvallis, OR, January, 1989.
[SC06] Allen, Heck, Kress
Kress, L.W., H.L. Allen, J.E. Mudano, and W.W. Heck. Response of loblolly pine to acidic
precipitation and ozone. Presented at the 81st Meeting of APCA, Dallas, TX, June 19-24,
1988. Report #88-703.
Shafer, S., S. Spruill, and M. Somerville. Responses of loblolly pine seedlings to ozone and
simulated acidic rain in controlled exposures; results of studies conducted by the Southern
Commercial Forest Research Cooperative, 1986-1987. Report to the Forest Response
Program, U.S. Environmental Protection Agency, Corvallis, OR, January, 1989.
[SC07] Richardson
Richardson, C J. and T.W. Sasek. Effects of gaseous pollutants and acid deposition on open-top
chambered seedlings in Duke Forest: Physiology and biochemistry. Report to the Forest
Response Program, U.S. Environmental Protection Agency, Corvallis, OR, November,
1988.
Shafer, S., S. Spruill, and M. Somerville. Responses of loblolly pine seedlings to ozone and
simulated acidic rain in controlled exposures; results of studies conducted by the Southern
Commercial Forest Research Cooperative, 1986-1987. Report to the U.S. Environmental
Protection Agency, Corvallis, OR, January, 1989.
70
-------
MPO#J
FRP SEEDUNO REPORT
PETERSON ETAL
WESTERN CONIFERS
[WC07] Turner and Tingey
Turner, D.P, D.T. Tingey, aad WJL Hogsett. Acid fog effectson conifer sradlings 1988. In:
Proceedings of the 15th International Meeting for.Specialists in Air Pollution Effects on
Forest Ecosystems "Air Pollution and Forest Decline*, in Interlaken, Switzerland, October,
1988.
[WC08] Hogsett and Tingey
Hogsett, WJL and D.T. Ungey. Sensitivity of important western conifer species to S02 and
seasonal interaction of add fog and ozone. Project Status Report to the Forest Response
Program, U.S. Environmental Protection Agency, Corvallis, OR, January 15,1988.
[WC09] Miller and Dunn
Miller, P.R., P.H. Dunn, TJD. Leininger, Si. Schilling, DA. Larson, D.C Herring, BA.
Heckmann, and AJ*. Gomez. Testing the sensitivity of five western conifer species to sulfur
dioxide alone, ozone alone, and ozone followed by acid fog. Report to the Forest Response
Program, U.S. Environmental Protection Agency, Corvallis, OR, October, 1988.
71
-------
MPO #3
FRP SEEDUNG REPORT
PETERSON ETAL
10 ABBREVIATIONS
ANCOVA Analysis of covariance
ANOVA Analysis of variance
CF Charcoal-filtered
CSTR Continuously-stirred tank reactor
df Degrees of freedom
DQO Data Quality Objective
DUKE Duke Forest
EPA Environmental Protection Agency
E/R Exposure/Response
FRP Forest Response Program
MS Mean sums of squares
MPO Major Program Output
NAPAP National Acid Precipitation Assessment Program
NCLAN .* National Crop Loss Assessment Network
NCSU North Carolina State University
NF Non-filtered
ORNL Oak Ridge National Laboratory
p Probability-value
ppb Parts per billion
QAJQC Quality Assurance/Quality Control
S&I Synthesis & Integration
SC Southern Commercial
SF Spruce-fir
SQ Scientific Question
TAMU Texas A & M U niversity
USFS United States Forest Service
USDA United States Department of Agriculture
WC Western Conifers
72
-------
MPO #3 FRP SEEDLING REPORT PETERSON ET AL-
II APPENDICES
11.1 Appendix A: Project Summaries
This appendix contains summaries for the following FRP projects:
EH01 (1987) DD. Davis and J. Skelly
EH01 (1988) DD. Davis and J. Skelly
EH01(1988 OTC) J. Skelly and DD. Davis
EH03 D. Karnoslcy and J. Witter
EH04 J. Skelly and D.D. Davis
EH06(1988) JCP. Jensen and LS. Dochinger
EH06(1989) JCF. Jensen and LS. Dochinger
SC02 J7. Fong
SC04 .S.B. McLaughlin
SC05 R.Reinert, C. Wells, & M. Schoeneberger
SC06 .H. Allen, W. Heck, & L. Kress
SC07 CJ. Richardson
SC12 .D. McGregor
SC13 J.Johnson
SC14 .R.B. Flagler
SC15 .B.G. Lockaby and A.H. Chappelka
SF06 J.S. Jacobson and J. Lassoie
SF07 JCF. Jensen and G A. Schier
SF10 .S.B. McLaughlin
SF11 C J. Richardson
SF13 J.Seiler
SF14 .M.H. Unsworth
SF16 X.Weinstein
SF27 J.Thornton
SF31 JL. Kohut
SF32 J. Rebbeck, M.S. Greenwood, and KP. Jensen
WC07 J). Turner and D.T. Tingey
WC08 .W. Hogsett and D.T. Tingey
WC09 J*. Miller
73
-------
MPO #3
FRPSEEDUNG REPORT
PETERSON ETAL
Project Number: EH01 (19X7)
Principal Investigator: D.D. Davis and J. Skelly
Cooperative: Eastern Hardwoods
Title: Testing the Sensitivity of Eight Eastern Hardwood Species to Ozone and Acidic Precipita-
tion.
Tree Species: 8 hardwood species
Objectives: The specific objective of this study is to screen seedlings of 8 eastern hardwood
species for relative sensitivity to O3, and acid precipitation.
Deliverables: Documentation of relative sensitivities of species to O3 and acid precipitation,
12/87,12/88, spring/89.
