United States Environmental Research May 1994
Environmental Protection Laboratory - Corvallis
Agency Corvallis, OR 97333
PROJECT PROGRESS AND
CURRENT STATUS
Project: Effects of C02
and Climate Change
on Forest Trees
For additional information contact:
Dr. David T. Tingey
Program Leader
ERL-Corvallis
(503)754-4621
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Project Progress and Current Status
TABLE OF CONTENTS
TABLE OF CONTENTS
I. OVERVIEW OF PROJECT
Peer Review Panel Report 1992
Reconciliation Memo for 1992 Review
II. EXPANSION OF PROJECT SINCE 1992
III. PROJECT QUALITY ASSURANCE PROGRAM
IV. PUBLIC EDUCATION PROGRAM
Page i
Effects of CO}and Climate Change on Forest Trees
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OVERVIEW OF PROJECT
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Project Progree and Current Status
OVERVIEW OF PROJECT
CHANGES TO PROJECT IN RESPONSE TO
PEER REVIEW, 1992:
At the end of this section of the booklet are the
Report of the Peer Review Panel on the Project
Plan "The Effect of C02 and Climate on Forest
Trees " for the 1992 review, and the reconciliation
memo drafted by the Project that was submitted to
ERL-C management during the EPA approval
process of the Research Plan. By reviewing these
documents the 1994 Review Panel will quickly
understand how we responded and revised the
Project as per the 1992 review.
The experimental tasks in the Project were modi-
fied from those presented to the 1992 Peer Re-
view Panel as per the suggestions provide in their
review document. In 1992, we designated ten
Tasks:
1. Photosynthesis and Stomatal Conductance
2. Shoot Growth, Maintenance, Phenology
3. Carbon Allocation
4. System Nutrients
5. Litter Layer
6. System Water
7. Root Growth and Phenology
8. Soil Biology
9. Computer-Aided Image Analysis
10. Experimental Facilities.
Briefly, in response to the 1992 review, the Project
was modified to have seven experimental tasks.
We moved the facilities-related tasks (9 and 10)
into the general area of Facilities which includes
Operations; Maintenance; and collection of data
related to non-hypothesis testing activities (e.g.,
flow rates and anti-freeze levels in chillers and
heaters) and data related indirectly to hypothesis
testing (e.g., weather station meteorological data).
We moved the synthesis task (3) into the synthesis
and inference module of the Project and expanded
the overall syntheses efforts to include budgets on
major resources, primarily carbon, water and ni-
trogen. This left seven tasks involved with direct
daily experimental activities to collect hypoth-
esis-testing data. We renamed some of the seven
tasks to indicate the parallel nature of activities
done above- and below-ground in the Terracosm
system (Figure 1).
OTHER CHANGES SINCE PEER REVIEW
1992:
Milestones, expansion and other changes in the
Project since the 1992 Peer Review can be sum-
marized as follows:
COMPLETION OF FACILITY CON-
STRUCTION
SEEDLINGS PLANTED AND CLIMATE
TREATMENTS IMPOSED
TREATMENT AND DEPENDENT VARI-
ABLE DATA COLLECTION
ACCEPTED AS A CONTRIBUTION TO
GLOBAL CHANGE & TERRESTRIAL
ECOSYSTEMS (GCTE) CORE RE-
SEARCH, CATEGORY1, INTERNA-
TIONAL GEOSPHERE-BIOSPHERE
PROGRAMME
Page ]
Overviw of Basic Project
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Pojecl Progress and Current Status
CORE PROJECT EXPANDED:
Supporting Field Studies (Cascades and
Corvallis)
Task 8: Soil Organic Matter
Addition of Federal and On-Site Contrac-
tor Project Staff
Aboveground Physiology
Analytical and Organic Chemistry
Quality Assurance
DEVELOPMENT OF MODELING
EFFORTS IN 1994:
Parameterize TREGRO for Douglas fir
Begin Work on System Models (TREGRO,
GEM, etc.)
COOPERATIVE WORK WITH OFF-
SITE COLLEAGUES EXPANDED:
Cooperators: OSU
Informal Relationships: Germany, Forest
Service, University of Utah
Post-Docs and Graduate Students: NRC/
DOE, OSU
QUALITY ASSURANCE PROGRAM
IMPLEMENTED:
Approved Quality Assurance PI an (Q APP)
for the Experiment
Standard Operating Procedures (SOPs),
Experimental Procedures (EPs), and
Other Procedures (OPs) Identified, Be-
ing Written, and Some Approved
Page 2
Draft of QAPP; and list of SOPs, EPs and
OPs for Field Studies Completed
QA Approaches for Operation of TERA
Facility Identified
INTERAGENCY AGREEMENT WITH
DOE/BATELLE NORTHWEST LABS
ENDED
CORE PROJECT MANAGEMENT:
The core Project is managed with a system of
identifying a Principal Investigator and Partici-
pants for the various Project modules and tasks/
sub-tasks within a module (Table 1). The Princi-
pal Investigator is responsible for ensuring that the
work in the module/task/sub-task is completed.
Participants have a strong interest in the activities
and contribute either by direct hands-on research
or in a consulting fashion.
The Principal Investigators meet once per week to
discuss progress and issues, provide technical
guidance, and plan. Any participant in the Project
may be present at Principal Investigator meetings
depending on the topics to be discussed. All
participants (on-site and Corvallis cooperators
when so inclined or needed) of the Project meet
every other week to summarize progress, raise
issues of concern to current and future progress,
receive technical guidance and plan
EXPERIMENTAL TASK OVERVIEW:
The new Task 8 (Soil Organic Matter) is presented
to the 1994 Review Panel for discussion and
evaluation.
The dependent variables measured in the eight
experimental Tasks, variables in both the core
project and in the greater project done in conjunc-
tion with various off-site colleagues, are summa-
rized in Figure 2. On-site and off-site personnel,
and affiliations, involved in each experimental
Task are summarized in Figure 3.
Overview of Basic Project
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Project Progree and Current Status
Experimental Research Tasks
Task 1
Shoot Carbon and
Water Fluxes
Task 3
System Nutrients
Plant Nutrients
Soil Nutrients
Task 2
Shoot Growth and
Phenology
Task 4
System Water
Plant Water
Task 5
Litter Layer
r
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Task 7
Soil Biology
Soil Water
Task 6
Root Growth and
Phenology
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Task 8
Soil Organic
Growth
;vyv
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Figure 1. Experimental tasks of Project. Task 8 is newly-added since the last Peer Review in 1992. Objectives, approach,
dependent variables and outputs described in the Research Plan, the Experimental Tasks and Facilities booklet, or the Task
8: Soil Organic Matter Booklet of the Peer Review Information Package.
Page 3 Overviw of Basic Project
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Poject Progress and Current Status
Table 1. Management program of Project for on-site staff and Corvallis Cooperators.
Principle Participant Participant Participant
Investigator
Project Management
MODULES/TASKS/Sub-tasks
SCOPING STUDIES
D Tinge}
EXPERIMENTAL TASKS
TASK 1 Shoot Carbon and Water Flux
Gas Exchange - Needle
Gas Exchange - Canopy
Needle Area Image Analysis
Stem Flow/Transpiration
TASK 2 - Shoot Growth and Phenology
Shoot Growth/Phenology
TASK 3 - System Nutrients
Biochemistry
Plant Nutnenis
Soil Nutrients
TASK 4- System Water
PJani Water
TDR - Soil Water
TASK 5 - Litter Layer
Litter Layer Addition
Decomposition
TASK 6 Root Growth and Phenology
Cores-to-depth
Minirhizotron
TASK 7 - Soil Biology
Microbiological Indices
Soil Fauna
Soil Enzymes
Mycorrhizae
Biogenic Gases
TASK 8 Soil Organic Matter
MODELING TASK
TREGRO
INTEGRATION AND INFERENCE
TERA OPERATIONS
D Olszyk
D. Tinge)
D Olszyk
D. Olszyk
D Olszy k
D. Tingey
D Tingey
M Johnson
D. Olszyk
M Johnson
M. Johnson
M. Johnson
P. Rygiewicz
M Johnson
P. Rygiewtcz
P. Rygtewicz
P. Rygiewicz
P Rygtewicz
M. Johnson
B Grtffis
D. Tingey
M. Johnson
D. Olszyk
P. Rygiewicz
D. Tingey
M Johnson
C. Wise
D Olszyk
B Baker
C Wise
C Wise
D Olszyk
P Rygtewicz
P Rygiewicz
P Rygiewicz
P. Rygtewicz
P Rygtewicz
M Johnson
M Storm
E Ingham
A Moldenke
B Caldwell
E Ingham
P. Rygiewicz
M Johnson
J. Weber
D Ols&k
R Waschmann
D Tingey
P Rygiewtcz
D Olszyk
B Grtffis
G Jarrell
M Storm
M Storm
M Storm
P Rygiewicz
M Johnson
M. Johnson
R Griffiths
M Johnson
P. Rygtewicz
M Johnson
P Rygiewtcz
B Baker
B Grtffis
B Grtffis
D Tingey
M Johnson
TERA Hardware/Software
TERA Database
M. Johnson
P. Rygiewicz
G. Jarrell
B Baker
R. Waschmann P. Rygiewicz
FIELD SITES
M. Johnson
P. Rygiewicz
D. Olszyk
M Storm
Page 4
Overview of Basic Project
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Project Progree and Current Status
VARIABLES
TASK 1: Shoot Carbon
and Water Fluxes
Canopy CO2 and HjO Flux
Whole Tree H20 Flux
Branch/Needle C02 and
H20 Flux
Stomatal Conductance
PAR
Needle Temperature
Dark Respiration
TASK 2: Shoot
Growth and
Penology
Stem Height and Diameter
Terminal Shoot and Bud
Length
Branch Count
Bud Count
Needle Area/Dry Weight
Tree Needle Area (Digital)
Phyllosphere Micro-
organisms
Task 3: System
Nutrients
Task 4: System Water
Chamber
Evapotranspiration
Irrigation
Humidification
Plant
Transpiration
Plant and Litter
C, N, Cations, Anions
Total
Soluble
Biochemistry
T
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Pojecl Progress and Current Status
PARTICIPANTS
TASK 1: Shoot Carbon
and Water Fluxes
Olszyk (EPA)
B. Baker (CSC)
G. Jarrell (METI)
D. Tingey (EPA)
Y. Vong (Ca!. State LA)
R. Waschmann (METI)
C. Wise (EPA)
Task 3: System
Nutrients
Tingey (EPA)
A. Fong (METI)
B. Griffis(EPA)
M. Johnson (METI)
R. King (METI)
TASK
Growth
Shoot
and
Phenoloev
Olszyk (EPA)
B.Baker (CSC)
R Crawford (USFS)
I. Ho (USFS)
C-Y Li (USFS)
E. Van Ess (OSU)
Y. Vong (Cal. State LA)
C. Wise (EPA)
D. Olszyk (EPA)
S. Otl (METI)
P. Rygiewicz (EPA)
M. Storm (METI)
Task 5: Litter haver
Johnson (METI)
P. Rygiewicz (EPA)
M. Storm (METI)
Task 4: System Water
Johnson (METI), Olszyk (EPA)
G. Jarrell (METI)
P. Rygiewicz (EPA)
F. Senecal (METI)
M. Strong (METI)
D. Tingey (EPA)
R. Waschmann (METI)
C. Wise (EPA)
r.... . .¦ .. . *... .> %v
Task 6: Root Growth and
Phenoloev
Johnson (METI), Rygiewicz (EPA)
E. Ingham (OSU)
D. Phillips (EPA)
M. Storm (METI)
D. Tingey (EPA)
A. Tuininga (OSU)
V . ¦. ..¦ ¦. . '¦ .¦ ¦... ¦. .¦ ¦. . ¦. .. ¦. . '¦ .. ¦ ,. ¦. . ¦
Task 7: Soil Bioloev
Rygiewicz (EPA)
N. Baumeister (OSU)
N. Buchmann (U. Utah)
B. Caldwell (OSU)
K. Cullings (Indp Contr.)
J. Ehleringer (U. Utah)
J. Filser (Germany)
R. Griffiths (OSU)
E. Ingham (OSU)
M. Johnson (MET!)
G. Lin (U. Utah)
C. MacQuattie (USFS)
A. Moldenke (OSU)
A. Tuininga (OSU)
J. Wemz (OSU)
I.1. . -j
*. *. ¦, *. ,. ,. ,... . .* .. % . ..
,
.* *.* *.*
. .*. *. .*. .*. .*, .*. .*. .*. .,*
" ,«*. ; *. ,.,; *. ; \ ; , *. , . ,, V
Task 8: Soil Organic
Griffis (EPA)
A. Fong (METI)
M. Johnson (METI)
R. King (METI)
P. Rygiewicz (EPA)
1
f /. /. /. *.* '.* '.*\
. *.*» *.* *.* *.
Figure 3. On-site and off-site personnel, and affiliations, involved in the experimental Tasks.
Page 6
Overview of Basic Project
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REPORT: 1992 PEER REVIEW PANEL
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Report of the Peer Review Panel
on Project Plan
"The Effect of COz and Climate Change on Forest Trees"
Review Date May 5 ¦ 6,1992
Review Panel Members
Kermit Cromack, Jr
James Ehlennger, Chairman
Alexander Fnend
Boyd Strain
Robert Zasoski
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Table of Contents
Introduction 1
Facilities and Engineenng 2
Draft Document Presentation 4
Long-term and Short-term Considerations 5
Evaluation of Tasks 6
Evaluation of Task 1 7
Evaluation of Task 2 ... . . . 8
Evaluation of Task 3 10
Evaluation of Task 5 ... 13
Evaluation of Task 6 14
Evaluation of Task 7 15
Evaluation oi Task 8 . . 17
Evaluation of Task 9 19
Need for External Review at Ihe End of Year 2 20
Interactions ... . . 22
Public Education ... 23
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Introduction
This report reviews the proposed Global Climate Research Program. "The Etlect of CO2 and Climate
Change on Forest Trees', at the EPA-ERL Laboratory at Corvallis. Oregon This review is based on
reading a draft version of the Research Plan, 'Effects of COj and Climate Change on Forest Trees', and
meetings and discussions with the staff on May 5-6,1992 There is no doubt that the proposed research
is central to understanding poss&le impacts of future environments on forest productivity and forest
structure. The investigators have chosen to work on a dominant forest species and one of major
economic importance By working on seedlings, that stage of the life cycle most Ikely to be first inrpacted
by any climate change. H seems clear that (he results of this study will have significant and immediate
impact on our understanding of the basic ecological consequences of climate change . It is the intent of
this report to provide a critical review that wifi help further refine the issues, approaches, and questions in
whai holds promise to be an outstanding research program by an excellent, interactive group of scientists
Overall, our review covers eight fundamental issues
growth facility and engineering
document presentation
shon-term aspects - how to get the best possible science with existing facilities
long-term aspects ¦ how to gel the best possible design for science, modeling, and facilities
critical review of specific tasks
need tor an external review at the end of second year
the need to insure regular interactions - both within the Corvallis group as well as with other groups
working in related areas (i.e. other EPA labs working on global climate change issues and other
national CO; programs directed at elevated CO2 effects), project management - division of labor and
responsibilities
public education
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Facilities and Engineering
At an international workshop. It was recently It was concluded thai a significant intensification of research
was required to develop an understanding of the long-term effects of atmospheric COj enrichment and
associated environmental change on terresinal vegetation and on ecosystems (Mooney. HA, E. Medina,
D W. Schmdier, E -0. Schulze, and B.H Walker (Eds), 1991, Ecosystem Experiments, SCOPE 45, John
Wiley & Sons, New York). Problems and appropnate research projects were Itemized and available, and
perceived technologies were described. Facilities from horticultural greenhouses to closed oommercial
growth chambers, opentop fumigation chambers, dosed ecocosms of various sizes (1 m* I00m2j placed
over existing vegetaiion in the field, free air CO2 release (FACE) arrays, and totally controlled
phtytorhizotrons (SPAR units) were descrbed and their possible applications discussed (Strain, B R
(ed ). Available technologies for field experimentation with elevated CO2 in global change research, In
Mooney, HA et al (Eds), Ecosystem Experiments, SCOPE 45, John Wiley A Sons, New York, p
245-261) The SCOPE volume 45 provides strong justification for the development ot the TERA
(Terrestrial Ecophysiolog
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Although the quality of environmental control in the battery of terracosms has not yet been determined,
the Corvallis group consulted extensively with the operators of the US DA SPAR facilities at the University
of Florida and Mississippi State University A technician from the Florida facility was assigned to the
Corvallis TERA facility to assist in construction, Installation and testing of the equipment. The system was
engineered using principles of overstzlng of refrigeration and heating components and redundancy of
critical flow control valves and pumps. Computer monitoring and control of the system was applied
throughout. The final proof of the concept and the adequacy of engineering will remain in the results of
the testing to be done in the summer of 1992 but the panel has no recommendations lor changes or
improvements in the system it appears to be an excellent design and to be adequately engineered
Strengths and Weaknesses of Approaches and Facilities There are strengths and
weaknesses in all experimental approaches and facilities The primary strength of the TERA facility is that it
provides excellent environmental control of both the atmospheric and edaphic components of terrestrial
ecosystems This will allow study of the feedbacks and linkages between ecosystem components
responding to global environmental change Strengths in the TERA facility also include the use of top-of-
the-line control components and monitoring equipment Once control algorithms are developed and
tested tor the computers which monitor and control the systems, the panel is confident that the desired
level of environmental control will be obtained The facility incorporated the design and component
experience of the Florida SPAR facility and thus can be expected to come on-line with a minimum o!
technical difficulty or delay Multiple sensors of environmental factors throughout the atmospheric and
edaphic halves of the terracosms will allow the characterization of environmental gradients and thus will
allow the environmental control to be optimized Modern sensors, analytical instruments ard imaging
equipment will allow adequate monitoring of plant and ecosystem responses to the experimental
treatments Excellent control of atmospheric gases and system temperature require completely
controlled facilities and justify the approach adopted by the Corvallis team
Weaknesses include the ever present expense of utilizing up-to-date instrumentation and engineering
components The relatively small size of the controlled environment created (2 m* bench space and 2 m3
of soil volume) in each terracosm seriously limits the numbers ol plants that can be enclosed in each
chamber. It must be pointed out that there will be edge effects which will cause environmental gradients
within the chambers. Unfortunately, a 3 X 7 plant array only encloses 5 individual plants not affected by
edge eflects The quality ol overall environmental control and spatial moniioring of actual conditions
within the chambers will compensate for this weakness The expense of the entire facility seriously limits
the number of terracosms that can be constructed The 14 units under construction only allow the
creation of a 2 COj X 2 temperature factorial experiment with 2 chamber replications and a partial water
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treatment and 2 unchambered control plots (2X2X2 + 4 + 2-14) A special effort will be required to
obtain statistically significant results with a minimum chamber replication of only 2 and experiments that do
not contain complete factorial design For example, the current design assumes that changes in water
regime will not occur without changes in temperature regime. If a simplifying assumption is needed to
reduce the number of treatments, It would seem safer to assume that elevated CO: would always be
associated with a change in water rather than elevated temperature.
Since it will require 2 3 years to complete one experiment with only 21 plants per cell, the panel
recommends that additional terracosms be added to the array at the earliest posstole time following the
testing phase of the original 12 terracosms A minimal experiment using three levels of two environmental
variables and three repiicatons would require 3 X 3 X 3 ¦ 27 terracosms.