Summary: Conducted in close coordination with the Jensen and Dochinger project. Controlled
environment chambers are used to expose seedlings to various levels of ozone, sulfur dioxide and
acidic precipitation. Response variables include foliar symptoms, fresh weight of roots and stems,
dry weight of roots and stems, shoot length, and number of leaves.
74
-------
MP0#3
FJU> SEEDLING REPORT
PETERSON ET AL
Project Number RH01 flOSSI
Principal Investigator D.D.Davis and J. Skelly
Cooperative: Eastern Hardwoods
Title: Relative Sensitivity of Four Eastern Hardwood Species to Ozone, Sulfur Dioxide, and/or
Acidic Precipitation.
Tree Species; 4 hardwood species
Objectives: The specific objective of this study is to screen seedlings of 4 eastern hardwood
species for relative sensitivity to O3, and acid precipitation.
Deliverables: Documentation ofrelative.sensitivities of 4 eastern hardwood species to O3 and
acid precipitation, 12/88,12/89, spring/90.
Summary: CSTR chambers are used to expose seedlings to various levels of ozone, sulfur dioxide
and acidic precipitation. Response variables include foliar, symptoms; fresh and dry weights of
roots, stems and leaves; stem base diameter growth; shoot diameter growth; height; and number
of leaves.
75
-------
MPO #3
FRP SEEDUNG REPORT
PETERSON ETAL
Project Number: EH01 C1988 Open-top Srudv^
Principal Investigator: J. Skelly and D.D. Davis
Cooperative: Eastern Hardwoods
Title: Testing the Sensitivity of Four Eastern Hardwood Species to Ambient Levels and Filtered
Levels of Air Pollution along the Pennsylvania Gradient.
Tree Species: 4 hardwood species
Objectives: The specific objective of this study is to study the response of seedlings of 4 eastern
hardwood species to ambient pollutants.
Deliverables: Documentation of relative sensitivities of species to ozone and acid precipitation,
12/88,12/89, spring/90.
Summary: Open-top chambers have been located at three rural sites in northern Pennsylvania.
Seedlings within the chambers are exposed to full-filtered, half-filtered, or non-filtered air.
Response variables include foliar symptoms, leaf area, fresh and dry weight, height growth, and
biomass at final harvest.
76
-------
MPO #3
FRP SEEDLING REPORT
PETERSON ETAL
Project Number EH03
Principal Investigator D. Karnosky and J. Witter
Cooperative: Eastern Hardwoods
Title: Effect of an Air Pollution Gradient on Northern Hardwood Forests in the Northern Lakes
Region: Open-Top Chamber Study.
Tree Species: 2 hardwood species
Objectives: To determine the response of trembling aspen and sugar maple to ozone along a
nitrate gradient.
Deliverables: Document response of trembling aspen and sugar maple to ozone, 12/89.
Summary: Conducted as part of the Michigan gradient project Open-top clumbers are used to
expose^seedlings to various levels of ozone. Response variables include foliar injury, stem
diameter, stem height, and stein, root, and leaf dry weight.
77
-------
MPO #3
FRP SEEDLING REPORT
PETERSON ET AL.
Project Number: EH04
Principal Investigator: J. Skelly and D.D. Davis
Cooperative: Eastern Hardwoods
Title: Foliar Sensitivity and Growth of Four Hardwood Species exposed to Ambient Concentra-
tions of Ozone in Open-Top Chambers.
Tree Species: 4 hardwood species
Objectives: To determine the influence of ambient, half-ambient, and charcoal-filtered levels of
ozone on foliar sensitivity and growth of four hardwood species.r
Deliverables: Documentation of relative sensitivities to ozone, 12/88,12/89, and 12/90.
Summary: At three locations in rural Pennsylvania, seedlings are being exposed to ambient and
partially-filtered levels of ozone. Foliar sensitivity and growth parameters are being measured.
78
-------
MPO#J
FRP SEEDLING REPORT
PETERSON ET AL.
Project Number EHOfi flOM phased
Prindpal Investigator KJ\ Jensen and L.S. Dochinger
Cooperative: Eastern Hardwoods
Title: Response of Eastern Hardwood Tree Seedlings to Atmospheric Deposition.
Tree Species: 6 hardwood species
Objectives: To evaluate the effect of atmospheric deposition on eastern hardwood seedlings and
determine if drought retarded this effect
Deliverables: Document the effect of sulfate, ozone, and drought on three oak species and
dogwood, and the effect of ozone and drought on sugar and red maple, 12/88,12/89.
Summary: Conducted in close coordination with the Ohio Corridor Gradient project CSTRs
are used to expose seedlings to various levels of ozone, sulfate, and drought Response variables
include foliar injury, leaf area, stem height, and stem, root, and leaf dry weight Part two was
conducted in close coordination with the Michigan Gradient Open-top chambers are used to
expose seedlings to various levels of ozone and drought Response variables were the same.
79
-------
MPO #3
PR? SEEOUNG REPORT
PETERSON ETAL
Project Number: EH06 C1989 phasp/l
Principal Investigator: K.F. Jensen and L.S. Dochinger
Cooperative: Eastern Hardwoods
Title: Response of Eastern Hardwood Tree Seedlings to Atmospheric Deposition.
Tree Species: 2 hardwood species
Objectives: To determine the response of sugar maple and trembling aspen to ozone, nitrogen,
deposition and drought.
Deliverables: Document response of sugar maple and trembling aspen to ozone, drought, and
nitrate, 12/89,9/90.
Summary: Conducted in close coordination with the Michigan Gradient project. Open-top
chambers and CSTRs are used to expose seedlings to various levels of ozone, nitrate and drought.