Draft Document Presentation
it was our impression thai the Research Plan was to serve two distinct purposes
an agency/public document on policy issues and a program to get answers to relevant questions
* a research proposal detailing scientific approaches to understanding basic climate change questions
Overall, the Research Plan as developed is very good It dearly and succinctly documents the data for
anticipated climate change effects and then follows with a coherent, integrated research program for
assessing the impacts of possible climate change scenarios on forest trees However, several aspects of
the presentation require revision Revision of the Research Plan is essential as we see this document
playing a major role in several aspects of the research First, as a public and agency document, it
describes policy issues of relevance to the EPA and a plan or strategy for assessing possible climatic
changes on long-term performance of forest trees This is important for issues and interactions withm the
EPA, for clarification to the public about the relevance and need for the proposed research, and for
budget justification. Second. K provides a documentation of the logic behind why certain research
questions were addressed, the justification of one approach over another, and the rationale for specific
approaches and data collection to assess the possible impact of anticipated climate changes
We see several rectifiable weaknesses in the current draft version of the Research Plan. These concerns
should be addressed in order to produce a document that serves the research program at Corvallis at the
public, agency, and scientific levels Specifically we are concerned about'
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need for an Executive Summary, including a summary of overall project objectives and
hypotheses
inadequate justification of the geographic origin, tree species and populations to be used in the
experiments
inadequate development of the linkage between policy issues and research tasks
inadequate development of the linkages between possible changes in forest redistribution and
primary productivity studies
inadequate clarification of the linkages between experimental and modeling efforts, including the
different scales of the modeling efforts, and it and how Douglas-fir responses will aid in modeling
the responses of other forest types
inadequate description of the quality assurance plan
inadequate presentation of leam interactions
It would also be extremely useful to have a very brief document describing the project available to the
public, especially those that will be visiting the facility over the next several years
Long-term and Short-term Considerations
The proposal contains a description of several ecophysiological experiments on Douglas-fir tree seedlings
and associated soil organisms that are programmed to extend through 2-3 years In addition, the intent to
use models to extrapolate to field conditions and to long-term considerations of the response of Dougtas-
fir forest ecosystems to future global environmental change is slated With some improvements in detail
as suggested in the following section on the individual tasks, the short-term experiments are
conprehendible and justified The panel, however, found the description of the modeling component to
be contusing and inadequate The purpose ot this section is to summarize the response of the panel to
the shori- and the long-term aspects of the proposed research.
Shorwerm Experiments The array of ecophysiologicaf measurements proposed to be conducted is
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logical and, with the adoption of improvements suggested in the following section on the ten tasks, the
individual projects are well justified The panel feels, however, that the major short-term objectives of the
research should be clearly stated in the beginning of the document. More importantly, the hypotheses to
be examined in the proposed research should be presented and briefly explained in a new section near
the beginning of the document
Long-term Objectives. The intent to conduct the short-term experiments in a fashion that will allow
extrapolation to normal field conditions in Douglas-fir forests, in other perennial ecosystems, and which will
allow prediction of tree growth and forest ecosystem responses to future global environmental change Is
inadequately presented and justified. Although, n is stated that TREGROW will be linked to a stand-level
model such as ZELIG and undefined soil system models, confusing statements that the modeling will
guide the measurements to be made and that the data collected will be used io construct models force
the panel to seriously question if adequate thought has been given to the entire issue of extrapolation of
the results of the short-term efforts
II is recommended that the actual measurements to be made in the short-term experiments be
reconsidered relative to the specific extrapolations and predictive modeling attempts to be made
,; ¦
* /
0
Evaluation of Tasks
General Comments The orientation of the research efforts toward specific tasks greatly improved the
comprehensiveness of the draft report While the subject areas and planned measurements are provided
in some detail, the task descriptions need conceptual improvement ^It is recommended that specific
hypotheses be developed under each task and that the experimental approach be explicitly addressed
toward testing these hypotheses Outputs might be more effectively detailed as they will enable scientific
questions to be answered Given the planned integration of task outputs with modelling efforts, it would
be advisable to present the physiological parameters and environmental responses (including units) that
will result from each task as a part of the outputs section. A preliminary review of this quantitative section
by a modeler may greatly ax) n the future linkage of this information with models
The nature of the linkages between data collection and models needs to be more explicitly presented
Additionally, the nature of the models and the kinds of models to be used deserves more attention
Specifically models like TREGROW, which apply to a single tree, may have little relevance to canopy-level
dynamics
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Evaluation of Task 1
An understanding of carton and water fluxes through leaves is a key critical and essential component to
the overall research program While the proposed research makes a slrong move in addressing the
essential photosynthesis, transpiration, and respiration measurements, H was felt that the following points
need to be addressed
a need to more clearly state objectrves
clarification of the photosynthetic potential to maintenance respiration concept
possible reductions in proposed efforts in methods that require chamber intrusions
an increased emphasis on automated stem flow measurements
consideration of stable isotope measures as a tool for integrating long-term patterns
Clarification of objectives A much more crisp and coherent statement of the specific research
objectives is needed This will greatly aid in focusing the research and providing a logical link between
photosynthesis, carbon allocation, and modeling efforts
Clarification of the photosynthetic potential to maintenance respiration concept While
measurements of both photosynthesis and respiration are discussed here and m other sections of the
research plan, there is no place where it is clear why photosynthesis and respiration should be related to
each other Development of the concept that respiration 15 a maintenance process to maintain
photosynthetic machinery would provide the framework lor anticipating specilic relationships and patterns
between maximum photosynthetic potential and dark respiration rate Furthermore, it would provide the
basis for specific hypotheses to be tested on plants developed under contrasting environmental regimes
and might ultimately provide new insights into the carbon requirements for maintaining photosynthetic
tissues under anticipated environmental conditions
Possible reductions In proposed efforts In methods that require chamber Intrusions
Overall It would appear that there are too many photosynthetic measurements A major consequence of
this is to be too many intrusions into the terracosms Where possible, photosynthetic measurements
should be event oriented rather than calendar determined
As to the kinds of photosynthetic measurements, there would appear to be a potential overemphasis of U-
6200 observations In the long-term, K is not clear just how useful these spot measurements will be for
modeling and carbon balance efforts While some U-6200 observations are of course necessary, care
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should be put into the experimental design to minimize the number of these observations In the long-
term, much more useful information of the kind that might be collected by LI-6200 observations could
come from automated diurnal observations such as from the stem sap flow and the Ceres stem diameter
devices
The functional response curves will be important and should be kept intact, especially since there are
likely to be time dependent changes in these parameters.
Increased emphasis on automated stem flow measurements. Once calbrated, the stem sap
flow devices can provide both quantitative and diurnal information on total gas exchange activity by entire
saplings. Once photos ynthetic versus conductance regressions have been worked out, ft should be
possible to use dumai sap flow measurements to provide information on both diurnal water loss and
carbon gain patterns Such measurements do not require disruption of CO2 levels within the terraeosm
and can be collected in an automated fashion The Ceres device will provide water stress information at
the same time An increased emphasis on the integrating, automated approaches is suggested
Stable Isotope measures as a tool for Integrating long-term patterns A better
understanding of long-term gas exchange patterns would be obtained through analysis of the UC
composition of leaf materials By analyzing either leaf components or whole leaf materials, it is possible to
get integrated estimates of the intercellular CO2 concentration over the past several days (starch) to
months (entire leaf) The use of this approach would overcome variations in physiological patlerns
between terracosms that might arise on a day-to-day basis Since the isotope composition of the source
C02 will be so different from that of current atmosphere CO2, it will be possible to clearly distinguish
between tissues laid down before the entry into the terraeosm and that produced under elevated COj
conditions
Evaluation of Task 2
Shoot growth and phenology measurements will be a key component of evaluating growth responses to
simulated climate change Maintenance respiration will also be essential in determining the mechanism by
which climate changes are brought about The following suggestions are onented toward improving the
value of growth and respiration measurements to investigating climate change impacts on trees and to
using the physiological responses collected in these experiments for future modelling efforts Our
comments encompass five main points
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more rigorous approach to respiration is needed
the culture of plants outside of the terracosm for destructive harvest is advisable
frequency ol growth measurement may be reduced
timing of growth measurements should be refined
Respiration is an important aspect of the carbon balance and simplifying assumptions such as constant
Ol0 etc . may be a weak link in carton budgets H certain points are not considered. The current direction
of sampling darfc respiration during photosynthetically active and inactive periods is of great importance to
inferring treatment effects and extrapolating respiration to carbon gain. The possibility thai temperature
responses of respiration are different during daylight and nighttime periods should be rigorously
addressed Certain current investigations of CO2 responses are finding depressed rates of respiration in
association with elevated CO; regimes No clear mechanism exists for this response. It is recommended
that this response be incorporated into the hypotheses to be tested under this task When interpreting
the physiological significance of changes in respiration it will be important to separate maintenance
respiration from growth respiration
Parallel seedling culture. It is recommended that the investigators consider growing a separate set
of seedlings fumigated under experimental CO2 and temperature regimes This could have several
benefits to the terracosm experiment (1) plants could be used for the production of litter for
decomposition studies, (2) it would enable development of aiiometric equations for biomass prediction,
(3) the presence and timing of anatomical changes could be investigated in response to CO2 and
temperatur for similar conditions and compared with the experimental seedlings at the end of the
experiment for verification, (4) changes in tissue secondary chemistry ooukJ be evaluated in similar fashion
to anatomy, and (5) the dynamics of changes in tissue carbohydrate concentrations, including non-foliar
tissues, could be more rigorously evaluated In each case, measurements would not be strictly
comparable with the terracosm environments, however, these efforts would provide better information lor
purposes outlined In (i) through (5) than information obtainable from existing literature.
Sample frequency. Constoer reducing the frequency of growth measurements, especially in the early
stages of the experiment when treatment differences may be very subtle.
Timing of measurements. Growth measurements should coincide, whenever possible, with actual
phonological events and with environmental effects For example, Ceres determinations may indicate
times during which growth slow-down is taking place and signal a need for more intensive measurements
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Evaluation of Taak 3
information on carton allocation will be key to understanding and explaining the mechanisms (or growth
responses and to exploring the implications of climate change scenarios to indirect physiological effects
through changes in plant structure and metabolism. In addition, this task will need to provide many of the
key parameters used in process-based models such as TREGROW. The following comments address
strengths and weaknesses of the current experimental plan and suggest potential improvements that can
be implemented within existing constraints. Three main points Involve:
potential improvements in determination of whole-plant carbon allocation
ideas tor re fining the approach to soil carbon pools
consideration of soil carton fractionation
C Allocation and CO2 fluxes. Using the current design, most ot the carbon dynamics data will be
with respect to the net COs fluxes into and out ot the terracosm These data will be of value to developing
hypothetical responses for mmi-ecosystems but will be of little value in parameterizing the allocation
functions for simulation models The magnitude of carbon translocated beiowground will be the principal
measure of carbon allocation, unless estimation of aboveground biomass is used, the transfer coefficients
for the allocation of carbon aboveground tissues will be weak To correct this, it is recommended that
biomass ot tissues be estimated from regression equations developed from destructive sampling of
representative seedlings, as discussed under task 2
Soil carbon pools. The accuracy of estimates of carbon allocated to the root and mycorrhizai tip
components of soil carbon will be strong points In contrast, estimating the other components of soil
carbon pool should be approached with caution because treatment effects on the turnover rates of root-
hyphae. which can be considered as allocation to root processes, but which may have high turnover rates
and may be under-estimated from core sampling, and fall outside the resolution ol soil gas measurements
The resolution of soil carbon pools as indicated in Figure 5-4 will be difficult An important flux of carbon is
from fine roots and mycorrhizae to soil litter; this should be addressed in the context of soil carbon
allocation (e g, Figure 5-4). Regarding soil/root respiration, stable isotopes (e g, 13C) may be of use in
separating carbon evolved from the soil from root respiration versus that evolved from decomposition
Soil carbon fractionation. Consideration should be given to the potential for treatment effects on
the fractionation of carbon among compounds and density fractions within the soil, and its significance to
microbial processes
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Evaluation of Task 4
Climatic change may aft eel the magnitude and timing of plant growth, therefore interactions between plant
growth and nutrient supplies is one of the critical links among ecosystem components. Even In the
absence of a seedling growth responses, nutrient supplies may be altered It is reasonable to assume that
those nutrients which are linked more closely with biological activity (N,S,P) will be more directly affected
by the imposed treatments The panel feels that the overall the experimental plan addresses the major
issues associated with nutrient availability very well The construction of terracosms with separate soil
horizons and a litter layer is to be commended and will introduce an element of soil variability realism not
often incorporated into these kinds of systems The panel felt that the following points need to be
addressed
Since nutrient availability may change with the imposed treatments, an addition of nutrients
should be approached with caution
Sampling of tissue nutrient levsts three times a year is not necessary
Disturbance effects on nutrient availably should be considered
DRiS may be a useful technique for comparing and analyzing nutrient concentrations among
treatments
ionic balance of the lissue should be considered
Extractable tissue sulfate is a reasonable measure of sulfate availably in Douglas-fir and should
be measured
Consider measuring mmerahzable nitrogen as a indication of nutrient availability and organic matter
quality
Addition of nutrients to simulate rainfall is not necessary
Reduce the frequency of soil Ca. Mg, Na. and K analyses
Analysis of zero tension water from the bottom dram may be anomalous and should be avoided
Other NBS tissues (Pine needles for example) and geological samples are available for QA/OC
Nutrient additions. Because of the unknown and potentially complex interactions among the plants,
microbes and climate treatments, the availability and importance of nutrients is likely to change. Therefore
any nutrient addition should be carefully considered We recommend that only when a nuirient is
uniformly deficient among all treatments should it be added The criteria for determining a deficient
conditions should be carefully delineated
Sampling frequency of tissue nutrients. A savings in sampling time, tissue, and costs could be
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realized by sampling the tissue in the late fall. Such sampling would adequately indicate changes in
nutrient status as compared to initial and final nutrient content Literature values for this sampling time are
also available lor comparison Nutnent content (concentration bio mass) should be considered as the
comparative measure ol nutrient uptake as the treatments may develop different amounts of
aboveground biomass
Disturbance affects on nutrient availability. The removal and storage of the soil will result In a
flush of available nutnents In order to quantify this change, comparisons with soil from the collection site
should be undertaken Growing a cover crop and removing the above ground biomass in conjunction with
replacement of the surface 5 cm of the A honion when a fresh litter layer is applied (see task 8 Soil
Biology) will mitigate some of the disturbance effects
ORIS as a system of comparing nutrients among treatments. The DRIS system (Diagnostic
Recommendation Integrated System), should be considered as a technique to facilitate comparison
among treatment nutrient levels [Beaulits (1973, a rather obscure reference but cited in Jones, C A
(1981) Proposed modifications of the diagnosis and recommendation integrated system (DRIS) for
interpreting plant analysis Com in Soil Sc and Plant Anal 12 785-794 J
Ionic balance (Sum of inorganic cations - sum of inorganic anions) may be an important variable across
treatments and should be calculated If the source of inorganic nitrogen varies among treatments
(ammonium v nitrate) the ionic balance of the plant as well as rhizosphere pH and composition could vary
The ionic balance of the tissue will be an indicator of the change in nitrogen source In order to calculate
charge balance a total acoountmg of the inorganic cations and anions will be necessary Alternatively, the
organic anion composition of the tissue could be determined
Extraetable Sulfate Changes in mineralization of the organic matter would be anticipated in this study
The release and availability of N.P and S may change Extraetable suffate suffur (Turner, J , M J Lambert
and S P Gessel, 1977, Use of foliage sulfate concentration to predicl response to urea application by
Douglas-fir Can. J For Res. 7.476-480) has been related to available sulfur. Suffate would be needed
for ionic balance calculations
Mlnaralliabla nitrogen As noted above, mineralization is likely to change. In order to assess this
possibility mineralizabie nitrogen should be measured at the beginning and at the termination of the study
and compared with the original site values.
Rainfall nutrient Inputs. The amount ol nutrients likely to be present in local rainfall would be low
12
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compared to those m the system and additions to account for rainfall inputs is not necessary
Soli nutrient analysis. The exchangeable cations, Ca. Mg, Na, and K are not likely to change rapidly
Therefore the frequency of sampling for these components could be reduced to a yearly or even an mmal
and final comparison If there are great or interesting changes between the initial and final samples, then
(he more frequent samples collected in the conngs could be examined
Evaluation of Task 5
The litter layer is a critical component of tr>e experimental system and the component ol the substrate
which is most likely to be altered during the study The examination and comparison of the litter dynamics
needs to be modified m order to be of similar scope with the soil nutrient studies First and foremost the
botic activity of litter needs to be documented and compared to the litter at or near the collection site The
panel feels thai the following recommendations should be considered
Addition of fresh litter and 5 cm ot A honzon material
Leaf packs rather than iitier bags are suggested
There is a concern that lyophilization may influence decomposition
Comparison ot Terracosms litter decomposition 10 on-site litter decomposition is suggested
A third year ot the study may be necessary to asses the quainy of litter produced in the Terracosms
Liner generated m the Terracosms should be quantified
Addition of litter layer and fresh A horizon material. Since the meso ano macro biotic
component of the litter as well as the microbiotic suite is essential to a realistic assessment of
decomposition, fresh litter from a siie near the collection point as well as the surface 5 cm of surface sod
should be placed in the Terracosms io establish a flora and fauna consistent with those in the undisturbed
state
Ltaf packs Litter bags may exclude important fragmenting organisms. Since the litter will be relatively
undisturbed leaf packs could be used to determine decomposition and changes in litter A pilot study
during summer of 1992 should be undertaken as a way to examine this methodology
Lyophilization Lyophilized foliage may not have a full component of surface fungi and other
decomposing organisms Air dry material should be used with the correction lor moisture content and ash
content
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Controls for litter decomposition. Since the system is disturbed and is maintained at conditions
quite different from those where the litter was collected, decomposition at the collection site should be
monitored as a means to compare with ambient conditions.
Extending the study. Leaf and root material produced in the Terracosms will be valuable material for
subsequent decomposition studies and may justify the extra year of growth Some consideration of
growing ^C-and ^N-enriched, high carbon dioxide-grown material during the next two year* for use in
future experiments would appear to be a high priority. This could be combined with the growth of
additional seedlings to establish allometric relationships.
Evaluation of Task 6
Site moisture balance is not as well defined for the future climate as is the increase in temperature and
carbon dioxide Consequently, it seems reasonable to depart from the ambient conditions on both the
positive and negative side in order to develop system responses to a wider range of moisture conditions
The panel felt the following aspects of site water should be addressed
A larger difference in moisture conditions should be imposed
Routine soil water measurements are too frequent
Schoiander Pressure Bomb measurements should be minimized
The conddions of bottom drainage should be avoided
A wider range of soli moisture conditions We do not believe that the waier contents represeni a
large enough difference Drying to -0 1 MPa (-1 bar) does is not a very stressed situation We suggest
that the scenarios in Table 4 1 i e *4 C ± 20% moisture would create a greater treatment effects
consistent with the intent of evaluating climatic differences on seedling growth Some guidance on the
soil moisture regimes could be taken from the ordination of Pacific north west vegetation on moisture and
temperature. For example, movement into a Ponderosa pine regime would seem appropriate
Constrict toll moisture measurements. Hourly measurement of soil water appears to be excessive
for routine measurements Daily measurements would be adequate with occasional measurements of the
daily course of soil water to provide detailed understanding
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Reduce pressure bomb measurements. Alternative methods of measuring plant stress * attached
tissue psychrometers, stem (low and diameter measurements calibrated to plant stress will require less
destructive measurements, less time and can be automated
Avoid toll saturation. As the original soil does not show signs of poor drainage, the conditions of
zero tension water indicative of saturation at the gravel-soil interface should be avoided A hanging water
column or a vacuum on the drainage would avoid this complication. Alternatively, the judicious application
of water would also be a means of avoiding saturation The implications for trace gases and solution
composition of soil saturation suggests efforts be taken to avoid saturated conditions.
Evaluation ot Task 7
The role of rool growth and rhizosphere processes in direct effects of elevated CO2 and climate change is
of great interest Also of interest is the potential for indirect effects of atmospheric changes on ecosystem
processes through their effects on root structure, function, and the interactions of rhizosphere processes
with soil biogeochemical processes Irrespective of these current concerns, root and rhizosphere
processes remain a substantial knowledge gap in basic understanding of environmental-plant
interactions The emphasis of root studies and the intensity of investigation in this task are well-placed
efforts Comments in the following areas are directed toward improving studies within the existing
expenmental framework The main points are the following
' smaller soil cores may improve root estimates
wf- dry sieving 1$ not recommended for root-soil separation
vertical stratification of root samples is important
\ consider analyzing roots for secondary compounds
£#- use caution with, and further justify the use of. ingrowth cores
\ consider separating tine roots into further categories
0^. outputs need further elaboration
Soil cores. The use of more numerous soil cores of smaller diameters should be considered Soil
cores would be the most reliable means of quantifying standing and vertical distribution of fine roots in the
terracosm An additional sampling to those Indicated in Figure 5-2. before the onset of rapid root growth,
may be advisable. It could provide interesting information on root modality and turnover and would be of
value in estimating the magnitude of growth rates during the periods of rapid growth.