Response variables include foliar injury, leaf area, stem height, and stem, root, and leaf dry weight.
80
-------
MPO JJ FRP SEEDUNG REPORT PETERSON ETAL
Project Number; SCP2
Principal Investigator F.Fong
. Coonerative: Southern Commercial
Title: Growth Response of Loblolly Fine Seedlings to Ozone and Low-water Stress.
Tree Species: Loblolly pine
Objectives: 1) Assess responsiveness of loblolly pine as a species to ozone; quantify the genetic
variation in responsetoozone, and characterize;symptomatology and mjyJianjsni.s (physiological
responses) of ozone phytotoxicity; 2) characterize physiological responses of seedlings to ozone
x low-water stress interactions.
Deliverables: Reports on responses of loblolly pine to ozone and water stress, 7/87,12/88.
Summary: Uses fumigation chambers to quantify the genetic variation in the response of loblolly
pine to ozone and to chmcterize the'symptomatoloigy and mechanisms of ozone phytotoxicity.
Thirty half-sib families are exposed to different levels of ozone and moisture stress. Response
variables include plant height,' root collar diameter, total fresh and dry weight of needles, stem,
and root, photosynthesis, respiration, and total non-structural carbohydrate.
81
-------
MP0#3
FRP SEEDUNG REPORT
PETERSON ETAL.
Project Number: SC04
Principal Investigator: S.B. McLaughlin
Cooperative: Southern Commercial
Title: Comparative Sensitivity, Mechanisms, and Whole Plant Physiological Implications of
Responses of Loblolly Pine Genotypes to Ozone and Acid Deposition
Tree Species: Loblolly pine
Objectives: 1) Quantify growth and physiological responses of 53 loblolly pine genotypes to ozone
and acid rain in the field and laboratory, compare lab vs. Geld results. 2) Develop protocols to
quantify physiological and growth responses of large trees in the field.
Deliverables: Interim report during summer of 1987. Final report 12/88.
Summary: The objectives are met by implementing closely related field and laboratory studies
designed to incorporate many common cultural and experimental protocols both within the
studies at ORNL and across collaborating sites within the Southern Commercial Coop.
Laboratory studies have focused on testing the physiological responses of 8 common families to
ozone while using the approximate ambient rainfall pH level as a common background irrigant.
Field studies have used open-top chambers to examine individual and interactive effects of ozone
and simulated acid rain. Response variables include growth, photosynthesis, carbon metabolism,
and mycorrhizal development.
82
-------
MPO #3
FRPSEEDUNO REPORT
PETERSON ET AL.
Project Number SfifK
Principal Investigator R. Reinert, C Wells, & M. Schoeneberger
Cooperative: Southern Commercial
Title: Comparative Responses of Loblolly Pine Families to Ozone and Simulated Acid Rain
Tree Species: Loblolly pine
Objectives; Evaluate loblolly pine "sensitivity" to ozone and acid rain.
Deliverables: Dose-response model, 3/88; Quantification of growth and physiological responses
of various half-sib families to ozone and acid run, 12/88
Summary: This is a genotype screening study using short-term seedling exposures in the green-
house. It seeks to determine relative responses of different half-sib families of loblolly pine. The
primary response variables are needle injury, stem diameter and height, and biomass.
83
-------
MPO #3
FRP SEEDLING REPORT
PCTERSON ET AL.
Project Number: SC06
Principal Investigator: H. Allen, W. Heck, & L. Kress
Cooperative: Southern Commercial
Title: Response of Loblolly Pine to Acidic Precipitation and Ozone Stress
Tree Species: Loblolly pine
Objectives: 1) Determine responses of a number of loblolly pine families to ozone exposure. 2)
Estimate effects of ambient ozone concentrations on young loblolly pine. 3) Study ozone x acid
rain interactions.
Deliverables: Quantification of loblolly response to ozone and acid rain. Development of 15'
open-top chambers. Assessment of exposure effects on internal nutrient content and mycorrhizal
development. Interim report 12/87. Final report 12/90.
Summary: The approach is to study plant responses over a range of ozone concentrations. A
range of acidic precipitation treatments is used in a factorial design with the ozone concentrations.
Phase I uses 10* chambers to test experimental protocols, assess physiological responses (see
project SC07, PI: Richardson), and provide initial dose-response estimates. Phase II will use 15'
diameter chambers to study larger trees for longer experimental periods.
84
-------
MPO03
FRP SEEDLING REPORT
PETERSON ETAL
Project Number SOT7
Principal Investigator CJ. Richardson
Cooperative: Southern Commercial
. ... '
Title: Effects of Gaseous Pollutants and Add Deposition on Open-top Chambered Loblolly Pine
Seedlings
Tree Species: Loblolly pine
Objectives: 1) Characterize physiological effects of loblolly genotypes exposed to ozone and add
rain. 2) Establish dose-response relationships: ozone vs. physiological and biochemical respon-
ses. 3) Develop mathematical relationships between physiology, biochemical responses, and
growth across 5 levels of ozone and 2 levels of add rain. 4) Study relationship between ozone
exposure and status of the antioxidant defense system. 5) Develop diagnostic measurements for
ozone exposure (e.g^ light response curves).
Deliverables: Annual Reports 1788, 12/88; Quantification of physiological and biochemical
responses and relationship to growth.
Summary: Trees taken from Project SC06 (PI: Kress), uses open-top chambers, a range of ozone
treatments, and two levels of simulated add rain. Physiological measurements indude survey
measurements of photosynthesis; transpiration, and stomatal conductance. Monthly measure-
ments of photosynthetic responses at both high and low irradiation levels for each trrateent group
will be coupled with measurements of transpiration, stomatal conductance, chlorophyll and
carbohydrates. Dose-response relationships will also be developed.