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Root-toll separation. The use of dry sieving is not recommended due to the potential lor the loss of
root fragments Other techniques are hand sorting, or gentle agitation by air and water followed by wet
sieving it is recommended that rigorously-defined visual critena be used for separation of live and dead
roots with staining used as a verification of visual criteria Root stains may respond to certain microbes and
result in an incorrect interpretation ot vitality. Vogt and Persson (1991 in: Lassoie and Hinkley
Techniques and Approaches in Forest Tree Ecophysiotogy. CRC Press. Bocaraton, Florida) provide a
review of root sorting and of live/dead differentiation.'
Vertical stratification. The current plan to stratify root biomass estimates by depth within horizons is
essential to the interpretation of these data.
Secondary compounds. The effects of climate-change treatments on the concentration ot
secondary compounds in roots should be considered as a potential feedback in the soil system
ingrowth cores. Caution is recommended in applying the rate of root ingrowth from ingrowth cores to
root growth rates in intact soil, potential artifacts are associated with adventrtious root formation and with
delays m the appearance of roots in the cores Higher moisture content of ingrowth cores, associated with
root-free soil, may be an additional artifact with ingrowth cores Without further justification, this method is
of principal value as a means of closure, or for verification of rates of change in growth and monality
determined trom mmirhizotrons
Fine roots. In addition to distinguishing root tips from mycorrhizal root tips, it may be of value to
separate line roots into the branching orders ot primary laterals, secondary laterals, ternary laterals, and
short roots It appears that the separation of fine roots from other roots is based on diameter class (e g ,
<2 mm are fine) If the above branching orders or morphological root lypes are not easily distinguished,
then diameter class separation within the <2 mm dia fraction (e g , 1 -2 mm, 0 5-1 mm, and <0 5 mm) may
improve your ability to resolve treatment effects on roots
Outputs. Although not mentioned explicitly, the following root parameters would be important to report
Root length density (cm cm-3]
¦ Specific root length [cm mg')
Ash-free root b«omass [g m-*]
Ash-free myoorrtiizal tip biomass Ig m 2]
Root growth rates by horizon [cm cm 3 wk-1)
Mycorrhizal colonization [per unit root basis]
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Root branching [root tips per unit length, or other)
Root carbon concentration [%]
Root nitrogen concentration [%]
Root turnover rates !g m-2 mo*1, or other]
Evaluation of Task 6
The soil biology research should consider using an array of current methodological approaches in the
following general areas determination of soil microbial biomass, soil enzyme activity, root biomass and
associated rhizosphere botic activity, soil animal populations and cumulative effects of their activities on
sod structure, and nutrient availability At the same time, provision for inoculation of the reconstituted soil
profiles by boih soil animals and soil microflora needs to be considered, preferably using a geographic
source at or near the srte where the original soil was obtained A general recommendation is that the soil
biology and litter decomposition methods be tested in preliminary research scheduled to be done in the
terracosms in the next few months It is recommended that smaller sample sizes and less frequent
sampling should be done in order to be less intrusive to the terracosms, this places an emphasis on
obtaining as many complementary measurements as practical for each sample series within a context
provided by seasonal and phenoiogical events The following are specific recommendations
Studies of Mycorrhizai Fungi Ectomycorrhizai fungal biomass associated with the fine roots can be
estimaied in conjunction with percentage of mycorrhizai inleciion It may be possible to inoculate the soil
in each terracosm with known species of Douglas-fir ectomycorrhizae. and later identify these through
hyphai characteristics and use of DNA sequencing techniques Some species may also produce fruiting
bodies, which would help in identification and in helping confirm adequacy of terracosm resources for
mycorrhizai reproduction Recent work has indicated the sensitivity of mycorrhizai colonization to factors
affecting canopy function, such as inseci defoliation (Gehring and Witham, 1991, Nature 353.556-557) It
may also be of value to consider bioassay of fine roots for N or P uptake using the methods given by Van
Cteve and Harnson (1985, Can J For Res 15156-162)
Microbial Studies Microbial biomass of mineral soil bacteria and fungi can be estimated with direct
counts using well established procedures lor both live and dead cells, as outlined in the current research
plan using the methods given in Lodge and Ingham (1991. Agric Ecosys Environ. 34 131-134.) Soil
fumigation microbial biomass and/or extractabie microbial biomass C, N and P could be estimated from
seasonal soil cores, provided that there are adequate quantities of material left over from soil cores taken
to estimate microbial biomass by direct counts and cores taken to estimate biomass of fine roots and
17
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associated myoorrtiizai fungal colonization and biomass For some purposes composite samples could be
used from several cores A variety of general methods have been developed in estimating soil microbial
biomass and extractable biomass C, N, P such as those detailed in the following.Jenkinson (1988 In.
Advances in Nilrogen Cycling E. A. Paul and J. M Ladd (Eds) pp 368-386). Parkinson and Coleman
(1991. Agnc, Ecosys and Environ. 343-33): Jordan and Beare (1991. Agric. Ecosys. and Environ. 34.
35-41); Vance et al (1987. Soil Biol. Biochem. 19.703-707); some are sensitive lo soil water content,
shown by Sparling and West (1989. Soil B«l. and Biochem. 21.245-253). A current example of adapting
microbial biomass measurements for small samples is given by Heilman and Beese. 1992 Soil Sci. Soc
Amer J. 56.596-598). If different ratios of bacterial to fungal biomass ratios are evident from direct counts
in the different treatments, this may influence proporiions of labile microbial biomass C retative to cell wall
constituents Changes in soil and litter microbial biomass and composition will affect availability of nutrients
such as N and P immobilized in microbial components Soil enzyme assays could include phosphatase
and protease for P and N, respectively, an interesting recent example is provided by work testing
phosphatase activity in ectomycorrhizae of Norway spruce forests impacted by pollution(Rejsek 1991
Soil Biol Biochem 23 667-671) For C compounds, ceiiuiase, phenol oxidase and peroxidase could be
considered (Sinsabaughet al ,1991, Agric Ecosys Environ 34 4 3-54) Careful consideration needs to
be given to sampling of gaseous emissions, such as methane and nitrous oxide, provided that the soil is
adequately drained in the terracosms (see recommendations under the soil hydrology section for waier
regimes) Reinforcing our earlier suggestion that a significant dichotomy of watering regimes be
considered, Gestet et al (1992, Soil Biol Biochem 24 103-111) have recently discussed soil microbial
responses to seasonal drying versus moist winter conditions
Restoration of Soil Profiles Remoculation of the soil with soil microflora and fauna will be necessary
This could perhaps be done best using sections of intact to rest floor and the upper A mineral soil horizon
from the original site or from old growth forest sites adjacent to those where the original soils were
collected Additionally, it may be desirable to add some native earthworm species to the soil in each
terracosm since they may influence soil structure and function (Spiers et al 1986, Can J For Res
16 983-989. Graham and Wood. 1991. Soil Sci. Soc Amer J 55 1638-1646) The assistance of soil
animal biologists and taxonomists is suggested, such as the kind of soil animal expertise exemplified by
Moldenke et al (1991. Agric Ecosys., and Environ. 34 177-185) Likewise, characterization of initial
microflora and fauna present in the newly added forest floor and mineral soil as well as those present at the
end of the general experiment, would be desirable Recent work has shown the value of comparing
stored soils compared with those recently sampled (Ross, 1991, Soil Biol Biochem 23 1005-1007).
Litter Decomposition Research. Litter decay studies could be made more realistic by using needle
litler packs, rather than enclosed litter bags (Tnska and Sedell, 1976, Ecology, 57.783-792). with the
18
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caution that polyester thread may be necessary for needle pack construction due to possible UV light
deteriorating efiecls on nylon monotiiameni line placed in the terracosms, but much diminished in the
stream environment where Triska and Sedetl worked litterbags can alter microbial colonization ot litter,
particularly affecting rhizomoiphs produced by higher lungi (St John, i960, Oecologia (Berlin) 46130-
132) Enough litter packs should be added lor the anticipated full three year duration ot the initial
lerracosm studies, sampled on a seasonal basis Litterbags with 1 mm mesh are Btefy to completely cover
the forest floor in the terracosms, affecting evaporation and surface temperature of the forest floor, and
would exclude larger soil animals. Utter nutnent content and carbon substrates should be done using
current accepted methods, K may be worthwhile to consider using near infrared reflectance spectroscopy
of dned samples to determine nitrogen, lignin and cellulose content (McCletlan et al., 1991, Can J. For
Res 21 1664-1688) Phenol content may also be important in affecting utter decay and N mineralization
Low temperature drying of litter sarcples may be desirable to minimize alteration of litter chemistry which
can occur at temperaiures above 50° C (Palm and Sanchez, 1991, Soil Bel Biochem 23.83-88)
Evaluation of Task 9
Since tissue growth is of key interest in climate change responses and since destructive measurements
are to be minimized m the current experimental design, image analysis techniques will enable non-
destructive measures of growth to be collected and will be an essential part of the proposed project
Specific comments and concerns on the application of these techniques are the following
mmirhizotrcn data may be best used as a relative indicator of root growth
consider reducing the dependence upon automated techniques until technology is proven
analysts of mimrhizotron data might be prioritized according to key plant/environmental events
aboveground image analysis plans need clarification and elaboration
Application of mlnlrhtzotron data. As indicated in the repon. these data win be of most appropriate
value in determining the rate of change in rool length and root turnover between two soil core samplings -
not for determination of biomass or standing crop
Automation of mlnlrhlzotron Image analysis. Effective use of automated techniques lor root
image analysis seems unlikely at present. Sampling intensity may be reduced by usjng intensive sampling
periods only when needed to investigate specific questions Scoping studies may enable sensitive time
periods to be determined Consideration should be given to collecting adequate datB to answer specific
questions and test hypotheses, but to avoid overemphasis ol time-consuming minirhizotron image
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analysis, due to the relatrve ease with which Images can be obtained. If there is a scientrfic justification for
collection of data that can only be analyzed with automated techniques, then images might be collected
and stored, the optimum subset analyzed by manual techniques, and the remainder saved to use when
automated techniques are available A key concern of the panel, which applies here, is that analysis of
data should be able to keep up with its acquisition. If excessive analytical delays occur, the flexibility of the
experiment is severely compromised.
Scheduling of mlnlrhlzotron sampling. In support of the previous point, specific environmental
conditions should be used as the criteria for mimmizotron sampling at frequeni intervals versus infrequent
intervals. The current research plan was undear in this regard Factors such as soil temperature and water
potential could be of value in guiding sampling intensity.
Aboveground Image analysis. Foliar area density [cms m-3] should be considered as an additional
means of expressing leaf area (in addition to leaf area index (m? m-zj) Estimated canopy volume, from
height and diameter measurements, may be of use as an independent vanable in predicting leaf area per
tree Conventional means of leaf area estimation (e g . Li-Cor devices) may be of use after canopy
closure Preliminary experiments for technique development are particularly advisable here
Need for External Review at the End of Ysar 2
Our panel recommends that another review panel be convened at the end of the first two years of
experimentation to review progress of the terracosm COj/iemperature experiments, prior to the curreniiy
planned termination of the initial experiments at three years At that point serous consideration should be
given to 1) terminating the current experiment and planning for the next round of experiments or 2)
proceeding with one-two more years of data collection Concerns of plant size in the terracosms versus
the valuable kinds of information that could be collected If the experiment should proceed tor one more
year should be the focus of that external review. Obviously for long-term studies there is inherent value m
having plant material that has been exposed to two years of continuously altered environmental
conditions, such material becomes progressively more valuable each year. On the other hand, there are
Ifcely to be concerns over the size of the plant material in the terracosms and the ramifications of this
crowding on plant behavior.
Should the decision be made to terminate the first round of experiments and harvest the material, then
consideration should be given to the following possible observations that could be conducted A number
of opportunities exist for detailed terracosm sampling at the end of the initial experiments. These include
fractionation of seedling biomass components for structural composition and for nutrient pool estimates
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Structural composition can include cellular morphology of wood and needle tissues, ratios ol cellulose and
lignin in woody tissues, and could include isotopic composition of wood cellulose, reflecting
combined etlects of C assimilation and allocation Detailed sampling of soil biology and biochemistry will be
possible, as will be censusing of soil fauna Soil structural analyses could be done in conjunction with
possible changes in soil nutrients and soil organic matter distribution, such work could include
micromorphie analyses of son structure and pore size distribution (West el al.. 1991. Agric Ecosys and
Environ 34 363-370} Long-term changes In other soO properties such as soil bufc density of the upper
root zone, may change also (Wild. 1986. Russets's Soil Conditions and Plant Growth. Longman, London)
A detailed sampling of root colonization by soil horizons, root size distribution and blomass, Including
mycorrtuzal root tp and root component nutrient analyses could also be done, as could a comprehensive
sampling of rhizosphere associated microflora and fauna Forest floor structure and composition may
change also, rrtcrobial activity, such as fungal rhuomorph and hyphal networking and nutrient pooling may
occur more extensively in the forest floor with greater decomposer biomass and activity Fungal
rhizomorph tissues in the forest floor could be sampled for both macronutrient and micronutrient
concentrations (Cromack et al. 1975. Soil Bio Biochem 7:265-268}. Opportunities will exist for
investigating changes in soil organic matter (SOM) chemistry and distribution using chemical and physical
fractionation methods Currenl methods include partitioning of SOM into light and heavy fractions
representing particulate and mineral associated SOM density fractionation procedures (Elliot and
Cambardella, 1991, Agric Ecosys and Environ 34 407-419)
The use of MC isotope studies to estimate C input and turnover tn soils has been given a new impetus by
Jenkinson et al (1992. Soil Biol Biochem 24 295-308] .which could be done at bolh the start and end of
the experiment This may reinforce the view lor a longer-duration experiment of at least three years The
initial 14C is Mope results could be available early Work with UC isotope chemistry from fine roots will
continue to be worthwhile as a separate effort, as shown by Balesdent and Balabane (1992, Soil Biol
Biochem 24 97-101). especially If the UC composition in the terrocosm can be controlled by the CO2
source The root isotope data could then be compared with the 1JC data from soil particle size fractions
(Bonde et al, 1992, Soil B«l Biochem. 24.275-277)
Nutrient analyses ooukl be done on the different density fractions (Spycher et al, 1984, Soil Science
135.79-87) Chemical fractionation could include changes in humic and fulvic acids of SOM (F. J
Stevenson, 1986, Cycles ol Soil. John Wiley and Sons, New York) There may also be changes fn other
basic soil properties, such as pH, cation exchange capacity, and exchangeable cations and anions,
particularly in the A soil horizon. Where applicable, the above analyses also should be done jusi prior to
Initiation of the main experiments. In order to document major changes occurring in the forest floor and the
mineral soil during the subsequent 2-3 years Provision should be made for archiving plant and sod
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material for comparison with future experiments and for calibration of fulure analysis methods
Interactions
Responsibility for overall project management needs to be clearly assigned, responsibilities for each of
(he individual scientists invoked in carrying out major project tasks was not evident in the research plan It
is recommended that team interactions be given careful thought, as they will be important in planning and
carrying out the scientific research and in communicating the results In a timely manner. It was not clear
from the Draft Document just how efforts among the various tasks would be team integrated Nor was it
evident just how mooeling and expenmental efforts would be melded
With respect to modeling, it is recommended thai modeling efforts include work on conceptual models at
the single plant level on production, water relations, and nutrient dynamics. At the ecosystem or canopy
level, consideration must also be given to nutrient cycling and canopy-level interactions Such efforts
were not evident in the draft proposed research plan It is also recommended that all of the scientific
disciplines represented in the research be involved in modeling efforts, as well as interacting on a regular
basis to assess progress of the scientific research Expertise and assistance in modeling should be
uMized from other EPA laboratories, where appropnate, as well as from ihe scientific community at large
Interactions between EPA-Corvallis and other EPA labs were not evident in the Research Plan Nor were
possible interactions with other groups around the United States working on global climate change
research Development ol such interactions is strongly encouraged
Other relevant research programs to be added to those identified in the Research Plan which should be
acknowledged are
Richard Norby at Oak Rdge National Laboratory
He is using open tops to study deciduous and coniferous species of the SE deciduous forest
Peter Curtis at Ohio Slate University.
He is using open tops and rtiizotrons to study deciduous species of the Great Lakes region His
facaities are at Douglas Lake Biological Station.
Fakhri Bazzaz at Harvard Forest
22
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He is using controlled chambers and is trying to get into open tops tor the study of NE deciduous
forest species
Detlef Schulze and John Tenhunen at Bayreuth Germany
They are using open tops and are setting up FACE rings to study European conifers and
deciduous species.
Sune Under of Sweden
He is coordinating a national program on climate change to be conducted at various forested sites
in Sweden
Public Education
A considerable opportunity exists for public education from the COj climate change experiment, through
facility tours and preparation of appropriate educational materials, both written and visual (such as videos,
etc) This experiment is likely to be viewed as an important component ol climate change research being
ai both the national and international levels, and release of information to the general public will be an
important benefit to both the project and the public's appreciation of the need for global climaie change
research
23
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RECONCILIATION MEMO: 1992 PEER REVIEW
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UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
ENVIRONMENTAL RESEARCH LABORATORY
200 S.W. 35TH STREET
CORVALLIS, OREGON 97333
April 11, 1994 - copy made from electronic storage. Original date: 1993
SUBJECT. Reconciliation Memo of Project Plan Effects of C02 and Climate Change on
Forest Trees
The Peer Review Panel met in Corvallis OR, May 5th and 6th, 1992 to conduct the review of the
draft Research Plan, Effects of C02 and Climate Change on Forest Trees. The panel was
composed of the following members.
Dr. James Ehleringer (Chairman), Biology Department
University of Utah
Salt Lake City, UT 84112
Dr. Boyd Strain, Botany Department
Duke University
Durham, NC 27708
Dr Alexander L. Friend, Department of Forestry
Mississippi State University
Mississippi State, MS 39762
Dr. Robert J. Zasoski, Department of Land, Air and Water Resources
University of California
Davis, CA 95616
Dr. Kermit Cromack, Department of Forest Science
Oregon State University
Corvallis, OR 97331
Overall the recommendations of the Peer Review Panel were very supportive of the research and
recommend that the research proceed However, a number of suggestions and modifications were
provided for consideration. These comments from the Panel were grouped into 8 separate areas
and our response to those comments follows the same organization. In general, we agree with the
suggestions of the Panel and have attempted to implement them in the revised research plan.
FROM David T. Tingey
TO.
File
Growth Facility and Engineering
The Panel concluded that the facility was well engineered and that it provides excellent control of
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both atmospheric and edaphic components of terrestrial ecosystems The panel stated that: "The
final proof of the concept and the adequacy of engineering will remain in the results of the testing
to be done in this summer of 1992 but the panel has no recommendations for changes or
improvements in the system It appears to be an excellent design and to be adequately
engineered "
The Panel expressed three concerns
1. The planting design provided only a few plants that were not edge plants. To reduce the
problem of edge plants, shade cloth will be used outside the chambers to simulate the reduced
light intensity that interior plants may receive. This should reduce the bias of edge plants
2. The Panel expressed concern about the original experimental design The experimental design
has been simplified to provide 3 replications of each treatment. This will increase the
statistical power of the experiment
3. The TERA site should be expanded to 24 chambers at the earliest opportunity. This action
will not be considered until the current chambers are adequately tested and made operational.
Document Presentation
The Panel expressed seven concerns which are addressed as follows*
1 Need for Executive Summary One was prepared and included in the plan Also a brief
description of the Project was prepared for general public consumption;
2 Justification for species selected The section was rewritten to provide a stronger justification,
3. Improve linkage between policy issue and research tasks;
4 Improve linkage between changes in forest productivity and vegetation redistribution issues;
5 Improve linkage between experimental and modeling task A section was added to each Task
outlining the experimental measurements that will be used as model inputs,
6 Improve description of Quality Assurance Plan,
7. Improve team interactions A chapter was added to Plan documenting team responsibilities
and interactions.
Short-term Aspects peri mental Tasks!
The Panel found the short-term experiments are comprehensible and well justified
The Panel recommended that objectives and science hypotheses be defined for each of the
experimental tasks. This has been done.
Long-term Aspeet* ^Modeling Tasks!
The Panel found the modeling component to be confusing and inadequate.
The Modeling section has been completely refocused and rewritten to provide a better linkage
and justification between the experimental and modeling tasks. The revised section was reviewed
by Tim Weber to check if the changes were responsive to the reviewers suggestions.
2
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The section now focuses on the TREGRO model only The proposal to link TREGRO to the
ZELIG model was dropped from the plan, because there are insufficient resources to conduct this
research Initial soil studies will use the soil components of TREGRO
The linkages between the experimental and modeling tasks have been improved by clearly
establishing the data that the experimental studies will provide for the modeling studies
Critical Review of Scientific Tasks
The task numbering in the final version of the Plan is different than in the version reviewed by
the Panel. The Tasks are listed below as numbered by the Panel with notes to show the changes
made in organization.
Task 1
More clearly state objectives
Objectives have been developed for all tasks
Qarify the photosynthetic potential and maintenance respiration
Tasks 1 and 2 were rewritten to respond to this request
Reduce intrusions into the chambers
This is being implemented through both the increased use of supporting studies and more
reliance on non-intrusive measurement techniques, e g, CERES automated stem diameter
meters
Increase emphasis on automated stem sap flow
Being implemented.
Consider stable isotope measures
We would like to do this but do not have sufficient personnel or financial resources to do
this at this time. We will continue to work towards a future implementation of stable
isotope tracer analysis for measurement of carbon flow within the system.
Task 2
Respiration section improved
This was improved and moved to Task 1
Culture of plants outside of Terracosms
A section on supporting studies was added to the Experiment to provide for more
destructive harvests and additional data to establish die comparability of the experimental
data with field results.
Reduce frequency of growth measurements
To the extent possible, this has been done.
Timing of growth measurements
The timing of above- and belowground measurements have been more closely coordinated
and linked to phenologjcal events. The changes are reflected in Figure S.2 in the Research
Plan
3
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Task 3
Task was eliminated The material on C allocation was placed in the Integration and
Inference section and the portions on soil carbon are now in Task 6.
C allocation
The TREGRO model does not require transfer functions for carbon allocation The
supporting studies will permit a series of destructive harvests that can be used to develop
transfer functions
Soil carbon pools
We accept the caution raised by the Panel and will be aware of their concern when the
data are analyzed
Soil carbon fractions
Initially we will not be able to consider soil carbon fractions but will attempt to expand
the study to include these measurements in subsequent years
Task 4 (now Task 3)
Nutrient additions
The Panel recommended that nutrients not be added unless it is uniformly deficient We
do not propose to add nutrients except at the low levels that occur in remote areas of
Oregon Nutrients are being added at a minimal level to reduce the corrosive nature of
distilled water which would artificially stimulate soil mineral weathering
Tissue sample frequency
The sampling frequency of both tissue and soils was reduced
Disturbance effects on nutrient availability
To minimize the disturbance effects, the soil has be conditioned for a year in the
terracosms and cover crops have been grown to remove excess nutrients
DRIS systems for comparing nutrients
The suggestion was accepted and has been added to the research plan
Ionic balance
The suggestion was accepted and has been added to the research plan
Extract* ble sulfate
The suggestion was accepted and has been added to the research plan.
Mineralizable nitrogen
Initially we will not be able to consider mineralizable nitrogen but will attempt to expand
the study to include its measurement in subsequent years.
Rainfall inputs
The nutrients in rain water are being added to reduce the corrosive nature of distilled
water which would artificially stimulate mineral weathering
4
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Soil nutrient analysis
The Panel suggested that the frequency of soil analysis be reduced This suggestion was
accepted and has been added to the research plan
Task 5
Addition on litter and fresh A horizon
We agree with the Panel's suggestion and will add fresh A-horizon soil at time of planting.
Leaf packs
Leaf pack will be used in addition to litter bags to estimate decomposition
Lyophilization
Litter will be air-dried instead of lyophized as originally proposed
Controls of litter decomposition
Litter decomposition will be monitored at 3 sites in the Cascades, including the one where
the soil and litter were collected so it will be possible to estimate the impact of
disturbance on decomposition
Extending study
The Panel suggested that we grow high-carbon of and 1 enriched tissue The
suggestion will be considered but at this time we do not have the facilities to enrich tissue
with 13C and 1$N. But needles from the high-C02 treatments will be saved for future
studies
Task 6 (now Task 4)
Wider difference in soil moisture levels
This suggestion was not implemented because we have deleted the soil moisture treatment
to increase the replication of the C02 and temperature treatments
Constrict soil moisture measurements
Because the collection of soil moisture data is automated, the collection frequency will
remain at one hour but will be aggregated to 4-hour to daily values for data analyses.
Reduce pressure bomb measurements
This was accepted and greater reliance will be placed on the non-intiusive measurements
Avoid soil saturation
We agree with suggestion, water addition will be monitored to prevent soil saturation.
Task 7 (now Task 6)
Soil cores
One soil core, 5 cm in diameter, will be collected from each Terracosm annually, as shown
in Figure 5.2. From these samples, subsamples will taken and used in the soil nutrient
(Task 3) and soil biology (Task 7) tasks. The roots will be used in the root phenology
task (Task 6). The minirhizotron data (Task 6) will be used primarily to assess root
dynamics.
5
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Root-soil separation
We agree with the panel that dry-sieving to separate soil and roots should be avoided We
will use a hand sorting technique.
Vertical stratification
We agree.
Secondary compounds
We hope to add this component in the 2nd or 3rd year of the study.
Ingrowth cores
Ingrowth have been deleted from the study.
Fine roots
We will segregate roots by size in both the destructive harvests and the minirhizotron
data, but at this time there is no plan to segregate by branching order as the personnel are
not available for this task.
Outputs
The Task has been rewritten to indicate that most of these variables will be measured
Task 8 (now Task 7)
We redesigned the frequency of sampling and the number of soil cores taken per sampling
time to minimize disturbance to the soil system as suggested by the Panel, and discussed
above
Studies of mycorrhizal fungi
The Pane! suggested that we be prepared to identify any fruit bodies that might develop
to help confirm our nucleic acid analyses work to track myconhizal fungi. We will watch
for fruit bodies
We are including in the mycorrhizal fungal diversity analyses an initial segregation of
mycorrhizae along "morpho-types* based on gross morphology. After this first sort,
additional analyses will be done to track "downward" (nucleic acid "fingerprints") the
changes in the community structure of the fungi forming these "morpho-types", and also
to track "upward" (soil foodweb) the organisms in the rhizosphere and soil immediately
surrounding the various mycorrhizal fungal "moipho-types".
The Panel suggested that we consider inoculating the seedlings with specific mycorrhizal
fungi known to colonize Douglas fir seedlings, and then track these isolates. We feel that
the fungal community resident on roots of bareroot nursery stock originating from a well-
prepared, fumigated nursery bed is likely to be limited to a handful of morpho-types. By
doing the initial morpho-type separation in conjunction with the fingerprint analyses, we
believe we can adequately follow a selected sub-group of fungi as they continue to
colonize roots under the various climate treatments.
The Panel's comments on recent work on sensitivity of mycorrhizal fungi to canopy
processes is exactly in line with our basic approach to following the changes in the
mycoTThizal community structure due to climate treatments and is duly noted.
6
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We do not plan at this time to perform nutrient bioassays of fine roots for N or P uptake
The time constraints imposed by taking samples of soil and roots, separating samples for
several analyses, etc, and these items coupled with personnel limitations do not allow us
to pursue this line of investigation
Microbial studies
We agree with the Panel to determine total and active bacterial and fungal biomass by
direct counts as originally planned. If sufficient soil is available after partitioning samples
for all other analyses, and personnel are available, we will undertake some limited work to
make comparisons between biomass values determined from direct counts versus other
methods as suggested by the Panel.
Dr Elaine Ingham will be part of this effort through her Microbial Biomass Service.
Additional arrangements are being finalized to cooperate with students from the OSU
Botany and Plant Pathology Department to become involved with the mycorrhizal fungi
morpho-type separations and foodweb analyses
Restoration of soil profiles
The addition of fresh A horizon (Task 5) will be done to help restore the biology of the
terracosms soil. Dr. Andy Moldenke, OSU Entomology Dept will cooperate with us to
ensure a complement of essential soil fauna will be reintroduced to the terracosms as now
included in the Research Plan
Litter decomposition research
Both litter bags and leaf packs (as suggested by the Panel) will be used This will permit
a better understanding of the range of decomposition possible under the treatment
conditions Litter chemistry will be determined and is now more fully explained in the
Research Plan
Soil animal populations
Dr Andy Moldenke will participate in the project through the OSU Microbial Biomass
Service to analyze the changes in the soil fauna introduced as part of the "Restoration of
soil profiles" discussed above
Additional arrangements are being finalized to cooperate with him and students from the
OSU Entomology Department to become involved with correlating changes in
populations of soil animals and physical and chemical properties of the litter layer.
Soil enzyme activities
Five soil enzymes, involved in the processing of phosphorus, carbon and nitrogen
(including proteins, and humic and lignin materials) were selected. Dr. Robert Griffiths
and Bruce Caldwell from OSU Forest Science Department will participate in determining
the activity of these enzymes through their Microbial Analysis Service.
Tusk 9 (primarily Appendix B and Task 6)
The experimental portions of this Task were moved to Task 6 and the rest placed in an
Appendix.
7
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Application of minirhizotron data
We concur with the finding of the Panel and it is implemented in the Plan
Automation of image analysis
We concur with the conclusion of the Panel and have dropped our attempt to automate
image analysis of minirhizotron data Data analysis will be based on the manual
"ROOTS" program so that we can keep current with the data.
Scheduling of minirhizotron data
Sampling will occur monthly so that the root-growth cycle can be determined and linked
to above ground phenological events and also to edaphic conditions.
Above ground image analysis
The measures suggested by the Panel were be added to the Plan.
Task 10 (primarily Appendix A and Experimental Design)
The introductory description of the Task was added to the Experimental Design Section
to provide the reader with an early description of the experimental facility
The extensive description of the facility is now in an Appendix.
Need for External Review After 2nd Year
The Panel recommended that a review be conducted after 2 years This will be done as it is
required by Laboratory policy. The plants will become more valuable as they become larger
(older), however, the experiment will be terminated before the plants outgrow the chamber
Externa] science input will be requested before the experiment is terminated to insure that we
obtain the maximum data and use possible from the study.
Interactions
Project interactions
A section was added to the Plan to clearly define the interactions
Interactions with other groups
This section was included in the Plan to recognize the research groups with whom we have
established research interactions rather than a listing of groups conducting climate change
research. We appreciate the additional groups suggested by the Panel and are working to establish
contacts with them.
Public Education
The potential for education from the Experiment will take 2 directions.
Training Cooperative Agreements
We are in the process of establishing Training Cooperative Agreements to provide education
opportunities for studies in the environmental sciences. The results from these agreements will
also extend the experimental portion of the study.
Community education
We have developed a brochure for the general public and are conducting tours of the experimental
site for various public education groups.
8
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PROJECT EXPANSION SINCE 1992
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Project Progree and Current Status
PROJECT EXPANSION
EXPANSION OF CORE PROJECT
Since the last Peer Review Project Staff have spent
time developing the supporting field studies com-
ponent (i.e., Cascade Mountain elevational plots,
the Large Lysimeters at ERL-C, and the RS-
BIOME) of the Project. We developed the sup-
porting studies to strengthen the Project's model-
ing efforts; provide additional types of "control
treatments" for the Terracosm experiment; and
create the opportunity to link video camera images
(minirhizotron work) to harvested root and myc-
orrhizae samples to address questions on mycor-
rhizal biodiversity and life histories (cohort analy-
ses)
After Project Staff expanded to include additional
plant physiological and chemistry expertise we
modified our approach to aboveground responses
in the Terracosms (Tasks 1 and 2) and also more
fully developed the analysis of soil organic matter.
The analysis of soil organic matter is designated to
become a separate experimental task. Writing
experimental Task 8: Soil Organic Matter, was
initiated in 1994 and is presented in detail as a
separate Booklet in the Peer Review Information
Package. It will be introduced and discussed at the
peer review; your evaluation of its scientific qual-
ity and appropriateness is requested so that it can
be revised and implemented.
Supporting Field Studies
Cascade Elevational Plots: In conjunction with
the experiment at the TERA facility we decided to
compare growth of Douglas fir seedlings under
natural conditions with seedling growth in the
Terracosms to provide:
another form of control treatment, and
allometric data from annual seedling harvests
to parameterize and validate
TREGRO for use with Douglas fir.
We established three small, temporary Douglas-
fir plantations on the west side of the Cascade
Mountains in the Sweet Home Ranger District of
the Willamette National Forest, OR. The three
plantations were established in June 1993, and lie
along an elevational gradient from the foothills to
the upper reaches of the growth of Douglas fir
(Low-site: 537 m; Mid-site: 951; High-site: 1220).
Each plantation has five 1 m by 2 m plots. Trees
were planted at the same density as were seedlings
planted in the Terracosms. Litter bags and needle
packs were buried at 3 cm depth in the 6-cm deep
litter layer placed on the plots. Seedlings will be
excavated in the fall from one plot per year (1994
through 1998) for whole plant allometric data
collection; mycorrhizal colonization and fungal
diversity estimates; and soil, litter and plant chemi-
cal analysis. The harvests will occur shortly
before or after seedling harvest from the Large
Lysimeters (below) and extracting soil cores from
the Terracosms. Additionally, the plots are used to
gather data on seedling growth and phenology,
and decomposition of plant litter. We established
a weather station at the low elevation site in 1993
and will establish similar weather stations at the
mid- and high elevations sites in the summer 1994.
The weather stations will provide year round en-
vironmental data. The field sites are also being
used by some of our Cooperators (litter/soil fauna,
microbial biomass, bulk soil enzymes, stable iso-
tope analysis).
Large Lysimeters: One of the limitations of the
TERA facility is that destructive harvests can only
occur at the end of the experiment. Since the last
peer-review we planted Douglas-fir seedlings in
four large lysimeters (pots) sunken into the earth
adjacent to the TERA facility. We planted the
lysimeters so that annual destructive seedling har-
vests (one lysimeter per year) can be done to
obtain intact seedling systems for allometric analy-
sis. The same soil, seedling density and litter layer
depth used in the Terracosms was used in the
lysimeters. The lysimeters are not climate-con-
Page I
Project Expansion
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Poject Progress and Current Status
trolled and so the seedlings are growing under
ambient Corvallis climate. The four annual fall
harvests of seedlings from the lysimeters will
occur shortly before or after seedling harvest from
the three elevational plantations and extracting
soil cores from the Terracosms.
RS-BIOME (Root/Soil-Biota Interaction Ob-
servation and Manipulation Experiment): We
constructed an experimental area in 1993 adjacent
to the TERA facility to examine soil biota effects
on root turnover; and improve our ability to link
the video camera images (the minirhizotron work)
to actual, collectable root and mycorrhizae samples,
and questions concerning mycorrhizal biodiver-
sity and life histories (cohort analyses) Within the
RS-BIOME, we can assess the effects of (1) colo-
nization of roots by mycorrhizal, saprophytic and
pathogenic fungi, (2) successional changes in
mycorrhizal colonization of roots, (3) interactions
of predators, parasites, mycorrhizae and perhaps
other root-colonizing fungi, and (4) interactions of
other rhizosphere organisms with mycorrhizal
colonization and succession. The RS-BIOME
offers the opportunity to assess, monitor and ex-
perimentally manipulate the tree seedling-soil
system. The RS-BIOME is not intended to be a
highly-controlled and monitored facility. The
intention is to have available a place to conduct
work where samples (mycorrhizae, roots, etc ) can
be harvested and compared with similar samples
found in the Terracosms using video camera and
minirhizotron technology
The RS-BIOME consists of a 2 m by 10 m
aboveground planting bed oriented in a N-S direc-
tion. The planting bed is approximately 70 cm
deep and constructed of redwood covered with
black plastic sheeting. The RS-BIOME is divided
into ten, 2 m by 1 m planting areas that are
separated by Plexiglas panels. The soil in the RS-
BIOME is the same as in the Terracosms, and its
depth profile closely matches that in the Terra-
cosms down to 70 cm [litter layer (6 cm), A
horizon (10 cm), and B horizon (43 cm); placed
over a coarse gravel bed covered with geotextile
cloth]. Two minirhizotron tubes (scored for length)
are located in each planting area: one at 10 cm
depth, the other at 40 cm depth. The top tube is
located 30 cm from one north edge of each plant-
ing area, and the lower tube is located on center.
The origin of the tube scoring is fixed to one end
of the planting area, so that the exact location in the
planting area can be assigned using the scoring
marks on the tubes. We can then loc ate and sample
any objects of interest. A soil berm was placed
against the walls of the entire RS-BIOME to
reduce temperature gradients across each planting
area. Extensions were added to the minirhizotron
tubes so that there is access to the tubes through the
berm. In January 1994, two seedlings from the
same set of seedlings used to plant the Terracosms
were planted in the center of each half of the 1 by
2 m planting areas (N= 20 seedlings).
The strength of the RS-BIOME is its flexibility.
Since root positions can be precisely determined,
roots can be excavated and the appropriate roots or
root tips removed for closer examination. The RS-
BIOME is going to be utilized by several collabo-
rators (students, Post-Docs). The exact manipula-
tion experiments have not been identified, but the
work done in the RS-BIOME will be monitored to
assure compatibility among users.
Tark 8: Soil Organic Matter
Processing of carbon in the seedlings and in soil is
one of the important research foci of this Project.
One experimental task area that was not included
in the Research Plan due to personnel limitations
was a detailed study of soil organic matter. Task
8 - Soil Organic Matter is being added as an
experimental area to investigate whether or not
elevated C02 and climate change affect the pro-
cessing and storage of various forms of carbon in
soils. Another objective of the task is to examine
the linkages between soil organic matter stability
and nutrient cycling. As for all the experimental
tasks, the results of Task 8 will be used to evaluate
treatment effects directly and as inputs to Project
modeling efforts.
Page 2
Project Expansion
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Project Pragree and Current Status¦
EXPANSION WITH OFF-SITE COL-
LEAGUES
After determining the core portions of the Project
and after the 1992 Peer Review, the Project began
expanding to form linkages with off-site investi-
gators to:
undertake work the Project would fund to
collect data of direct relevance to Project ob-
jectives,
undertake work not precisely specified within
one of the experimental Task areas, or
work in an area that is not part of the core
program, but is of mutual interest and benefit
to all collaborators.
The various types of work interactions fall roughly
into the categories below. Cooperators are recipi-
ents of Project funds (e.g., Cooperative Agree-
ments, Purchase Contracts). The Informal Rela-
tionships involve work done by colleagues whereby
the Project Staff work directly with the off-site
colleagues and the funding sources range from
provided exclusively by the off-site colleague to
shared financial support. The Post-Docs and
Graduate Students are supported in a variety of
ways including Project funds through Training
Co-operative Agreements, Purchase Order Con-
tracts, Department of Energy Fellowships, and
Project funds through the National Research Coun-
cil. Descriptions in greater detail of these expan-
sion efforts are presented in the most appropriate
booklet of this Peer Review Information Package.
Cooperators
Dr. Elaine Ingham, Department of Botany and
Plant Pathology, Oregon State University
analysis of soil foodweb structure
Drs. Andrew Moldenke and James Wernz,
Department of Entomology, Oregon State
University speciation and enumeration of
litter and soil faunal community
Dr. Robert Caldwell and Mr. Bruce Caldwell,
Department of Forest Science, Oregon State
University Carbon, nitrogen and phospho-
rus processing enzyme activity in bulk soil
To-be-named: an individual to parameterize
the TREGRO process model for Douglas fir
Informal Relationships
Dr. Juliane Filser, Institut filr Bodenokologie,
Neuherberg Germany speciation of
Collembola collected by Drs. Moldenke and
Wernz, and Ms. Nancy Baumeister (see Post-
Docs and Graduate Students below)
Dr. Carolyn McQuattie, US Forest Service,
Northeast Forest Experiment Station, Dela-
ware OH ultrastructural changes in promi-
nent mycorrhizaJ morphotypes obtained from
the semi-annual Terracosm soil cores using
light and electron microscopy
Drs. Ralph Crawford, Iwan Ho and Ching-
Yan Li, US Forest Service, Pacific Northwest
Experiment Station, Corvallis OR, com-
munity structure of phyllosphere microorgan-
isms using needle samples taken from Terra-
cosm seedlings.
Drs. Jim R. Ehleringer, Guanghui Lin and
NinaBuchmann, University of Utah, Salt Lake
City.UT 13c/18q techniques to partition
root and microbial respiration, and deep root
and shallow root respiration using root and
litter samples, and soil gas and solution samples
from the Terracosms and from field sites in
Oregon and Utah.
Post-Docs and Graduate Students
Dr. Ken Cullings, Department of Botany and
Plant Pathology, Oregon State University
Drs. Cullings was trained in the molecular
ecology of mycorrhizae at the University of
California at Berkeley. He is developing the
molecular techniques to assess the diversity of
Page J
Project Expansion
-------
Poject Progress and Current Status
the mycorrhizal fungal community. Dr.