85
-------
MPO#3
FRP SEEDLING REPORT
PETERSON ETAL
Project Number: SC12
Principal Investigator: D. McGregor
Cooperative: Southern Commercial
Title: Response of Shortleaf Pine Families to Acidic Precipitation and Ozone in South Carolina.
Tree Species: Shortleaf pine
Objectives: Determine the influences of acidic precipitation and ozone on the growth, nutrition,
and physiology of shortleaf pine under field conditions.
Deliverables: Quantification of Shortleaf Pine Response to Acid Precipitation and'Ozone, 6/91.
Summary: One-year-old loblolly pine seedlings are grown in 15* open-top chambers. They are
exposed to 3 levels of pH and 4 ozone concentrations during the growing season. Treatment
effects are quantified on plant dry weight, stem diameter, cumulative height growth, fascicle
length, and leaf area.
86
-------
MPO#3 FRPSEEDUNG REPORT PETERSON ETAL.
Project Numhen SCI 3
Principal Investigator. J.Johnson
Cooperative: Southern Commercial
Title: Response of Slash Pine Families to Acidic Precipitation and Ozone in North Florida.
Tree Species: Slash pine
Objectives: Determine the influences of acidic precipitation and ozone on the growth, nutrition,
and physiology of slash pine under field conditions.
Deliverables: Quantification of slash pine response to acid precipitation and ozoiie, 6/91.
Summary: One-year-old seedlings are grown in open-top chambers. They are exposed to 3 levels
of pH and 4 ozone concentrations. Treatment effects are quantified on plant dry weight, stem
diameter, total height, average fascicle length of the current flush, total leaf area, and visible
87
-------
MPO #3
FRP SEEDLING REPORT
PETERSON ETAL
Project Number: SC14
Principal Investigator: R.B. Flagler
Cooperative: Southern Commercial
Tide: Response of Shortieaf Pine Families to Acidic Precipitation and Ozone in East Texas
Tree Species: Shortieaf pine
Objectives: Determine the influence of acidic precipitation and ozone on the growth, nutrition,
and physiology of shortieaf pine under Geld conditions.
Deliverables: Quantification of shortieaf pine response to acid precipitation and dzone, 6/91.
Summary: One-year-old seedlings are grown in open-top chambers. They are exposed to 3 levels
of pH and 4 ozone concentrations. Treatment effects are quantified on plant dry weight, stem
diameter, total height, fascicle length, visible injury, foliar chlorophyll content, photosynthesis,
and concentration of selected nutrients.
88
-------
MPO#J
PR? SEEOUNO REPORT
PETERSON BT AL
Project Number SC15
Principal Investigator B.G. Lockaby and Ail. Chappelka.
Cooperative: Southern Commercial
Title: Response of Loblolly Pine Families to Acidic Precipitation and Ozone in Alabama.
Tree Species: Loblolly
Objectives: Determine, the influences of acidic precipitation and ozone on the growth, nutrition,
and physiology of loblolly pine under field conditions.
Deliverables: Quantification of iobloBy pine response to acidic precipitation and Ozone, 6/91.
Summary: One-year-old loblolly pine seedlings are grown in IS* open-top chanibers. They are
exposed to 3Jeyels of pH and 4 ozone concentrations during the growing season. Treatment
effects are quantified on above-ground plant biomass by component part, stem diameter,
cumulative height growth, and visible injury.
89
-------
MPO#J
FRP SEEDLING REPORT
PETERSON ETAL
Project Number: SP06
Principal Investigator: J.S. Jacobson and J. Lassoie
Cooperative: Spruce-Fir
Title: Test of the Nitrogen Fertilization Hypothesis of Red Spruce Decline.
Tree Species: Red spruce
Objectives: Determine if combinations of sulfate and nitrate acidic mist alter the growth,
development, cold tolerance, or water relations of red spruce seedlings in ways that might
contribute to premature decline.
Deliverables: Dose-response relationship of acid mist vs. seedling growth, development, phenol-
ogy, water relations, nutrient, balance, and biochemistry, 3/89.
Summary: Red spruce seedlings are exposed repeatedly, for extended durations, to simulated
acidic mist at levels of acidity, sulfate, and nitrate concentrations that range from above to below
those found in ambient wet deposition at high elevations in spruce-fir forests. Cold tolerance
tests are performed in the fall as seedlings enter dormancy and in the early spring as they break
dormancy. Needles and roots are analyzed for total nitrogen and sulfur concentration. Seasonal
measurements are taken of needle water potential. Other measurements include needle diffusive
conductance and transpiration, total chlorophyll, and cuticular wax content.
In six different experiments, Jacobson et al. [8] exposed seedlings of varying ages (one to three
years) obtained from varying locations (from New York to Nova Scotia) to acid mists ranging in
pH between 25 and 4.5 over varying time intervals (41 to 116 days). Their objective was to
determine the effects of continuous and intermittent exposures to sulfate, nitrate, and combined
sulfate-nitrate acidic mists on seedlings. Seedlings were scored visually in a variety of ways in an
attempt to quantify the foliar damage (needle discoloration per seedling, the percent area of
discoloration per affected needle, and needle abscission). Stem diameters, shoot lengths, shoot
displacement volumes, and root displacement volumes were measured on seedlings over the
course of the treatments. Sample sizes were not given.
90
-------
MPO#J
FUP SEEDLING REPORT
PETERSON ETAL
Project Number SPQ7
Principal Investigator KJF. Jensen and G A. Schier
Cooperative: Spruce-Fir
Title: Impact of Ozone and Acid Deposition on Foliar I caching and Growth of Red Spruce
Seedlings
Tree Species: Red spruce
Objectives: 1) Determine effects of acid rain and ozone on photosynthesis and water relations
of spruce srcdlmgs 2) Determine if add rain and ozone reduce carbohydrate concentrations of
red spruce roots.
Deliverables: Dose-response of add rain and ozone on red spruce needle leachate, 3/88.
Dose-response of ozone and add rain on red spruce needle, development and physiology, 9/88.
Effect of ozone and add ram on spruce budworm development, 9/88.
Summary: Red spruce seedlings are exposed to ozone and add rain treatments in CSTRs.
Photosynthesis is measured periodically and the composition of foliar leachate is analyzed. At
four periodical harvests, needle, stem, and root dry weights, total starch;, and sugar contents are
measured. Supplemental tests will involve infestation of treated and untreated seedlings with
spruce budworm larvae.
Patton and Jensen [2] exposed one-year-old seedlings (from a common seed source) to three
levels of ozone exposure (charcoal-filtered air, either alone or with additions of ozone for varying
time periods) and three levels of simulated add rain (pH 3.5,4.0, and 4.5) over a six-month interval
(first week in May - first week in November, 1988). Seedlings were harvested over the course of
the treatments; heights, diameters, and dry masses of components (current-year needles, old
needles, current year stems, old stems, and roots) were measured and 12 parameters of nonstruc-
tural carbohydrates in needles and roots were examined. Sample size was three seedlings per
ozone-add treatment for each of five harvests.
91
-------
MPO#J
FRP SEEDLING REPORT
PETERSON ET AL
Project Number: SF10
Principal Investigator: S.B. McLaughlin
Cooperative: Spruce-Fir
Title: Interactive Effects of Natural and Anthropogenic Factors on Growth and Physiology of
Red Spruce
Tree Species: Red spruce
Objectives: 1) Determine dose/response of HN03, H2O2, Al, and Mn on spruce seedlings. 2)
Characterize differences in gas exchange, carbon allocation, and growth patterns across a
"gradient" in decline and presumed deposition.
Deliverables: 1) Characterization of physiological changes associated with declining red spruce.
2) Determination of effects of nitric acid vapor, hydrogen peroxide, Al, and Mn, 10/88.
Summary: This project includes both Geld and laboratory components. The field study is a
comparison of one high and one low elevation site in the Great Smoky Mountain National Park.
Growth is estimated and monitored on canopy and sapling trees at each site. Photosynthetic
capacity, respiration, and water relations are estimated on saplings. Carbon metabolism studies
are also conducted using C14 techniques.
In the laboratory, red spruce seedlings are exposed to various levels of H2O2 and NO2. Height,
diameter, r.' ..^thesis, and nitrogen reductase activity are all measured. Screening techniques
are being developed "or examining the toxicity of red spruce to individual and combined trace
metals.
92
-------
MPO #3
FRP SEEOUNO REPORT
PETERSON CTAL
Project Number: SF11
Principal Investigator CJ. Richardson
Cooperative: Spruce-Fir
Title: Effects of Atmospheric Deposition on Red Spruce: A Free Radical Based Approach
Tree Species: Red spruce
Objectives: To determine if the key mechanism for atmospheric imposed stress in forest
vegetation is the generation of oxygen-based free radicals by photochemical oxidants in the tissues
of affected plants.
Deliverables: Evaluation of radical formation in leaf tissue of red spruce, 11/87/
Summary: Seedlings from the Wcinstein project that have previously been exposed to different
ozone treatments are used.- Analyses include activities of superoxide dismutase and peroxidase,
and concentrations of glutathione and malondialdehyde.
93
-------
MPO#3
FRP SEEDLING REPORT
PETERSON ETAL.
Project Number: SF13
Principal Investigator: Seiler
Cooperative: Spruce-Fir
Tide:
Tree Species:
Objectives:
Deliverables:
Summary:
Tseng etal. [12] exposed 3-year-old Fraser fir seedlings (obtained from a commercial seed source
in North Carolina) to three levels of ozone (< 20,50,100 ppb) and three levels of moisture stress
over a 10-week interval. This study differed from most in that shorter exposures were employed
(4 hrs/day, 3 days/week). Dry ma«L< of shoots, dry mass of roots, root-to-shoot ratios, and stem
diameters were measured on the seedlings at the end of the treatment interval. Photosynthesis,
transpiration, and needle conductance (using a LI-COR 6000 portable photosynthesis system)
were measured on the seedlings, after overnight rehydration, throughout the ozone and moisture
stress treatments. Sample size for physiological measures was six seedlings per ozone-moisture
treatment for each of three measurement dates.
94
-------
MPO #3
FRP SEEDLING REPORT
PETERSON BT AL.
Project Number: SF14
Principal Investigator. M.H. Unsworth
Cooperative: Spruce-Fir
Tree Species; Red spruce
Title: Frost Hardiness of Red Spruce in Relation to Forest Decline and Effects of Winter
Exposure to SO2 and NO2.
Objectives: Determine if SO2, NO2, SO4 acidic mist and O3:0 alter frost hardening and ii) result
in winter accumulation of phytotoxic substances that alter metabolism and growth of red spruce.
Deliverables: Evaluation of risk of frost injury based on weather data and field observations.
Experimental evaluation of relationship of S, N, and acidity to physiology and biochemistry of
frost hardiness. Model relating deposition to risk of frost injury, 1/89.