Cullings is supported jointly by OSU and
through the Project. He has applied for De-
partment of Energy and National Research
Council Fellowships to determine the speci-
ficity of mycorrhizal fungi forming the sym-
biosis in Douglas fir forest, silver fir forests
and the transition between these forests types.
He will test the specificity hypotheses to con-
tribute to answering the question on the rate of
vegetation redistribution under altered cli-
mates. Various models indicate that Douglas
fir is will invade silver fir zones based on
climatic variables. However, is it uncertain on
whether the soil microbial community will
support the species change due to conflicting
hypotheses on fungus-plant specificity require-
ments.
Dr. Robert McKane, The Ecosystems Center,
Marine Biological Laboratory, Woods Hole
MA has applied for a Post-Doctora) Fellow-
ship through the National Research Council.
Dr. McKane will contribute to the modeling
efforts of the Project. He proposes to utilize
TREGRO, GEM and data from the experi-
ment to test some basic concepts on how
carbon and nitrogen interactions control whole-
system changes in C storage.
Ms. Nancy Baumeister, Department of Ento-
mology, Oregon State University Ms
Baumeister is studying for the Ph.D. degree.
Ms Baumeister received a National Network
for Environmental Management Studies
(NNEMS) Fellowship funded through the
Project. She is a first-year student interested in
soil fauna, nutrients, diversity and climate
change. She is developing her dissertation
research project during 1994 .
Ms. Amy Tuininga, Department of Botany
and Plant Pathology, Oregon State University
Ms. Tuininga is supported by a Project
Training Cooperative Agreement. Ms.
Tuininga is a first-year graduate student inter-
ested in decomposition of woody debris, and
linkages with the soil foodweb structure and
nutrient cycling. She is developing her re-
search project during 1994 to utilize the Terra-
cosms, and perhaps the RS-BIOME.
Page 4
Project Expansion
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PROJECT QUALITY ASSURANCE PROGRAM
-------
Project Progree and Current Status
QUALITY ASSURANCE
INTRODUCTION:
The philosophical approach of Project Staff was to
establish and implement an efficient and manage-
able organizational structure for QA/QC (Quality
Assurance/Quality Control) within the Project,
and between the Project and Lab Management.
The multidisciplinary nature of the project en-
couraged and required careful evaluation of the
requirements of the QA (Quality Assurance) pro-
gram of ERL-C and the Agency. Although the
Project Quality Assurance Project Plan (QAPP)
represents a departure from typical ERL-C proce-
dures, it fully conforms to all QA/QC require-
ments.
The QAPP describes how the QA program of the
Project is managed, audited and results of audits
reported to Lab Management. The QAPP also
explains where various QA/QC information can
be found. All QA/QC aspects concerning envi-
ronmental measurements are addressed in detail
in the numerous Standard Operating Procedures
(SOPs defined below) that support the QAPP. If
the traditional approach to QA used at ERL-C
would have been taken, the numerous QA/QC
details required to be in the QAPP would have
made it an unwieldy document; minimizing its
usefulness as a reference and guide to Project
Staff. Rather, a loose, general-guidance QAPP
was written, and major reliance was placed on
individual SOPs. A standardized SOP format was
designed for all measurement activities to estab-
lish a uniform code that addresses all key QA/QC
requirements. We designed the SOPs to serve as
guidelines to accomplish both the scientific and
QA/QC procedures simultaneously; the QA/QC
procedures are blended into the description of
Standard Operating Procedures. The SOPs are
written by Project Scientists and Principal Inves-
tigators and must be reviewed within the Project
before the SOPs are reviewed by the ERL-C QA
Staff.
The SOPs describe activities concerning taking
environmental measurements related to treatment
variables and hypothesis testing in the core project.
Other Protocols (OPs) are written about activities
related to measurements not directly involved in
treatment variables and experimental hypothesis
testing (e.g., meteorological data, calibration of
the Terracosm measurement systems). Experi-
mental Procedures (EPs) are documents that de-
scribe activities that do not collect data, qz data
collection activities of work done by our collabo-
rators in cases where they are providing their own
funds. General examples of the former activities
are descriptive documentations of procedures such
as initial handling of seedlings or soils, sample
handling, preparation, etc.
Standard Operating Procedures are given an iden-
tifying number that is coded to one of the eight
research tasks of the project. Operating Proce-
dures and EPs are also given a distinguishing
number, but because they may describe activities
not related to specific tasks they are not linked to
any task(s). A version number is also assigned to
track revisions. Finally 8 serial number is as-
signed to each official copy of all SOPs, OPs and
EPs. The serial number links the document to a
specific authorized user and enables proper man-
agement of the QA documentation and procedures
so that all Project Staff are following the most
recent, approved procedures. The recipient of an
official copy automatically receives a new version
of the document.
The Project Quality Assurance Coordinator, a
project-wide position, is responsible for all docu-
Page 1
Quality Assurance
-------
Poject Progress and Current Status
ment tracking. The Coordinator works through
the Project Leader on all matters concerning imple-
mentation of Project QA activities (see Booklet
Quality Assurance Program). The information
provided in the booklet includes the QAPP, stan-
dard SOP format, and an example of an approved
EP and SOP. Also included is the current version
of the List ofSOPs for the Terracosm experiment.
QUALITY ASSURANCE PROJECT PLANS
(QAPP):
Terracosm Experiment
The QAPP for Che portion of the Project in the
Terracosms was approved on May 11,1993. Only
a few SOPs, OPs and EPs have been approved.
However, many of the others are in various states
of development, preparation or in the review and
approval process.
Field Site QAPP
A rough draft of the QAPP and a list of SOPS, OPs
and EPs for the supporting studies in the Cascade
Mountains and in Corvallis have been prepared.
Many of the environmental measurements per-
formed in the field will be identical to those done
in ;he Terracosms. The philosophical approach to
the QAPP is similar to that of the Terracosm
experiment. All applicable SOPs, OPs and EPs
from the Terracosm experiment QAPP will be
reevaluated and rewritten for inclusion into the
Field Site QAPP. Appropriate new SOPs, OPs,
and EPs will be developed as necessary.
TERA Facility
A Quality Assurance program will be implemented
for the TERA facility. The program may take one
of two forms:
a QAPP describing all data collection activi-
ties (hypothesis-testing and non-hypothesis-
testing), with TERA operations imbedded
within as an SOP, or
a QAPP for the hypothesis-testing data collec-
tion activities of TERA and a separate TERA
Operations Manual that describes how to op-
erate the facility and collect data not related to
hypothesis-testing (e.g., levels of anti-freezer
in chillers and heaters).
Project Staff have not decided which option to
follow. Lists of procedures related to both kinds
of data collection are being identified, and in some
cases are being written. The descriptions will be
placed in the appropriate document(s) when the
philosophical approach to QA/QC issues of the
facility is decided.
Page 2
Quality Assurance
-------
PROJECT PUBLIC EDUCATION PROGRAM
-------
£% CDA United States Environmental Research May 1994
Environmenta] Protection Laboratory - Corvallis
Agency Corvallis, OR 97333
EXPERIMENTAL TASKS
AND FACILITIES
Project: Effects of C02
and Climate Change
on Forest Trees
For additional Information contact:
Dr. David T. Tingey
Program Leader
ERL-Corvallis
(503)754-4621
-------
Experimental Tasks and Facilities
TABLE OF CONTENTS
TABLE OF CONTENTS
I. TASK 1: SHOOT CARBON AND WATER FLUXES
II. TASK 2: SHOOT GROWTH, MORPHOLOGY, ALLOMETRY, PHENOLOGY,
AND CARBON PARTITIONING
III.
TASK 3:
SYSTEM NUTRIENTS
IV.
TASK 4:
SYSTEM WATER
V.
TASK 5:
LITTER LAYER
VI.
TASK 6:
ROOT GROWTH AND PHENOLOGY
VII.
TASK 7:
SOIL BIOLOGY
Task 7A: Soil Biology - Microbial Biomass, and Protozoan and Nematode
Numbers
Task 7B: Soil Biology-Ectomycorrhizae Colonization and Diversity
Task 7C: Soil Biology - Soil Gases
Task 7D: Soil Biology - Soil & Litter Fauna
Task 7E: Soil Biology - Microbial Transformation Rates/Enzymatic Activities
VIII. COLLABORATIVE RESEARCH EFFORTS
Part A: Collembola Speciation
Part B: Light and Electron Microscopy of Ectomycorrhizae
Part C: Community Structure of Phyllosphere Microorganisms
Part D: 13C/180 and Soil Respiration
IX. TERA: A STATE-OF-THE-SCIENCE-RESEARCH FACILITY
X. DATA ACQUISITION AND DATABASE MANAGEMENT
Page i Effects of COJ and Climate Change on Forest Trees
-------
TASK 1: SHOOT CARBON AND WATER FLUXES
-------
Experimental Tasks and Facilities
TASK 1: SHOOT CARBON AND WATER FLUXES
PARTICIPANTS: D Olszyk and D. Tingey (EPA) - Principal Investigators
C Wise (EPA)
B. Baker (CSC)
R Waschmann (METI)
OBJECTIVES:
The overall objective for this task is to determine
whether increased C02and temperature will alter
the net carbon flux and water fluxes of plants, and,
therefore, their water-use efficiency. This objec-
tive is addressed by measuring-
at the canopy level- photosynthetic, respira-
tion, and transpiration rates;
at the needle/branch level- photosynthetic,
respiration, and transpiration rates and sto-
matal conductance changes;
at both the canopy and needle/branch levels-
diel and seasonal patterns in photosynthetic,
espiration, and transpiration rates and sto-
matal conductance changes in response to thee
individual and combined effects of increased
C02 and temperature.
APPROACH:
To determine carbon (C02) flux in the terracosms,
photosynthesis and respiration are measured at
two scales: (1) continuously for the tree canopy,
and (2) periodically for a limited number of needle/
branch samples. Photosynthesis and respiration
rates are determined by carbon flux in the light and
dark, respectively. Both canopy and needle/branch
measurements are made under ambient climate,
and, therefore, reflect diel and seasonal differ-
ences in photosynthetically active radiation (PAR)
and air temperatures.
At this time canopy carbon flux data are obtained
by operating the terracosms as closed systems for
the first 20 minutes of each two hour period. The
resulting increase or decrease in C02 concentra-
tion in the terracosm over this period is used to
calculate photosynthetic or respiration rates, re-
spectively Carbon flux for the canopy will be
corrected for terracosm leak rates, soil C02 fluxes,
and needle area in individual terracosms. Needle
area at the start and end of the experiment will be
calculated based on destructive harvests to deter-
mine weight of needles per tree and area/weight
for a representative sample of needles per tree.
Intermediate, needle area will be estimated
nondestructively based on digital photographs
taken to determine needle area of a subsample of
individual trees, and total planar area covered by
the canopy in each terracosm.
Needle/branch level data are obtained with a LI-
COR 6200 portable gas exchange system. Mea-
surements are being made for both age classes of
needles currently present on the trees, and at two
C02 levels for each needle sample (approximately
350 and 550 |il 1"). Measurements are made
monthly from approximately April-October and
every two months from November-March.
To determine water flux in the terracosm, transpi-
ration is measured at two scales: (1) continuously
for the tree canopy, and (2) periodically for a
limited number of needle/branch samples. Canopy
data are based on daily collections of water con-
densing from the terracosm air stream using a
tipping bucket, and will be adjusted for water
vapor added to the air through the humidification
Page I
Task I Shoot Carbon and Water Fluxes
-------
Experimental Tasks and Facilities
system and for needle area in individual terra-
cosms. Needle/branch data are based on data
taken during the photosynthetic measurements
with the LI-COR system.
To compare the data from the terracosms with
forest trees in more natural settings, additional
needle/branch carbon and water flux measure-
ments are being made at three field sites in the
Willamette National Forest. The sites are at three
altitudes (with corresponding different microcli-
mates) in the Cascade mountains. Measurements
are from trees from the same population as the
terracosm trees, but planted in protected plots in
forest soil.
STATUS/RESULTS:
Canopy level carbon and water fluxes Carbon
dioxide and water vapor data for the terracosms
have been collected since fall, 1993. Figure 1 is an
example of mean daily terracosm C02flux data for
the four treatments. These data are preliminary,
and have not been corrected for chamber leaks or
soil C02 flux. These data also assume there are no
differences in tree canopy size among chambers,
which is reasonable at the present time given the
lack of differences in shoot growth among treat-
ments as described for Task 2.
The data suggest that C02flux to plants (decrease
in C02 concentration in the closed chamber) dur-
ing the day is greater in the elevated C02 com-
pared to ambient C02 treatments (Figure 1). They
also suggest that C02 flux from plants at night is
less in the elevated C02 compared to ambient C02
treatments The higher C02 fluxes for the elevated
C02 treatments may be due to physiological re-
sponses of the trees to C02 resulting in higher
photosynthetic rates in the day and lower respira-
tion rates at night. However, the greater apparent
decreases in C02 concentration in the terracosms
with the elevated C02 treatments, likely are, at
least in part, also associated with greater leak rates
due to the greater chamber vs. outside C02 gradi-
ent for the elevated compared to ambient C02
chambers. Figure 1 also shows that elevated tem-
perature has little effect on photosynthesis or
respiration as the elevated temperature and ambi-
ent temperature treatments had similar means (con-
sidering the high variability during the day for the
ELV C02 AMB TEMP treatment)
Needle/branch carbon and water fluxes. Prelimi-
nary data for average photosynthetic rates for the
terracosms per treatment and date (all for 1993
needles) are shown in Figure 2. The rates were
measured at the same atmospheric C02 concentra-
tion as the seedlings were grown, i.e., approxi-
mately 550 ^il 11 for the elevated C02 treatments
(ELV C) and 350 jil l'1 for the ambient C02
treatments (AMB C, CHAMBERLESS). The
data indicate no apparent effect of the elevated T
treatment (ELV T) on photosynthesis either at the
elevated or ambient C02 level. Photosynthetic
rates generally are similar in the open plots and
ambient terracosms A comparison of photosyn-
thetic rates for the two age classes of needles is
shown in Figure 3. Needles from the older, 1992,
age class had higher photosynthetic rates than
1993 needles.
In addition to the above needle/branch photosyn-
thetic data, other data were taken but are not
shown here These data include: limited respira-
tion measurements for the open terracosms on
1 /12/94, needle/branch data were taken at the field
on 8/25/93, and needle/branch transpiration and
stomatal conductance data taken simultaneously
with all photosynthetic data.
FUTURE PLANS:
The terracosms are successfully collecting raw
C02 and water flux data necessary to calculate
canopy photosynthetic, respiration, transpiration,
and stomatal conductance rates in the future
Improvements are being made in the system to
Page 2
Task 1 Shoot Carbon and Water Fluxes
-------
Experimental Tasks and Facilities
insure continuously calculation of chamber leak
rates using SF6 as at tracer gas. Improvements
also will provide C02 free air for better control of
C02 concentrations in the ambient C02terracosms,
which will especially provide for better nighttime
respiration measurements. Soil C02 emissions
measurements are being taken to quantify that
component of terracosm C02 fluxes. Digital pho-
tographs have been taken of the trees for calcula-
tion of needle areas in individual terracosms.
Needle/branch measurements will continue, with
additional needle/branch data will be taken at the
field sites. Stem flow gauges are being developed
and will be put in place for continuous measure-
ment of whole-tree transpiration rates for the terra-
cosms All of these data, along with literature
values, will be used to derive photosynthesis,
respiration and stomatal conductance input vari-
ables for the TREGRO model.
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Figure 1 Diel variation in C02 flux among treatments for 2/21/94 Data are averages (mean standard with deviation across
lime periods in parentheses) for three repl icate chambers for A C02 A Temp (0 17) and A C02 E Temp (0 24), and two replicate
chambers for E C02 A Temp (1 18) and E C02 E Temp (0 43) The fluxes have not been corrected for terracosm leak rates,
soil C02 flux or needle areas
Page 3
Task 1 Shoot Carbon and Water Fluxes
-------
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DATE
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11 CHAMBERLESS
Figure 2. Photosynthetic rates for 1993 age class of needles with different treatments and measured on at different time
periods. Photosynthetic measurements are made at approximately 350 and 550
pi 11 CO, for each seedling, however, only the data for the CO., level at which the plants were grown are shown. Data taken
with LI-COR 6200 portable gas-exchange system with preliminary corrections for chamber leak rates. Bars are averages
for up to six single plants, two from each of three replicate chambers per treatment.
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Figure 3. Photosynthetic rate for 1992 vs. 1993 age class of needles across all treatments and both 350 and 550 (il I"1 C02
cuvette measurement levels for each seedling. Data taken with LI-COR 6200 portable gas-exchange system with preliminary
corrections for chamber leak rates. Bars are averages for up to six single trees with two trees from three replicate chambers
per treatment, and two CO: levels per tree.
4
-------
TASK 2: SHOOT GROWTH, MORPHOLOGY, ALLOMETRY,
PHENOLOGY, AND CARBON PARTITIONING
-------
Experimental Tasks and Facilities
TASK 2: Shoot Growth, Morphology, Allometry, Phenology, and Carbon
Partitioning
Participants: D. Olszyk (EPA) - Principal Investigator
C. Wise (EPA)
OBJECTIVE:
The overall objective for this task is whether
elevated CO2 and climate change will alter shoot
biology for young Douglas Fir trees. This question
is addressed by measuring at the individual plant
level the effects of elevated CO2 and climate
change on shoot:
biomass (dry weight) by age class of the
main stem, branches, needles, and buds;
allometric parameters, i.e., stem diameter,
height, and needle elongation;
morphology by age class, i e., numbers
and rank of branches, needles, and buds,
with, needle areas taken to determine spe-
cific needle weights;
phenology, i.e., the dates of key events
such as primary bud break, any secondary
bud break, and onset of first frost will be
carefully noted, and;
biochemical partitioning of carbon between
structural and nonstructural compounds in
different tissues in response to elevated
CO2 and climate change.
APPROACH:
The task focusses on the long-term effects of
elevated CO2 and climate change (elevated tem-
perature with no change in dewpoint) on Douglas-
fir seedlings growing in the terracosms. Baseline
measurements were taken at an initial destructive
harvest of 50 bare root seedlings. Intermediate,
nondestructive, measurements are being taken to
follow the course of tree growth over time. Final
destructive measurements will be made to look at
the cumulative effects of the treatments and ex-
perimental conditions on overall tree growth Ini-
tial, intermediate,and final tissue samples will be
used to characterize biochemical partitioning of
carbon among tissues.
In addition to the terracosm studies, in supporting
studies trees are being grown in large lysimeters at
ERL-C and in the field at the three Cascade Moun-
tain sites These trees are from the same popula-
tion as the terracosm trees, and will be used to
evaluate shoot growth under different climatic
and soil conditions and to obtain intermediate
destructive harvesting data for comparison to the
chamber trees.
STATUS/RESULTS:
Initial architecture and growth measurements made
on a sample of the tree population obtained in
Spring, 1993, from the Weyerhauser company
and used in the terracosms and supporting studies.
These were 1+1 trees, i.e., grown from seed sown
in Spring 1991. These are being used to establish
the baseline biomass and architecture of the trees.
Measurements included main stem diameter and
height, area of a subsample of needles, number
and order (primary, secondary) of branches, and
dry weights of needles, stems, and buds. Needle
and stem samples were collected for chemical
analysis.
Intermediate measurements for terracosm trees
Page I
Task 2 Shoot Growth and Phenology
-------
Experimental Tasks and Facilities
were made on 6/30-7/7/93, 7/29-30/93, 8/18/93,
9/14-16/93, 10/13-19/93, 12/6-9/94, 2/1-3/94, 3/
1-3/94, and 3/29-4/3/94. Measurements included
main stem diameter and height, terminal shoot
length, needle length, and terminal bud length.
Architecture measurements were made for a sub-
set of the trees in the terracosms on 10/28/93.
Needle samples were taken for chemical analysis
on 10/5-7/94. Phenology (bud break) is being
monitored closely in each chamber during March
and April, 1994.
Preliminary analysis of the intermediate data col-
lected to date indicates no difference in tree growth
among the terracosm climate change treatments in
terms of stem diameter between either June and
October 1993 (Figure 1A) or October 1993 and
March 1994 (Figure IB). There also was no differ-
ence among terracosm treatments in number of
buds set in 1993 (Figure 2). Further measurements
and analysis are continuing.