Summary: Shoots excised from red spruce growing at Whiteface Ml, NY, were tested in Scotland
for frost hardiness. Results indicate that susceptible trees were likely to be damaged in 6 of the
last 7 years at Newfound Gap and in 4 of the last 7 years at Whiteface ML An exposure facility
for simulated acid mist has been established. Filtered and unfiltered chambers will be used to
test for interactions with air quality. Preliminary results indicate that shoots exposed to SO2 +
NO2 were more damaged than controls after freezing to -4 and -7°C. Ion chromatographic
analyses of xylem sap from red spruce shows increased^ trate, nitrite, sulfite, and sulfate in sap
from trees exposed to SO2 + NO2. The study has also developed NMR techniques to measure
intracellular pH in spruce needles.
Murray et aL [10] developed a method whereby frost damage to foliar tissues may be assessed.
In August of 1987, they cut shoots from one-year-old red spruce wiling? and exposed sections
of each shoot to a range of temperatures (unfrozen control, -3° C, -6°, -9°, -12° C, and immersion
in liquid nitrogen). After the treatment, the rate of leaching of electrolytes from the tissues into
deionized water was measured.
Fowler et aL [9] exposed two-year-old (grown from seeds from New Hampshire) to six
levels of acid mists (pH IS, 17,3.0,33,4.0, and Si)) over a 22-week interval (July 24 - December
18,1987). Sections of shoots were cut from seedlings over the course of the treatments and further
exposed to six levels of simulated overnight frosts (the range of temperatures was changed in
order to reflect the developing frost hardiness of the seedlings). After the frost treatments, foliar
cell damage was assessed by placing the shoot sections in deionized water whereby conductivity
measurements were used to derive an index of leaching rate. Sample size was 20 shoot sections
per acid-temperature treatment for each of six measurement dates. I .caching rate in unfrozen
control shoots was two-fold higher in pH 25 versus pH 4.0 or 5.0.
95
-------
MPO #3
FRP SEEDUNG REPORT
PETERSON ET Ai-
Project Number: SF16
Principal Investigator: L. Weinstein
Cooperative: Spruce-Fir
Title: Effect of Ozone and Soil Nutrient Status on the Physiology of Photosynthesis, Car-
bohydrate Allocation, Nutrition, and Winter Hardiness in Red Spruce
Tree Species: Red spruce
Objectives: Evaluate effects of ozone on the physiology, growth, and development of spruce
seedlings using open-top chambers. Study effects on chloroplast function, respiration, carbon
assimilation and allocation.
Deliverables: Dose-response relationships between ozone exposure and various physiological
response parameters, 1/88.
Summary; Red spruce seedlings are exposed to various levels of ozone in open-top chambers.
Variables measured include those related to photosynthesis, carbohydrate production and
translocation, and shoot and root growth.
Alscher et al. [5] exposed two-year-old seedlings (grown from seeds from Nova Scotia) to levels
of ozone (1,2, and 3 times ambient concentrations, and charcoal-filtered air) over a seven-month
interval (May 30 - December 15, 1987). Seedlings were harvested over the course of the
treatments, and dry masses of components (roots, current-year stems, ? .1 s'ems) were
measured. Sample size was three seedlings per ozone level for each of four bimonthly harvests.
Cumming et al. [7] exposed four-year-old seedlings (grown from seed from Vermont) to three
levels of ozone (1 and 2 times ambient concentrations, and charcoal-filtered) over a 16-week
interval (beginning in late June, 1986). They also exposed three-year-old seedlings (grown from
seed from Nova Scotia) to five levels of ozone (1, 2, 3 and 4 times ambient concentrations, and
charcoal-filtered) over a 16-week interval (beginning June 1,1987). Growth measures were not
described. Sample size in 1986 was six seedlings per ozone level for each of four monthly harvests;
sample size in 1987 was three seedlings per ozone level for each of 12 biweekly harvests. Statistical
analyses were not discussed.
Fincher et al. [4] exposed two-year-old seedlings (grown from seeds from Nova Scotia) to five
levels of ozone (1, 2, 3, and 4 times ambient concentrations, and charcoal-filtered air) over a
seven-month interval (May 30 - December 20,1987). Photosynthetic rates on seedlings (LiCor
6000 photosynthesis unit) were measured throughout the growing season. Needles were ex-
amined microscopically for cell damage and analyzed for photosynthetic pigments. Sample size
was nine seedlings per each of four ozone levels (0.4,1,2,3 times ambient) for microscopy and
pigment analyses. (Sample size was not given for photosynthetic measures).
96
-------
MPO«
FRPSEEDUNO REPORT
PETERSON ETAL
Project Number SF27
Principal Investigator F. Thornton
Cooperative: Spruce-Fir
Title: A Field Chamber Study of the Response of Red Spruce to Cloud Interception and Ozone
Tree Species: Red spruce
Objectives: Determine the effects of acidic cloud water and O3, alone and in combination,, on
red spruce seedling root and shoot growth, photosynthetic rates and tissue nutrient concentra-
tions..
Deliverables: Evaluation of use of chambers for cloud exclusion studies, 11/87. Results of
exclusion experiments, 1/88,1/89.
Summary: Uses open-top chambers on Whitetop ML, VA. The chambers are used to exclude
ambient ozone, clouds, or both. Throughout the growing season'periodic evaluation of
physiological response is determined by measuring photosynthetic rate, stomatal conductance,
and needle water potential. Periodic event sampling of foliar interception of cloud water is also
conducted. Root and shoot biomass and. tissue nutrient concentrations are determined from a
subset of destructively sampled seedlings.