For the supporting studies, intermediate shoot
growth measurements were made at all field sites
during the 1993 growing season over the dates of
6/10/93, 7/2/93, 7/21/93, 8/4/93, 9/23/94; and
during the winter only at the low field site on 1 /12/
94,2/17/94, and 3/17/94. Shoot growth measure-
ments were made for the large lysimeters on 2/7/
94.
A CHANGE IN STEM DIAMETER JUNE-OCT 1993
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Figure 1. Preliminary data on average change in stem diameter between (A) June and October, 1993;
and (B) October 1993 and March 1994. Treatments: CL=Chamberless, A=ambient C02 and elevated
temperature, C=elevated C02 and ambient temperature, CT=elevated C02 and elevated temperature,
and T=ambient C02 and elevated temperature.
Page 2
Task 2 Shoot Growth and Phmology
-------
Experimental Tasks and Facilities
Figure 2 Preliminary data on average total buds/tree after first growing season. Treatments: CL=chamberIess,
A=ambient C02 and elevated temperature, C=elevated C02 and ambient temperature, CT=elevated C02 and
elevated temperature, and T=ambient C02 and elevated temperature
TOTAL BUDS PER TREE, OCT. 1993
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Future Plans
The trees in the terracosms currently are putting
out a new flush of growth (1994 needles). Addi-
tional phenology measurements have been initi-
ated to characterize the effects of elevated C02
and/or temperature on phenology at this critical
time Preliminary evaluation of the phenology
data indicates earlier bud break for the elevated
temperature treatments, which needs to be veri-
fied through statistical analysis. Needle samples
are being collected for the spring chemical analy-
sis. Phenology measurements will be made at the
field sites and lysimeters for comparison to the
terracosms. All of the previously collected and
new shoot growth data will be analyzed statisti-
cally and used as input for modeling of Douglas fir
tree growth using the TREGRO model.
Page 3
Task 2 Shoot Growth and Phenology
-------
TASK 3: SYSTEM NUTRIENTS
-------
Experimental Tasks and Facilities
TASK 3: SYSTEM NUTRIENTS
PARTICIPANTS: D Tingey (EPA) - Principal Investigator
A. Fong (METI)
B Gnffis (EPA)
M. Johnson (METI)
R. King (METI)
S. Ott (METI)
P. Rygiewicz (EPA)
INTRODUCTION
This task has two distinct functions: 1) a research
function specifically to evaluate the effects of
increased CO2, increased temperature, and al-
tered soil moisture on the size and relationships
among plant and soil nutrient pools and 2) a
support function to provide chemical analysis of
plant, soil, and soil solution samples as required by
other tasks.
OBJECTIVES
Monitor changes in inorganic nutrient concen-
trations in above- and belowground plant tis-
sues, litter, soil horizons and soil solutions as
a function of CO2 and temperature change.
Measure C and N, S, and TNC concentrations
in above- and belowground plant tissue, litter,
soil horizons, and soil solutions
Evaluate the effects of elevated CO2 and cli-
mate change on inorganic nutrient balance in
Douglas fir seedlings
APPROACH
Sample Handling
To insure continuity among sample compart-
ments and to deal with potentially small size,
priorities for sample analyses (Table 1) were
established as well rules for combining small root
samples (Table 2).
The soil coring procedure will yield 4 separate
soil core samples for each sampling event which
will be used for a variety of chemical and biologi-
cal analyses. If there is insufficient root tissue in
an individual core for the analyses the cores are to
be combined as shown in Table 2. To illustrate
the sample combining procedure two examples
are provided. Example 1 illustrates the general
rules of combining samples. In the first case (Plan
A) none of the cores (by horizons) have sufficient
root material for chemical analyses; in this case
the A and B1 horizons are combined into a single
sample and the B2 and C horizons are combined
for another sample (Plan B). If there are insuffi-
cient samples for chemical analyses these two
samples are combined again (Plan C). Example
Priority
Plant Tissue
Litter
Soil
Soil Solution
1
C &N
C & N
C&N
DOC, DIC
2
Aber Fractions
Nutrients
Nutrients
Nutrients &pH
3
Nutrients
Aber Fractions
Aber Fractions
Organic Acids
4
Isotopic Ratios
Isotopic Ratios
CHO & AA
Table 1. Analytical Priorities for Various Sample Types
Page I
Task 3 System Nutrients
-------
Experimental Tasks and Facilities
2 illustrates the case when some cores (A and B1)
have sufficient sample weight for analyses and
others (B2 and C) do not.
Aber Fractions
The plant tissues and litter samples will be analyzed by
Aber Fractionation Method (Aber et al., 1990) as it
provides operationally defined fractions that fit directly
into TREGRO and models soil and litter processes that
we are evaluating for use. Using the Aber Fractionation
Method (Figure 1), the three fractions are operationally
defined as: (1) Extractives - defined as the sum of the
nonpolar (CH2CI2 extractable) and polar (H2O extract-
able) fractions, (2) Cellulose - defined as the soluble
fraction of an acid hydrolysis of residue from (1), and (3)
Lignm - defined as the unhydrolized residue of (2).
Although the Aber Fractionation method has been
selected we have several things to resolve before we can
apply the method on samples: (1) We need to refine the
method for small samples; (2) We need to develop
methods to analyze the extracts for carbohydrates and
nitrogen compounds need to be selected; and (3) analyze
the residues for C & N contents.
Nutrient Analyses
Plant nutrients will be analyzed in plant, litter, soil
and soil solution samples. The methods that are
being used are shown in Table 3.
Table 2. Rules for combining small root samples.
Horizon
Sample
(Plan A)
Combination
(Plan B)
Combination
(Plan C)
Example 1
A
B 1
B 2
C
Example 2
A
B t
B 2
C
Q
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G
b
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Page 2
Task 3 System Nutrients
-------
Experimental Tasks and Facilities
Figure 1. Example of the Aber Fractionation Method that wil be used.
Ease of
Decomposition
Readily
Moderate
Slowly
Plant/Litter
Sample
Residue
t
Residue
Extractant - CH2CI2
Waxes, Fats, Terpenes, Pigments
Extractant - H2O
TNC, AA, Organic Acids
Extractant - H2SO4
Hemicellulose, Cellulose
Lignin
RESULTS
With the addition of chemists to the Project, we
have been able to initiate sample preparation and
analyses.
Plant and Litter Samples
Plant samples from planting and the fall 1993 are
ground and awaiting analyses. Samples of the
initial litter placed on the terracosms is awaiting
grinding and analyses.
Soils
Soil Type and Mineralogy The Douglas fir in the
Cascade Mountains of Oregon and Washington is
found primarily on two kinds of soils. One type,
found on about 70% of Douglas fir sites, is a
heavy-textured soil derived from colluvium and
residuum is classified as Site Class II and III. In
general these soils are found at elevations between
300 to 1100 m. The second soil type, found on
about 30% of Douglas fir sites, is coarser-textured
(sandy loam) and derived from volcanic ejecta and
glacial till. In general, these soils are found mainly
at higher elevations (900 to 1400 m), yielding
higher Site Classes of III or IV. We selected the
higher elevation soil (Type II) because it was
better suited for use in the terracosms, primarily
because of the ease with which it could be exca-
vated and reconstituted, its resiliency to distur-
bance and its hydraulic conductivity.
Samples of each master horizon (A, B, and C)
were sent to the Laboratory of Dr. Lucian Zelazny
at Virginia Tech for mineralogical analysis. A
particle size analysis indicated that all horizons
fall within the sandy loam textural class. The
mineralogical findings were consistent with the
Page 3
Task 3 System Nutrients
-------
Table 3. Description of the chemical analyses that will be conducted on plant, litter, soil and
soil solution samples from the terracosms.
Sample Type
Analysis
Plant Tissue I Litter
Air-Dried Soil
Field Moist Soil
Soil Solution
Total C, N
Carlo Erba CHNS Elemental Analyzer
Total Na, Ca, K, Mg
HC104 - Acid Digestion - ICP
H202 - Acid
Digestion - ICP
Total Zn, Fe, Cu, Mn
HC104 - Acid Digestion - ICP
H202 - Acid
Digestion - ICP
Total P, S, CI (soil
only)
HC104 - Acid Digestion - IC
(anion)
H202 - Acid
Digestion - ICP
Soluble Na, K, Ca,
Mg
HC104 - Acid Digestion - IC
(anion)
ICP
Soluble CI, S04,
HP04
H20 Extraction IC - (anion)
IC (anion)
Soluble NH4, N03
H20 Extraction Auto Analyze:
Auto Analyzer
Soluble Zn, Fe, Cu,
Mn
ICP
pH
H20 & 0.1 M CaC12 -
pH electrode
pH electrode
Extractable K, Na,
Ca, Mg
NH4Ac Extraction -
ICP
Extractable Zn, Fe,
Cu, Mn
HC1 Extraction - ICP
Extractable NH4,
N03
KC1 Extraction - Auto
Analyzer
Extractable S04
500 m/1 P Extraction
IC (anion)
Extractable P04
Bray P Extraction -
Auto Analyzer
-------
Experimental Tasks and Facilities
origin of the parent material. After removal of soil
organic matter, the less than 2 mm fraction of soil
is approximately 30% noncrystalline material by
weight The noncrystalline material is high in
NH4-oxalate extractable A1 and has calculated
Si02/Al203 ratios of 0.5 to 0.6.
Using standard mineralogical techniques the min-
eralogy of the clay sized material (< 2 Jim) found
little layer silicate material and only a small amount
of K-feldspar in the B and C horizons. Trace
amounts of kaohnite, mica, vermiculite in addi-
tion to K-feldspar were found in the A horizon clay
samples. The medium and fine silt (2 - 20 |im) and
the coarse silt (20 - 50 jim) fractions are dominated
by K-feldspars > plagioclase feldspars » quartz.
Because the mineralogy in all horizons is similar,
the weathering of the parent material is slight and
is relatively uniform throughout the profile.
The origin of the parent material (volcanic ejecta
and glacial till) and the low degree of weathering
constrains the fertility of the Terracosm soil The
mineralogy is dominated by noncrystalline miner-
als but abundant weatherable minerals are present.
These conditions may limit primary production,
but may also create a situation m which the Dou-
glas-fir seedlings may increase the production of
exudates to promote mineral weathering in the
rhizosphere or increase the allocation of carbon to
symbionts to increase nutrient acquisition. El-
evated CC>2 and elevated temperature may also
help to stimulate nutrient acquisition.
Soil Chemistry Soil samples were collected and
initially analyzed when the soil horizons were
removed from the forest floor and as the B and C
horizons were placed in the terracosms. The B and
C horizons were also analyzed after the soil had
been in the terracosms for 4 months and the fresh
t
A horizon was analyzed at planting. The results of
these latter analyses are shown in Table 4. Soils
samples from the fall coring are currently being
analyzed.
Soil Solutions Soil solutions are sampled every
4 weeks, at the same time that soil gases (Task 7)
are determined and video tapes of roots (Task 6)
are made. Individual cations and anions are deter-
mined via ion chromatography and trace metals
via ICP. The sum of the cations (Ca, Mg, K, Na),
determined by ion chromatography, were consis-
tently larger than the sum of the anions (S04 and
CI) (Figure 2). The cations and anions were most
nearly balanced in the B horizon of the covered
terracosms. The analyses of the soil solutions
found that nitrate and phosphate were below de-
tection limits in all horizons and all treatments.
Ammonium was found only in the ambient terra-
cosms, exposed to ambient rain) and not in any of
the covered terracosms. Given the animal opera-
tions in the vicinity, the ammonium have come
from atmospheric deposition.
CONCLUSIONS:
Most of the methods needed for chemical analyses
have been selected and developed. The analyses of
the soil solutions and the analyses on moist soil are
current. The dry soil, plant and litter samples will
be analyzed during the summer.
Page 5
Task 3 System Nutrients
-------
Table 4. Soil characteristics prior to planting.
Horizon
Extractable Cations
Ca
pH - water pH - KC1
Mg
K
meq/100 g
Na
CEC
A
B
C
6.33
6.50
6.60
5.00
5.30
n.a
2.70
2.30
2.30
0.60
0.98
0.95
0.22
0.27
0.27
0.06
0.10
0.07
18.50
n.a.
n.a
Horizon
A
B
C
Organic Matter Total Nitrogen
% %
9.88
3.27
3 27
0.105
0.067
0.062
NH4-N
2.12
6.10
4.18
N03-N
~7T
-------
Experimental Tasks and Facilities
Sum Anions meq/1 ~ Sum Cations meq/I
Treatment
A
Ambient g
c
TA CA
TA CE
TE CA
A
B
C
A
B
C
A
B
C
A
TECE B
c
1II I I I I II I I I I I I I I I I
I 1 1 1 ' I
I 1 1 1 1 I
Treatment
A
B
C
TA CA
TA CE
TECA
TECE
A
B
c;
A'
B
c;
A'
B
C
0.00 0.05 0.10 0.15 0.20
(Cations - Anions) - meq/1
0.00 0 05 0 10 0.15 0.20 0.25 0.30 0.35 0.40
Concentration - meq/1
43
24
' 56
17
47
12
47
24
51
B 45
\y
35
0.25
Figure 2. Cation and anion concentrations in soil solutions from the terracosms. The samples were
collected from paired tension lysimeters located in each of the soil horizons which are illustrated on the
left axes of the graphs The top graph illustrates the sum of the cations (Ca, Mg, K, Na) and anions (CI,
S04) while the bottom graph illustrates the difference between the sum of the cations and anions and the
numbers to the right of the bar shows the percent of charge unaccounted for. The ambient treatment refers
to the chamberless controls.
Page 7
Task 3 System Nutrients
-------
TASK 4: SYSTEM WATER
-------
Experimental Tasks and Facilities
TASK 4: SYSTEM WATER
PARTICIPANTS: M.G. Johnson (METI), Soil Water, D. Olszyk (EPA), Plant Water - Principal
Investigators
D T. Tingey (EPA), P.T. Rygiewicz (EPA), R. Shimabuku (EPA), G. Jarrell
(METI), M.J. Storm (METI), F. Senecal (METI)
OBJECTIVES:
To measure the effects of elevated CO2 and
climate change on the relationship between
plant and soil water.
To develop a daily water budget for each
Terracosm.
To monitor the water content of each Terra-
cosm soil horizon to schedule Terracosm wa-
tering (timing and quantity of water addi-
tions).
APPROACH:
A soil water regimen was developed for the Terra-
cosm soils that mimics the wet winter and dry
summer pattern of Pacific Northwest forests. The
regimen (discussed below in detail) provides tar-
get values of soil water content on an annual basis.
The soil water conditions in the ambient CO2 and
ambient temperature treatment Terracosms are
compared to the target values to determine whether
or not water is added to all the Terracosms. If
water needs to be added to raise soil moisture in
the ambient CO2 and ambient temperature treat-
ment Terracosms, then the same amount is added
to all the Terracosms. Even though water addi-
tions will be the same in all the Terracosms, soil
water conditions are likely to be different between
treatments because water use by seedlings and
evaporative demands will be a function of treat-
ment.
The approach we have taken to monitor system
water in the Terracosms is to measure water inputs
and outputs, the amount of water that individual
seedlings transpire, the amount of water held in
the soil, and the amount of water that drains
through the soil. These measurements provide the
water data needed to meet the objectives stated
above Below we develop the equations and
concepts that are being used to calculate water
budgets and to determine the timing and amounts
of water addition.
Hydrologic Cycle
Because the Terracosms are closed systems, water
use in the Terracosms is governed by a modified
hydrologic cycle. Here we develop the equations
that describe the Terracosm hydrologic cycle un-
der various conditions. We also describe how the
various parameters are measured.
The hydrologic cycle of open terrestrial systems
can be written as:
P + I + Ron + Sdep = Et + Roff + Srec + Dr
Equation 1
where P is precipitation, I is irrigation or other
water additions, Ron is ninon, Sdep is depletion of
soil water storage, Et is evapotranspiration [soil
evaporation (Es) plus plant transpiration (Tr)],
R0ff is runoff, Srec is recharge of soil water
storage, and Dr is drainage (Hanks, 1992). Water
supply is the group of terms on the left side of
Equation 1 and outputs, losses or sinks is the set on
the right.
Page 1
Task 4 System Water
-------
Experimental Tasks and Facilities
With the exception of the chamberless Terra-
cosms (Terracosms 1 and 8) the Terracosms are
closed systems and do not allow precipitation
inputs or any runon or runoff (i.e , P = 0, R0ff = 0,
and Ron = 0). To describe the hydrologic cycle of
the Terracosms these modifications of Equation 1
result in the following equation:
I + Sdep = Et + S|-££ + Dr
Equation 2
Equation 2 says that water in the Terracosms may
be lost as Et or Dr and that some may go to
recharge soil storage (Srec)- These losses plus
recharge must be equal to the inputs of water from
irrigation plus the amount of stored water that was
removed or depleted.
To put the Terracosm hydrologic cycle into its
proper context it is necessary to review the water
regimen that we are using in the Terracosms
Terracosm Water Regimen
As presented in the Research Plan we will follow
the wet winter and dry summer soil moisture
pattern of Pacific Northwest forests in the Terra-
cosms. Figure 4-17 (in the Research Plan) shows
the general drying and wetting cycles that are
being used. Figure 1 is similar to Figure 4-17 but
shows soil water content as a percentage of field
capacity instead of volumetric water content (in
the Addendum to this chapter, Figures 4,5, and 6
show the Terracosm water regimen in volumetric
water content and percent of field capacity for the
Terracosm A, B, and C horizons, respectively.).
This soil water versus time curve has four distinct
regions The first is the winter period in which soil
water is at 100 % of field capacity. The second
region begins in early spring and is the slow drying
period In this period water is reduced because of
decreased inputs and increased losses (i.e., evapo-
transpiration). This period corresponds to the end
of winter rains and the beginning of the dry season.
The third region occurs during the summer when
the soil is driest and remains dry for approximately
two months. In the northwest this is the dry
season. The fourth region signals the end of the
dry season and the return of fall precipitation
During this period soil water is recharged rapidly
and reaches 100 % of field capacity in early
winter.
Terracosm Water Measurements
Here we describe our approach to measuring in the
T erracosms each of the components listed in Equa-
tion 2.
Water Additions (I)- There are two ways that
water is added to the Terracosms. The first is
irrigation (Ir) additions which are added either by
a hand-held watering wand or through soaker
hoses that are installed in each Terracosm The
second form of water addition the water vapor
(Wv) that is used to adjust Terracosm dew-point
depression. The irrigation additions are of known
volume and the water vapor additions are quanti-
fied daily The "I" term can be broken down into
its components as shown in Equation 3:
I = Ir + Wv
Equation 3
Soil Water: Soil water is measured using an
automated Time Domain Reflectometry (TDR)
system on an hourly basis There are five 30-cm
TDR probes installed in each Terracosm (see
Figure 5-5 in the Research Plan). One is posi-
tioned vertically through the A-horizon and 20 cm
into the upper B-horizon (Bl). The other 4 are
placed horizontally in the middle of the A, Bl
(upper B-horizon), B2 (lower B-horizon), and C-
horizons.
The measurement of soil water depletion (Sdep)
or soil water recharge (Srec) depends upon the
measurement of soil water and its change over
time. For example, soil moisture data from each
probe location can be averaged over a period of
time (e.g., a four hour period beginning at noon on
Page 2
Task 4 System Water
-------
Experimental Tasks and Facilities
one day) and compared to data averaged over the
same period on the next day. The difference
between the numbers when weighted by the vol-
ume of soil is equal to Sdep if positive or Srec 'f
negative. This is described by Equation 4.
Change Soil Water Storage (ti .> t2)
[©vxl x Vsx] ¦ [©vx2 x Vsx]
Equation 4
Where 0Vx 1 is the volumetric water content of
horizon "x" at time " 1" and 0vx2 is the volumetric
water content of horizon "x" at time "2" (cm^
water/cm^ soil) When multiplied by Vsx, the
volume of soil horizon "x" (cm^), the volume of
water held in soil horizon "x" is calculated. The
difference between the volume of water at time
" 1" and time "2" is equal to depletion of stored soil
water and recharge of soil water if negative.
Evapotranspiration (Et): Evapotranspiration is
the sum of soil evaporation (Es) and plant transpi-
ration (Tr) as shown in Equation 5:
Et = Es + Tr
Equation 5
In the Terracosms we quantify whole chamber Et
and Es and by difference calculate Tr. Evapo-
transpiration is measured when moisture in the
Terracosm air is circulated over the cold water
heat exchanger coils and collects on the coils. This
water accumulates and drips into a tipping-bucket
rain gauge where it is quantified. The amount of
water collected over a period of time is the Et over
that period. The Et data are recorded automati-
cally by the TERA data collection system. When
the seedling stomates are closed (at night) Tr is
approximately equal to zero and Es can is approxi-
mately equal to Et This measurement of Es can be
used to calculate daytime whole chamber transpi-
ration rates.
We also plan to measure Tr at the whole plant level
using stem sap flow gauges similar to those de-
scribed by Steinberg et al. (1989) and Ham and
Heilman (1990). These gauges use a heat-pulse
technique to measure Tr. Stem sap flow gauges
will be attached to 2 or 3 seedlings in each Terra-
cosm and will provide continuous measures of
whole plant Tr.
Drainage (Dr) The Terracosms are open at the
bottom to allow free drainage of gravitational
water The quantification of these waters gives the
last term in the Terracosm hydrologic equation.
Rooting Volume
In the Research Plan we proposed calculating soil
water content on the rooting volume basis. We
define rooting volume as "the soil volume con-
taining roots." Rooting volume is verified by root
images obtained with the minirhizotron camera
system (monthly collection frequency) and by the
distribution of roots collected during the twice per
year soil coring events. At the time of planting the
seedling root systems were shallow and limited to
the A and B1 horizons Until there is root growth
into the B2 or C horizons, only the A and B1
horizons need be considered in soil moisture cal-
culations
STATUS/RESULTS:
Irrigation System
We have employed two kinds of irrigation sys-
tems in the Terracosms. In the winter months we
use a wand system to apply irrigation waters above
the seedling canopy. We adopted this system to
mimic precipitation additions in nature and to
insure that the forest floor/litter layer is moist
throughout the winter. During the Spring, Sum-
mer, and early Fall when the litter layer in natural
forests is drying, water additions will be made
through a soaker hose array that lies between the
forest floor/litter layer and the mineral soil. The
rationale for selecting the soaker hose system was
to facilitate irrigation without wetting the forest
Page 3
Task 4 System Water
-------
Experimental Tasks and Facilities
floor/litter layer during the period when it is nor-
mally dry. Currently, water is pumped into the
soaker hose array using a hose bib and a pressure
regulator. Later this year, a computer controlled
metering system will be installed on each Terra-
cosm to automate irrigation events.
Water additions as vapor (steam) are quantified on
a daily basis Over the past winter we realized that
using steam adds heat to the system that is difficult
to remove. Plans are being developed to replace
the steam system with an ultrasonic cool-water
vapor addition system. This new system will
allow more precise control of dew-point depres-
sion and reduce condensation on the chamber
walls. The plan is to exchange these systems this
summer.
Stem Sap Flow Gauges
We are manufacturing the stem sap flow gauges to
measure transpiration. Dr. Jay Ham of Kansas
State University, one of the key developers of
stem sap flow gauges, instructed us on their con-
struction and installation. Later this year the
gauges will be installed in each Terracosm. The
Allen-Bradley process control hardware and soft-
ware will be used to control the gauges and to
collect the transpiration data.
Drainage Waters
We are developing plans to quantify the amount of
water that drains from each Terracosm. One plan
under consideration is to install a reservoir to
collect drainage waters and periodically pump the
water out of the reservoir and into the tipping
bucket rain gauge used to quantify Terracosm
evapotranspiration. Another possibility is to use a
reservoir that sits on a pressure transducer. Peri-
odically the weight of the reservoir plus drainage
water will be recorded and then the reservoir is
pumped dry. The weight of empty reservoir is also
taken. The difference between the two numbers is
the quantity of drainage water. Periodically we
will be collecting a subsample of the drainage
waters for nutrient and soluble carbon analyses.
Soil Water Measurement Systems
In Spring 1992 the Terracosm B and C-soil hori-
zons were placed in the soil compartments. At that
time three horizontal TDR probes were installed
(one each in the middle of the Bl, B2 and C-
honzons). When the seedlings were planted (June
1993) a fourth TDR probe was placed horizontally
in the middle of the A-honzon. The fifth TDR
probe, with a vertical orientation, was placed in
the A and Bl-horizons prior to the placement of
the forest floor/litter layer. With 5 TDR probes per
Terracosm, there is a total of 70 probes, or soil
water measurement points, in the 14 Terracosm
field.
Based upon vendor recommendations, the Re-
search Plan calls for installing a single TDR unit
and an array of nested multiplexors to measure soil
water content for the entire Terracosm field. As it
turned out, one unit was not sufficient to provide
complete coverage. This was primarily due to the
long distances between the TDR unit and some of
the probes. We decided to install a second TDR
unit and to divide the Terracosm field between the
two units Overall, this dramatically improved the
soil moisture data being collected However, even
with two TDR units there were problems with high
noise data from several of the Terracosms. We
decided to install a third TDR unit. The first TDR
unit came on-line in November 1993 (Terracosms
1 - 7) and the second in January 1994 (Terracosms
8-14). The third unit will come on-line in Spring
1994.
Irrigation Scheduling
Prior to having this in situ soil moisture informa-
tion the Terracosms were watered based on a
subjective analysis of soil and plant water status
When TDR data became available in November
1993, it was quickly established that the Terra-
cosm soils were much dryer they should have been
according to the water regimen we had established
(See Figure 1). Since achieving and maintaining
field capacity was our objective over the winter
months, 20 and 30 liter (depending on soil mois-
Page 4
Task 4 System Water
-------
Experimental Tasks and Facilities
ture in a particular Terracosm) increments of wa-
ter were added to the Terracosms once or twice a
week to raise the soil water content up to the
prescribed winter moisture levels.
During the spring, summer and fall soil moisture
will be allowed to go below field capacity (Figure
1). The strategy and rules for adding water are
discussed below. An addendum follows this chap-
ter that contains addition information on the soil
water properties of the Terracosm soils and shows
example soil water calculations.
Irrigation During the Wet Period: Soil moisture
during the wet period, as shown in Figure 1, is to
be held at 100 % of field capacity. Field capacity
was estimated from soil moisture release curves
shown in Figure 2 and summarized in Table 1.
Water was added when the TDR data indicated
that the soil water content was below field capac-
ity
Irrigation During the Slow Drying Period1 Be-
ginning in April of each year the Terracosm soils
will be allowed to dry. The objective here is to
allow them to dry slowly over a five month period.
Since the soils are at field capacity when this
period begins the only losses of soil water will be
losses through Et. The TDR data will be used to
assess the soil drying process in conjunction with
Figure 1. As specified m the Research Plan,
Terracosm watering will be based upon soil mois-
ture in the ambient CO2 and ambient temperature
treatment (Terracosms 2, 11, and 13). If the soil
gets more than 5% of field capacity below the
target value of the water regimen curve (Region 2
of Figure 1), water will be added to bring the soil
back up to the target value.
Irrigation During the Dry Period: During the dry
period soil water is to be held at or near 50% of
field capacity When soil water drops to 45% of
field capacity in the rooting volume of the soil, the
amount of water added to all chambers will be the
amount of water required to bring the soil of the
three ambient CO2 and ambient temperature Terra-
cosms back to 50% - 55% of field capacity.
Irrigation During the Rapid Recharge Period:
The objective during this period is to rapidly
recharge soil water that has been depleted. The
recharge period lasts approximately 10 weeks.
Water will be added, based upon the TDR data, at
a rate that follows the recharge portion of the curve
in Figure 1.
FINDINGS/CONCLUSIONS/SUMMARY:
Figure 3 shows the data collected by each TDR
probe in Terracosm 5 for 9 days beginning 21
February 1994. These data were collected when
the soil moisture is to be held at or near field
capacity (Figure 1). The data collected at the A/Bl
Horizon 0y @ FC 0y @ 50% FC 0y @ WP Vs
A 23.58 11.79 9 72 20xl05
B1 26.36 13.18 8 68 5.6 xlO5
B2 26 36 13 18 8.68 6.0 xlO5
C 34.64 17.32 10.71 40xl05
Table 1 Volumetric water content (0V, cm3 waler/cm3 soil) at field capacity (FC), 50% of FC, and at the
permanent wilting point (WP)t and the volume of soil (Vs, cm3) of each Terracosm soil horizon
Page 5
Task 4 System Water
-------
Experimental Tasks and Facilities
and A probes, and to a lesser degree at the B1
probe, show two spikes about mid-day on the 25th
and 27th of February. The spikes correspond to
mid-day Terracosm irrigation events. The amount
of water held in the soil two days after irrigation is
approximately equivalent to field capacity. Field
capacity values estimated from Figure 3 corre-
spond nicely to those obtained from soil moisture
release curves (see Table 4 1 and Figure 4.2) for
each horizon.
Several items still need to be installed and brought
on-line before daily water budgets for the Terra-
cosms can be calculated on a routine basis. The
remaining items will be added in the Spring and
Summer of 1994 Currently the data processing of
the soil water data is not automated. Future plans
include automation of this and the entire water
budget.
REFERENCES:
Ham, J.M andJL Heilman 1990. Dynamics of
a heat balance stem flow gauge during high flow.
Agron. J. 82:147-152.
Hanks, R.J. 1992. Applied soil physics. 2nd
Edition. Spnnger-Verlag, New York.
Steinberg, S., C.H.M. van Bavel and M.J
McFarland. 1989 A gauge to measure mass flow
rate of sap in stems and trunks of woody plants. J.
Amer. Soc. Hort Sci. 114-466-472.
Page 6
Task 4 System Water
-------
Experimental Tasks and Facilities
ADDENDUM TO TASK 4: SYSTEM WATER
This Addendum contains additional information
about the water holding characteristics of the
Terracosm soil. It also contains a discussion of
how to calculate soil water content and to use that
information to decide whether or not to irrigate
Percent of Field Capacity and Volumetric Water
Content
Figure 1 depicts the soil water regimen established
for the Terracosms. The units on the y-axis are
"percent of field capacity " Having units of volu-
metric water content on the y-axis would be help-
ful in gaining some sense of how much water is
held in the soil. Here we graphically present the
soil moisture regimen for the three Terracosm soil
master horizons showing both "percent of field
capacity" and "volumetric water content" on the
y-axis for the A, B, and C-horizons, respectively
(Figures 4,5, and 6). The solid black line indicates
an estimate of soil water content at wilting point (-
1.5 MPa). The difference between the volumetric
water content at any point on the soil water regi-
men curve and the wilting point line, is the amount
of available water.
Calculating Soil Water Content
To calculate the water content of the A and B1
horizon data in addition to those obtained using
the TDR probes are needed. We get additional
data from moisture release curves and the volume
of each soil horizon. Figure 2 shows the moisture
release curves for the three Terracosm master
horizons (A, B=B1 & B2, and C). Using -0.01
MPa and -1.5 MPa as approximations of the ten-
sion at field capacity and at the permanent wilting
point, respectively, we can estimate the amount of
water held in each soil horizon. The volumetric
water content and the volume of each soil horizon
are reported in Table 1. Table 2 contains estimates
of the amount of water that is held in each horizon
and Table 3 contains estimates of the amount of
water held in four predetermined rooting volumes.
These three tables will be used to assess the water
status of the Terracosm soils and to determine the
irrigation schedule.
Table 2 Approximate amount of water held in each horizon at field capacity (FC), 50% of FC, and at the
permanent wilting point (WP) by Terracosm soil horizon.
Horizon
Liters @ FC
Liters @ 50% FC
Liters @ WP
A
47.2
23.6
19.4
B1
147.6
73.8
48.6
B2
158 2
79.1
52.1
C
138.6
69.3
42.8
Table 3 Approximate amount of water held in four rooting volumes delineated by the soil honzon, at field
capacity (FC), 50% of FC, and at the permanent wilting point (WP)
Rooting Volume1
Liters @ FC
Liters @ 50% FC
Liters @ WP
A
47.2
23.6
19.4
A+Bl
194.8
97.4
68.0
A+B1+B2
353.0
176.5
120.1
A+B1+B2+C
491.6
245.8
162.9
'Rooting volume (the volume of soil that contains roots) has been delineated here as the A honzon or incremen-
tal additions of subjacent soil horizons
Page 7
Task 4 System Water
-------
Experimental Tasks and Facilities
Below are two examples of how soil water content for rooting volumes of more than one horizon are
calculated and how the decision whether or not to irrigate is made
Example 1
Suppose that during the summer the rooting
volume was limited to the A and B1 horizons
and theTDR data indicated that 0V (volumet-
ric water content) for the A-horizon was 18 %
and the B1 horizon was 24 %. What is the
water content of the rooting volume and does
the soil need to be irrigated7
Calculations. The rooting volume consists of
2.0 x 105 cm3 ofA-honzon and 5.6 x 10^ cm3
ofB-honzon (volume data from Table 1). The
A-honzon contains 36 liters [(18% or 0.18
cm3 H20/cm3 soil x (2.0 x 10^ cm3 soil))/
1000] of water (Using the data in Table 2, the
amount of water held in the A-honzon at
wilting point is 19.4 liters. If there are 36 liters
of water held in the A-horizon only 36-19 4 or
16.6 liters that are plant available.). The B-
horizon contains 134 liters [(0.24 x (5.6 x
10^))/1000] of water (of which 86 2 are plant
available).
During the summer soil water is supposed to
be at 50% of field capacity according to Figure
1. To calculate the amount of water held in this
rooting volume at 50% of field capacity mul-
tiply the Ov at 50% of field capacity by the
volume of soil for both the A and B1 horizons.
Sum and convert that volume of water into
liters. In this case the volume is 96.8 liters
[(0.12 x (2.0 x 105 cm3))/1000 + 0.13 x (5.6 x
10^ cm3)/I000] Therefore, since the amount
of water held in the rooting volume (A+B1) is
greater (134 > 96.8) than 50% of the field
capacity for the rooting volume, no water
needs to be added at this point.
Example 2
Suppose again that during the summer the
rooting volume was limited to the A and B1
horizons and the TDR data showed that 0V
for the A-honzon was 9 % and the B1 horizon
was 12 %. What is the water content of the
rooting volume and does the soil need to be
irrigated7 If the rooting volume is below 50%
of field capacity, how much water is needed to
bring the soil up to 50% of field capacity?
Calculations: The rooting volume consists of
2.0 x lO^cm^of A-horizon and 5.6 x 10^ cm3
of B-horizon. The A-horizon contains 18
liters [(9 % or0.09 x cm3 H20/cm3 soil x (2.0
x 10$ cm3 soil»/1000] of water. The B-
honzon contains 67.2 liters [(0.12 x (5.6 x
10^))/1000] of water We know from Ex-
ample 1 that the water held in this rooting
volume at 50% of field capacity is 96.8 liters
Under the current conditions this rooting vol-
ume now only holds 85.2 liters.
The criteria for watering during the summer is
when the soil drops to 45% of field capacity.
The water held at 45% of field capacity in this
rooting volume is 87.6 liters. Since current
soil moisture conditions are below 45% of
field capacity, water needs to be added. Under
these conditions the addition of 11.6 liters
(96.8 - 85.2) of water are needed to bring the
rooting volume back to 50% of field capacity.
Page 8
Task 4 System Water
-------
Experimental Tasks and Facilities
Twenty-four to 48 hours after a watering event the
TDR data should be checked to see if the water
additions accomplished the intended objective. If
the objective is not met, more water is needed.
Adding water to obtain a certain soil moisture
content is likely to be an iterative process until we
understand the hydrological properties of the Terra-
cosm soils more fully. By documenting the soil
conditions, the amount of water added, and the
observed response we will compile a dataset that
will help us develop a nomograph of soil water
content and soil water additions to more precisely
control water additions.
Page 9
Task 4 System Water
-------
Experimental Tasks and Facilities
110
Winter
Spring
Summer
Fall
100-
90-
K 80-
70-
2 60-
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re
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~iiiiiir
10 12 14 16 18 20 22
\ i r
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Bi-Weekly Interval
Figure I. The annual Terracosm soil water regimen.
A-horizon B-horizon C-horizon
c 50
O 40
^ 30
i i n|
0.001
0.0001
0.01
0.1
1
10
Tension (-MPa)
Figure 2. Soil moisture release curves for the three master horizons of the Terracosm soil
Page 10 Task 4: System Water
-------
Experimental Tasks and Facilities
c
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0.25-
0 20
A/B1-Horizons
c\j
A-Horizon
0.35-
0.30-
0.25-
B1 -Horizon
-1r
B2-Horizon
C-Horizon
«y»>* i
¦
-------
Experimental Tasks and Facilities
Winter
*
o
&
(0
O
"O
"5
110
100
90-
80-
70-
60-
50-
40-
30-
20-
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0
A-Horizon
Spring Summer
Fall
Available Water
Wlltlno Point
Unavailable Water
tir
2
-iiiir
6 8 10
iiiiiiiIiiiiir
10 g
3
5 S?
12 14 16
Bi-Weekly Interval
18 20 22 24 26
Figure 4. Soil moisture regimen for the Terracosm A-horizon three individual horizons showing both "percent of field
capacity" and "volumetric water content" on the y-axis as a function of time. The solid black line indicates an estimate of
soil water content at wilting point (-1.5 MPa). The difference between the volumetric water content at any point on the soil
water regimen curve and the wilting point line, is the amount of available water.
B-Horizon
Winter
o
<0
a
(0
O
¦o
75
il
c
s
£.
110
100
90
80
70
60
50
40
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10-
Spring
Summer
Fall
Available Water
Wilting Point
Unavailable Water
-iiiiiiiiiiiiiiir
2 4 6 8 10 12 14 16
Bi-Weekly interval
h5 £
iiiiiiir
18 20 22 24 26
Figure 5. Soil moisture regimen for the Terracosm B-horizon three individual horizons showing both "percent of field
capacity" and "volumetric water content" on the y-axis as a function of time. The solid black line indicates an estimate of
soil water content at wilting point (-1.5 MPa). The difference between the volumetric water content at any point on the soil
water regimen curve and the wilting point line, is the amount of available water.
Page 12
Task 4: System Water
-------
Experimental Tasks and Facilities
110-
100-
o
re
a
re
O
2
a>
c
a>
o
u.
a>
a.
90-
80-
70-
60-
50-
40-
30-
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10-
0-
Winter
C-Horizon
Spring Summer
Fall
Available Water
Wilting Point
Unavailable Water
-iiiiiiiiiiiiiir~
2 4 6 8 10 12 14 16
Bi-Weekly Interval
IIIIi-
18 20 22
-35
30 <
-10 =
-5
24 26
Figure 6 Soil moisture regimen for the Terracosm C-horizon three individual horizons showing both "percent of field
capacity" and "volumetric water content" on the y-axis as a function of time The solid black line indicates an estimate of
soil water content at wilting point (-1 5 MPa) The difference between the volumetric water content at any point on the soil
water regimen curve and the wilting point line, is the amount of available water.
Page 13
Task 4 System Water
-------
TASKS: LITTER LAYER
-------
Experimental Tasks and Facilities
TASK 5: LITTER LAYER
PARTICIPANTS: M.G Johnson (METI) - Principal Investigator
P.T Rygiewicz (EPA), M.J. Storm (METI)
INTRODUCTION:
The forest floor, or Jitter layer, is an important
component of forest nutrient cycling. In the litter
layer plant litter comminution and decomposition
releases important plant nutrients. The litter layer
is a site of high faunal and microbial activity.
Also, a large proportion of fine roots is found in the
nutrient-rich litter layer. Task 5 was developed to
evaluate the effects of elevated CO2 and climate
change on the litter layer decomposition (chemis-
try and weight loss) The Task 5 research activi-
ties since the last peer review have followed those
outlined in the Research Plan.
OBJECTIVES:
The specific objectives of this experimental task
are-
To measure the rate of litter decomposition
with elevated CO2 and climate change.
To determine how elevated CO2 and climate
change affect nutrient cycling in the litter
layer.
To measure changes in litter quality
throughout the Terracosm study.
To determine the effect of elevated CO2
and climate change on net storage of carbon
in the litter layer.
APPROACH:
The litter layer Task has two primary components
The first is the addition of a litter layer to the
Terracosms and field site plots (Cascade field
plots and large lysimeters), and the second is a
study of litter decomposition in the Terracosms
and at the Cascade field sites using litter bags and
needle packs.