Thornton and Peir [3] exposed two wiling types of Unspecified age (native stock froih the study
site and phyton-grown from a seed source in the Great Smoky Mountains Natior$i jkkk),to
varying conditions on top of Whitetop Mountain, VA (1680 m) during 1988. This study is unique
in that effects were removed by chambers instead of added.- Exposures in chambers were ambient
air, ambient air with clouds removed, and charcoal-filtered air with clouds removed. A fourth
treatment (shade cloth) was conducted to assess the effect,of the chamber. Photosynthesis and
respiration measurements (LJ-COR LI-6200 Portable Photosynthesis System) were taken over
the course of the growing season. Sample size was generally nine per treatment-seedling type for
each measurement date.
97
-------
MPO #3
FRP SEEDLING REPORT
PETERSON ETAL.
Project Number: SF31
Principal Investigator: R. Kohut
Cooperative: Spruce-Fir
Title: Comparison of the Responses of Seedling and Sapling Red Spruce Exposed to Ozone and
Acidic Precipitation Under Field Conditions.
Tree Species: Red spruce
Objectives: 1) Assess effects of acid precipitation and ozone on photosynthesis and growth of
red spruce seedlings. 2) Produce dose/response functions using measures of photosynthesis and
growth as response variables for seedlings and saplings. 3) Develop quantitative; assessments of
the effects of previous exposure and tree age on the dose/response function.
Deliverables: Comparison of dose/response relationships of 1- to 3-year old spruce seedlings and
saplings for acid and ozone treatments, 9/88.
Summary: Red spruce seedlings and saplings are exposed to ozone and acidic deposition in
open-top chambers over a three year period. Response variables include foliar and root pathol-
ogy, net photosynthesis, stomatal conductance, growth, carbon allocation, projected leaf area,
leaf dry weight per age class, and chlorophyll content.
Laurence et al. [1] exposed one-year-old seedlings (grown from seeds from Maine) to four levels
of ozone (0.5,1,13, and 2.0 times ambient concentrations) and three levels of simulated acid rain
(pH 3.1, 4.1, and 5.1) over a three-month interval (June 26 - September 30). Seedlings were
harvested over the course of the treatments, and heights and dry masses of components (roots,
needles, and stems) were measured. Rates of photosynthesis were measured on the seedlings
(using an ADC portable photosynthesis unit) over the course of the treatments. Sample size was
three seedlings per ozone-acid treatment for each of three harvests.
98
-------
MPO#3
FRP SEEDLING REPORT
PETERSON ET AL
Project Number SF32
Principal Investigator J. Rebbeck, M.S. Greenwood, and KJ7. Jensen
Cooperative: Spruce-Fir
Title: Evaluation of the Impact of Ozone on Seedling and Grafted, Mature Red Spruce Using
Open-Top Chambers
Tree Species: Red spruce, balsam fir
Objectives: 1) Determine the influence of tissue maturation on red spruce response to ozone;
2) Determine, under controlled field conditions, the effects of oizone on the growth and morphol-
ogy of red spruce and balsam fir seedlings, via the mechanism of altered caibop allocation and
physiological processes.
Deliverables: Dose-response of ozone on red spruce as a 'function of tissue maturation,
12/88^/90.' Response of red spruce and balsam fir seedling growth and physiology to ambient
ozone at a low-elevation site, 12/88,2/90.
Summary: Open-top chambers are used to expose mature and juvenile grafted red spruce scions
to various levels of ozone. In a second study, balsam fir and red spruce seedlings are being grown
in open-top chambers to assess the impact of ambient ozone at the Howland, Maine, low-elevation
forest.-. Height, diameter, foliar injury, photosynthetic rate, and stomatal conductance will be
measured. Seedlings and grafts will periodically be destructively sampled and analyzed for root
and shoot (scion and rootstock) bioniass, chlorophyll, epicuticular wax, and leaf area.
99
-------
MPO #3
FRP SEEDLING REPORT
PETERSON ETAL
Project Number: WO)7
Principal Investigator: D.T. Tingey and D. Turner
Cooperative: Western Conifers
Title: The Effect of Acid Precipitation on Seedling Growth as a Function of Foliar Leaching,
Foliar Nitrate Uptake, and Cation Availability
Tree Species: Douglas-fir, Engelmann spruce
Objectives: To evaluate the role of foliar cation leaching and cation availability to roots of
Douglas-fir and Engelmann spruce in response to acidified mist.
Deliverables: Report on first-year effects of simulated rainfall chemistry'on throughfall
chemistry, and seedling biomass and nutrient content, 10/87. Final report, 10/88.
Summary: Results of a pilot study on Douglas-fir include:
- Biomass per plant increased at the end of the 12-week experiment in response to higher levels
of nutrient availability.
- Content of Ca, Mg, and K in second-year foliage increased in response to higher levels of nutrient
availability, but did not show a response to fog pH.
- Leaching of Ca, Mg, and K from foliage was considerably higher with fog of pH 3.1 than with
fog at pH 5.6, with K being most susceptible to leaching. However, the amounts of nutrients
removed at pH 3.1 are small relative to the observed uptake rates of these trees.
- Epicuticular waxes were not affected by either treatment.
Further studies will focus on foliar leaching in Engelmann spruce and on the cation exchange
capacity and buffering capacity of conifer needles.
100
-------
mpocj
TO SEEDUNO REFOKT
PETERSON ETAL
Project Number WCOS
Principal Investigator. W. Hogsett and D.T. Tingey
Cooperative: Western Conifers
Title: Sensitivity of Important Western Conifer Species to SO3 and Seasonal Interaction of Add
Fog and Ozone
Tree Species: Douglas-fir, ponderosa pine, lodge pole pine, western hemlock, western redcedar
Objectives: Assess relative sensitivity of western species to: 1) a seasonal pattern of acid fog and
ozone, and 2) SQ2 exposure during fall and winter.
Deliverables: Interim sensitivity ranking, 10/87. Final sensitivity rankings, KV881
Summary: The screening of various species for sensitivity in growth and visible needle injury is
accomplished with two deposition exposure scenarios: 1) acid fog (wintcr)/ozone (summer) as
a seasonal combination of pollutants, and2) gaseous SO2 deposition (winter). Seedling sen-
sitivities are being assessed as a growth' response over two growth periods with year-round
exposures. The fumigation regimes reflect the seasonality of deposition duration, frequency of
events, fog chemistry, and the seasonality of the frequency and distribution parameters of ozone
and SO2 characteristic of selected regions of the west. A range of treatment concentrations is
employed for each pollutant which is representative of the possible air quality conditions of the
west. .The fumigation periods are those months when these deposition patterns occur. Seasonal
interaction of the pollutants, rather than concurrent pollutant combinations, represent a realistic
exposure scenario for much of the climatic conditions of the coastal western U.S. and Cascade
and Sierra foothills.
101
-------
MPO #3
FRP SEEDUNG REPORT
PETERSON ETAL.
Project Number: WC09
Principal Investigator: P. Miller
Cooperative: Western Conifers
Title: Testing the Sensitivity of Five Western Conifer Species to SO2 Alone, and Ozone Followed
by Acidic Fog
Tree Species: Douglas-fir, ponderosa pine, white fir, subalpine fir, Engelmann spruce
Objectives: Using open-top chambers, determine the sensitivity of the five conifer species to SO2
alone, and acid fog and O3 in combination.
Deliverables: Report of preliminary rankings, 10/87. Report of final rankings, 10/88.
Summary: This project is conducted in coordination with the Hogsett and Tingey project.
Exposures are conducted year-round in modified open-top chambers under natural environmen-
tal conditions. Simulated ambient exposure profiles for acidic for, SO2, and ozone are employed.
Exposure regimes for the gaseous pollutants were developed by averaging air quality charac-
teristics from a number of sites across a region. These average values were then used to construct
a 30- or 60- day hourly concentration regime that reflects the ambient air quality characteristics
of the region of interest. A range of treatments for each pollutant is created by building additional
hourly concentration profiles within 1-2 standard deviations of the base profile. Development of
fog regimes follows a similar methodology reflecting the frequency, chemistry, and deposition
volume of selected regions.
102
-------
MPO#3
FRP SEEOUNO REPORT
PETERSON ETAL
112 Appendix B: Example of Power Curve
The following figure illustrates a power curve computed by Project WC08 for ponderosa pine
seedlings. Power was computed for treatments of acid fog (AF), ozone (OZ), and their interac-
tion (AFxOZ), with number of chambers and plots fixed (Len held constant). As might be
expected, there is a higher probability (p > 0.70) of detecting a treatment effect when the
differences are quite large ( > 40%). Conversely, the power of the experiment to detect differen-
ces of less than 20% is very weak.
Suppose, for example, the goal is to detect a 30% difference due to treatment, at an alpha level
of 0.05. The probability of detecting that effect from AF is about 0.40, whereas the probability
of detecting the same change from OZ is about 0.70. Both the alpha level and power desired for
the test can be set, and the sample size varied, as in Table Bl. From these computations, the
number of plants needed per treatment chamber to detect differences as low as 10%, with a high
level of probability (power = 0.90), can be determined.
Note that the example in Table 1 is specific not only to the species, but to the response variable
under consideration (in this case, biomass). Additionally, these are sample sizes for a given power
and alpha level, which assume a fixed or limited number of chambers. Similar tables could be
arrayed by number of plants per harvest, relative differences to detect, and the estimated
population coefficient of variation, in order to determine the number of chambers needed at a
specified level of power. This would require compiling such information for each response
variable used in both repeated measures and final biomass component data for the species or
family of interest.
1.0
0.9 -
0.7 -
0.6
0.4 -
03 -
0.2 "
40
60
20
80
0
^Difference
¦ OZTRTMT + AFTRTMT ~ OZxAF TRTMT
Figure Bl. Power curve constructed from ponderosa pine seedling data of project WC08, where
OZ=ozone, AF=acid fog, and OZxAF is the interaction.
103
-------
FRP SEEDUNC REPORT PETERSON ET AL
Table Bl. Example of sample size determination for a seedling study (project WC08) in the
Forest Response Program.
Ponderosa pine sample size calculations for biomass dry weight.
LN transformation used: power-.90; 2-sided t-test: a=.05.
6
error df for
the
OZ main effect
test;
3 OZ trt; 3
Ch/trt; MSE-.17.
3
error df for
the
AF main effect
test;
3 AF trt; 2
Ch/crt; MSE-.18.
36
error df for
the
OZxAF test; 9
OZxAF
trt; 6 Ch combo/trt; MSE-.13.
%
# Pits per
# Pits per
# Pits per
Difference
OZ Chamber
AF Chamber
OZxAF trtmt
10
188
460
318
15
88
214
74
20
52
126
43
25
34
84
,29
30
25
61
21
35
19
46
16
40
15
37
13
45
12
30
10
50
10
25
9
55
9
22
8
60
8
19
7
65
7
17
6
70
6
15
5
75
5
13
5
n - (t(T(.
025)-T(.90))2]*
[2*MSE/(DIFFLN2) ])
/ #Ch
Where: # Pits - number of seedling plants.
OZ ozone
AF - acid fog
Ch - chamber
n - sample size
T - Student's t statistic
MSE - mean squared error
DIFFLN - natural log of treatment difference
104
------- |