Litter layer
The litter layer, in addition to sequestering and
providing nutrients, also functions to store water
and to insulate the underlying mineral soil. To
complete the reconstruction of the forest soil in the
Terracosms we added a litter layer. At the site
where the soil for the Terracosms was collected
the mineral soil was overlain by a 6 cm thick litter
layer. This litter layer consisted of 4 cm of slightly
decomposed material (Oi horizon) on top of 2 cm
of highly decomposed material (Oa horizon). Ini-
tially we planned to reconstruct both of these
layers, but later realized that it would be impos-
sible. We opted to collect forest floor litter and use
that to create a 6 cm thick litter layer in the
Terracosms, large lysimeters, and at the Cascade
field sites. Two times a year (spring and fall) the
depth of the litter layer in the ambient CO2 and
ambient temperature Terracosms (numbers 2,11,
and 13) will be assessed. If the depth is less than
3 cm then the amount of litter needed to bring the
litter layer in these three chambers to 6 cm will be
added to all the Terracosms and proportionate
amounts (as per surface area) to the large lysim-
eters. Independent assessments are made of litter
depth at each of the Cascade field sites.
Page 1
Task 5 Utter Layer
-------
Experimental Tasks and Facilities
Decomposition
Litter decomposition is being assessed using litter
bags and needle packs. Litter bags consist of a
nylon mesh bag containing a known weight of
litter material. The litter bags are placed in the
litter layer in the Terracosms and Cascade field
sites. Needle packs (needle packs are akin to leaf
packs, Triska and Sedell, 1976) consist of needles,
collected from the forest floor, threaded on non-
decomposable thread. The needle packs are placed
in the litter layer Periodically a portion of these
bags and needle packs are removed, air-dried, and
reweighed. These litter bags and needle packs are
replaced by fresh litter bags and needle packs
Decomposition rates will be based upon the mass
of litter layer material lost
Needle packs will likely produce overestimates of
decomposition because grazing mesofauna may
clip a needle in two, leaving a non-decomposed
fragment no longer attached to the thread. When
the needle packs are collected and weighed, the
observed weight loss could be slightly exagger-
ated and not entirely attributable to decomposi-
tion Therefore, decomposition estimates using
needle packs may be greater than the actual de-
composition rate. On the other hand, litter bags
are likely to underestimate decomposition be-
cause the bags may limit grazing by soil mesofauna
that comminute litter. By using both litter bags
and needle packs, we hope to bracket the actual
decomposition rate.
The litter bags are also being used to assess changes
in litter layer chemistry. The chemistry (nutrients
and biochemistry) of the initial litter material will
be compared with that in subsequent samples over
the course of the study.
STATUS/RESULTS:
Litter layer
Following seedling planting in the Terracosms,
large lysimeters, and Cascade field sites, we col-
lected forest floor litter at a high elevation old-
growth forest site near the soil collection site.
Logging in and around the soil collection site
precluded collecting litter there. Litter was col-
lected by raking the forest floor. The material was
earned to a central collection area where it was
sieved to remove materials larger than 2.5 cm.
During the collection and sieving process the
material was mixed and homogenized. This sieved
forest floor material was either brought to Corvallis
for addition to the Terracosms and large lysim-
eters or carried into the Cascade field sites for
distribution (Note: After litter collection, the site
where the litter was collected was fertilized with
mineral fertilizer to replace the nutrients that were
removed)
The litter layer material was distributed at the
Cascade field sites, the Terracosms, and the large
lysimeters. At the Cascade field sites, 6 cm of
forest floor litter material was distributed over the
plots and planted buffer areas (see Cascade field
site write-up in this booklet for field site layout).
Six cm of weighed forest floor litter layer material
was placed in each Terracosm. A total of 24 kg of
fresh litter was added to each Terracosm. The
litter layer material that was added to the Terra-
cosms was subsampled to determine water con-
tent and for chemical analysis. The large lysim-
eters received 36 kg of litter (50% more than in the
Terracosms because the surface area of the large
lysimeters 50% greater than the Terracosms).
Visually, the addition of the forest floor litter layer
to the Terracosms mimics that found in natural
northwest forest stands. There are needles, cones,
and small woody debris in the litter layer. The
Page 2
Task 5 Litter Layer
-------
Experimental Tasks and Facilities
litter layer appears to be quite active The assess-
ment of litter layer fauna in the fall of 1993 found
large faunal populations distributed over a large
number of species (see Task 7, in this Booklet).
There is also evidence (i.e., the uppermost part of
the litter layer can be very dry while the lowest
part, adjacent to the mineral soil, can be quite
moist) that the litter layer is functioning as a mulch
and reducing evaporation of water from the soil.
In September 1993 a reservoir of air-dried forest
floor litter material was established near TERA
This reservoir of material will be used for biennial
litter layer additions. During late summer or early
fall this reservoir will be refilled in preparation for
subsequent litter layer additions If litter additions
are required in the fall, the litter for this will be
collected fresh and added to the various chambers
and plots.
Decomposition
Forest floor needle litter was collected in October
1992 following the fall needle drop for the litter
bags and needle packs. The site of collection was
the old-growth stand adjacent to the soil collection
site. A sufficient amount of litter for all the litter
bags and needle packs planned for the entire
experiment was collected This litter was air-dried
and frozen
Six inch by 6 inch litter bags were made from fine
nylon mesh. Approximately three grams of needle
litter (air-dried weight) was placed in each litter
bag and the overall weight of the litter bag and
contents determined. The needle packs consist of
about 0.5 grams of air-dried needles threaded on a
thread. The needle packs were air-dried and
weighed On both the needle packs and litter bags
plastic identification tags have been attached.
To place the litter bags and needle packs, the litter
layer material in an area slightly larger that a litter
bag was pulled back to create a hollow such that 2
cm of litter remained on top of the mineral soil.
The litter bag and needle pack were placed in this
hollow and covered with the excavated litter. At
the time of installation the litter bags and needle
packs had approximately 4 cm of litter on top of
them and 2 cm of litter beneath them. Additional
litter bags were used as controls to assess the loss
of liter from the litter bags that occurs during litter
bag placement in the Terracosms and at the Cas-
cade field sites.
In October 1993 twelve litter bags were placed
between the trees in each Terracosm (see Figure
1). A needle pack was placed adjacent to each
litter bag. A total of 168 litter bags and 168 needle
packs were placed in the Terracosms. At each of
the Cascade field sites 18 litter bags and 18 needle
packs were placed in one of the five plots.
Twice a year (spring and fall), following soil
coring in the Terracosms, two litter bags and two
needle packs will be removed from each of the
Terracosms. The first set of litter bags and needle
packs will be removed in May 1994. At the
Cascade field sites three litter bags and three
needle packs will be removed from each site to
correspond with removal at TERA. The harvested
litter bags and needle packs will be air-dried and
weighed. A subsample of the litter remaining in
the litter bags will be analyzed for nutrients and
biochemistry (see Task 3 in this Booklet for spe-
cific chemical and biochemical analyses). All
litter bags and needle packs that are removed will
be replaced with new litter bags and needle packs
containing non-decomposed needle litter (collected
in October 1992). All replacement litter bags will
be harvested at the end of the experiment.
REFERENCES:
Tnska,F.J.andJ.R. Sedell. 1976. Decomposition
of four species of leaf litter in response to nitrate
manipulation. Ecology 57:783-792.
Page 3
Task 5 Litter Layer
-------
Experimental Tasks and Facilities
Litter Bags
(1 mm2 mesh,
15 x 15 cm)
Minirhizotron Tubes
(5 cm dia. x 100 cm L)
Soil Fauna Sample
Collection Area
-2 Meters-
Litter Layer
A Horizon
B - Horizon
C - Horizon
Drainage Gravel
6 cm
10 cm
60 cm
20 cm
10 cm
1 Meter
L'VVVVS'S'N'S
a s
Figure 1: Cross section and top view of Terracosm soil chamber showing details of tree seedling placement, soil horizons, and location
of 12 litter bags (needle packs, not shown, are placed adjacent to the litter bags).
Page 4
Task 5: Litter Layer
-------
TASK 6: ROOT GROWTH AND PHENOLOGY
-------
Experimental Tasks and Facilities
TASK 6: ROOT GROWTH AND PHENOLOGY
PARTICIPANTS: Paul Rygiewicz (EPA), Mark Johnson (METI) - Principal Investigators
David Tingey (EPA), Marj Storm (METI)
OBJECTIVES:
To quantify root growth with numbers of
roots produced; their distribution and turn-
over, and the total weight of the standing
stock of roots under elevated CO2 and
climate change.
To quantify dynamics of root production,
development, and mortality under elevated
CO2 and climate change, and determine the
effects on root allometnes, i.e., distribution
of biomass among coarse roots,
nonmycorrhizal fine roots, and mycorrhizae.
To characterize effects of elevated CO2 and
climate change on root phenology.
To quantify biochemical partitioning of C
between structural and nonstructural com-
pounds in the various root fractions.
APPROACH:
Initial (To) Seedlings
Prior to planting the Terracosms, large lysimeters,
and Cascade field sites, a subset of seedlings were
randomly selected for physical and chemical analy-
ses to characterize the initial condition of the
seedlings. The root systems of these seedlings,
known as the To seedlings, were removed and
separated into three groups for specific kinds of
analyses The root tips from one group were
removed and morphotyped. The roots in the
second group were separated by diameter, dried,
weighed and ground, and will be used for chemi-
cal and biochemical analyses. The roots in the
third group were separated by diameter and their
total length measured.
Cores-to-depth
Two times a year a full profile soil core (core-to-
depth) is collected in each Terracosm. The soil
core is collected by horizon (A, B1, B2, and C) and
the roots are separated from each soil horizon
sample using sieves and hand-picking. Within 24
hours of collection the mycorrhizal and
nonmycorrhizal root tips are removed for
morphotyping and DNA fingerprinting (see Task
7 in this Booklet for more information). The
remaining root material is sorted into 4 size cat-
egories based upon root diameter (0.0-0.5 mm,
0.5-1.0 mm, 1.0-2.0 mm, and roots greater than
2.0 mm). The length, fresh weight and dry weight
of each root fraction are measured. The dry root
samples are ground and chemically analyzed (nu-
trients and biochemistry).
Minirhizotrons
In situ root and mycorrhizal observations are made
in the Terracosms using a minirhizotron camera
system. Root observations are recorded on video
tapes and root data are extracted from these tapes.
This technique provides a powerful tool for docu-
menting root dynamics because repeated root ob-
servations are made over the course of the study
and the growth and life history of individual roots
can be observed and quantified.
There are four minirhizotron tubes in each Terra-
cosm (see Figure 1), with one minirhizotron tube
in each soil horizon ( A, Bi, B2, and C). The
minirhizotron tubes have a horizontal orientation
and are located such that the top of each tube is in
the middle of the respective soil horizon. Root
images are collected on the uppermost surface
Page I
Task 6 Root Growth and Phenolohy
-------
Experimental Tasks and Facilities
(1 e., the minirhizotron camera view is in an up-
ward direction) of the minirhizotron tubes. Root
images are collected every four weeks, or 13 times
a year. The image collection events coincide with
soil gas analysis and soil solution collection.
Collecting these data at the same time will provide
information that may be useful for interpreting the
images collected in the rrunirhizotrons
Root data is extracted from the video tapes using
a software program called "ROOTS." The ROOTS
software was developed at Michigan State Uni-
versity and has been described by Hendnck and
Pregitzer (1992). With ROOTS, video images are
digitized and a mouse is used to trace out various
features (length and width). ROOTS creates a
database that includes root classification codes
and information about the image.
STATUS/RESULTS
Initial (To) Seedlings
The root tip results for the To seedlings are pre-
sented in Task 7 section of this booklet. The root
To length and weight data are reported in Table 1.
The greatest mass of roots was in the 0.5 -1.0 mm
category. More than a third of the total root mass
was in this category. The total root length mea-
sured on 16 seedlings was more than 220 meters.
More than half of the total root length was in the
0.5 - 1.0 mm category and more than 90% of the
total root length was due to roots less than 1.0 mm
in diameter.
Cores-to-depth
The first cores-to-depth were collected in October
1993 and the second set were collected in April
1994. The roots from the October coring have
sorted and classified. The weight and length data
have not been compiled Some of the preliminary
mycorrhizal tip results are reported in Task 7
section of this booklet.
Minirhizotrons
Root image collection began the week of July 13,
1993 and images have been collected every four
weeks since then. Each time images are recorded
more than 3,200 image frames are recorded. Over
a year, or 13 collection events, more than 42,000
total image frames will be collected. We have
observed roots, mycorrhizae, fungal hyphae, and
mesofauna in the video tapes.
Data extraction from the video tapes using the
ROOTS program has not started yet, but will
begin this Summer. Before extraction could start
we developed a scheme for classifying roots,
mycorrhizae and fungal hyphae in the Douglas-fir
system. This scheme is based upon the video
images that were collected in the Terracosms in
the Summer and Fall of 1993. The resulting
classifications, along with their codes and a brief
description of each classification are presented in
Table 2. We are planning to modify the ROOTS
software to accommodate our long-term, multi-
year study
REFERENCES:
Hendnck, R.L. and K.S. Pregitzer. 1992. The
demography of fine roots in a northern
hardwood forest. Ecology 73:1094-1104.
Page 2
Task 6 Root Growth and Phenology
-------
Experimental Tasks and Facilities
Camera
Monitor
Side View of Terracosm
Microphone
Camera Control Unit
Indexing Handle
Root
Observation
Tubes
Tube*1: A-Horizon
Tube *2: B^Horizon
Tube #3: B2-Horizon
Tube *4: C-Horizon
Figure 1: General layout of minirhizotron tubes in the Terracosms and set-up of minirhizotron camera
system.
Table 1. Root length and dry weight by root diameter category of To seedlings from lots planted in the Terracosms,
large lysimeters, and Cascade field sites.
Parameter
Root Diameter Categories (mm)
Totals
0.0 - 0.5
0.5 - 1.0
1.0-2.0
>2
Weight 1 (g)
41.0
63.7
38.2
44.5
187.4
% of Total
21.9
34.0
20.4
23.7
100.0
Length^ (m)
75.0
123.6
19.0
3.2
220.8
% of Total
14.0
56.0
8.6
1.4
1000
'number of seedlings used to obtain root weight data = 42
^number of seedlings used to obtain root length data = 16
Page 3
Task 6: Root Growth and Phenolohy
-------
Experimental Tasks and Facilities
Table 2 Root, Mycorrhizae and Fungal Hyphae Classification and Codes
ROOT CLASSIFICATION AND CODES
Code
Classification
Description
C
D
E
G
H
I
K
L
M
N
O
New white roots
New white roots with root hairs
White roots
White roots with root hairs
New tan roots
New tan roots with root hairs
Tan roots
Tan roots with root hairs
New brown roots
New brown roots with root hairs
Brown roots
Brown roots with root hairs
Missing roots
Woody roots
Decaying roots
Roots which were not previously present and which are predominately white in
color, without visible root hairs
Roots which were not previously present and which are predominately white in
color, with visible root hairs
Roots which are predominately white in color, without visible root hairs
Roots which are predominately white in color, with visible root hairs
Roots which were not previously present and which are predominately tan in
color, without visible root hairs
Roots which were not previously present and which are predominately tan in
color, with visible root hairs
Roots which are predominately tan in color, without visible root hairs
Roots which are predominately tan in color, with visible root hairs
Roots which were not previously present and which are predominately brown
in color, without visible root hairs
Roots which were not previously present and which are predominately brown
in color, with visible root hairs
Roots which are predominately brown in color, without visible root hairs
Roots which are predominately brown in color, with visible root hairs
Point label that identifies roots that were classified in one of the root categories
in an image from a previous sampling but are no longer visible
Roots that show secondary thickening and growth
Roots that show obvious signs of decay These include loss of root margin
integrity, holes or spots of decay, signs of grazing by soil fauna, or loss
(dieback) of portions of the root
Table 2. is continued on Page 5
Page 4
Task 6 Root Growth and Phenology
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Experimental Tasks and Facilities
Table 2 , continued
MYCORRHIZAE CLASSIFICATION AND CODES
Code
Classification
Description
P
1-5 Mycorrhizal root tips
Point label to identify 1-5 mycorrhizal root tips per root
Q
6-10 Mycorrhizal root tips
Point label to identify 6-10 mycorrhizal root tips per root
R
>10 Mycorrhizal root tips
Point label to identify >10 mycorrhizal root tips per root
S
Missing mycorrhizae
Point label that identifies mycorrhizal root tips that were classified in one of
three mycorrhizal categories in an image from a previous sampling but are no
longer visible
FUNGAL HYPHAE CLASSIFICATION AND CODES
Code
Classification
Description
T
Fungal hyphae w/roots <25%
Point label that indicates the presence of fungal hyphae in apparent contact with
a root or roots, present in 25% or less of the screen
U
Fungal hyphae w/roots
>25%
Point label that indicates the presence of fungal hyphae in apparent contact with
a root or roots, present in more than 25% of the screen
V
Fungal hyphae w/o roots<25%
Point label thai indicates the presence of fungal hyphae not in apparent contact
with a root or roots, present in 25% or less of the screen
W
Fungal hyphae w/o roots>25%
Point label that indicates the presence of fungal hyphae not in apparent contact
with a root or roots, present in 25% or more of the screen
X
Fungal hyphae with mycorrhizae
Point label that indicates the presence of fungal hyphae in apparent contact with
mycorrhizal root tips
Y
Missing fungal hyphae
Point label that indicates the loss of previously identified fungal hyphae
z
1-5 Rhizomorphs
Point label to identify the presence of 0-5 rhizomorphs per screen
i
6-10 Rhizomorphs
Point label to identify the presence of 5-10 rhizomorphs per screen
2
>10 Rhizomorphs
Point label to identify the presence of >10 rhizomorphs per screen
3
Missing Rhizomorphs
Point label that indicates the loss of previously identified rhizomorphs
Page 5
Task 6 Root Growth and Phenolohy
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TASK 7: SOIL BIOLOGY
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Experimental Teaks and Facilities
TASK 7 A: SOIL BIOLOGY - MICROBIAL BIOMASS, AND PROTO-
ZOAN AND NEMATODE NUMBERS
PARTICIPANTS: Paul Rygiewicz (EPA), Mark Johnson (METI) - Principal Investigators
Elaine Ingham (OSU)
Tuk 7: Sea Biology
-------
Experimental Tasks and Facilities
TASK 7 A: SOIL BIOLOGY - MICROBIAL BIOMASS, AND PROTO-
ZOAN AND NEMATODE NUMBERS
PARTICIPANTS: Paul Rygiewicz (EPA), Mark Johnson (METI) - Principal Investigators
Elaine Ingham (OSU)
OBJECTIVES:
To quantify the effects of elevated CO2 and
climate change on total and active soil micro-
bial populations (bacteria and fungi), nema-
todecommunity structure, and protozoan popu-
lations
APPROACH:
Sampling
Forest soils: Three soil samples from each depth
(O, A, B, and C horizons) were obtained from the
high elevation Cascade field site where the soil
was removed for placing into the Terracosms
(Summer 1993). Another set of three cores was
taken in the Fall 1993, approximately one week
before soil cores were taken from the Terracosms.
Soils were transported to the lab and analyses (see
below) began within one day of field collection.
Terracosm sampling: In October 1993, one soil
core was removed from each Terracosm, split into
A, the upper half of the B (B i), the lower half of
the B (B2), and the C horizons. The soils were
sieved and subsamples for microbial and nema-
tode analyses placed into zip-lock plastic bags.
These bags were transported to the lab and analy-
ses began within 18 hours of sampling (see be-
low).
Mass Determinations/Enumeration
Active and total bacterial and fungal biomass:
Active bacterial and fungal biomass were esti-
mated by determining the numbers, diameters and
lengths of fluorescein diacetate (FDA)-stained
bacteria, and length and diameter of FDA-stained
hyphae, in soil-agar films using epifluorescent
microscopy (Ingham and Klein, 1984). Total
fungal biomass was estimated by determining
length and diameter of all hyphae in soil-agar
films using phase contrast microscopy. Total
bacterial biomass was estimated by determining
numbers and size of fluorescein isothiocyanate
(FITC)-stained bacteria on membrane filters
(Babiuk and Paul 1977). Bacterial and fungal
volumes were converted to biomass using an av-
erage bacterial density of0.33gcm"^, and average
hyphal density of 0.41 g cm"^ (Van Veen and Paul
1979, Van Veen et al. 1987).
Protozoa: Numbers of protozoa were determined
by examining 10-fold dilutions of soil incubated
in 24-well microliter plates according to the tech-
nique of Darbyshire et al. (1974). Conversion of
numbers to biomass was based on the following
equivalents: 10"g per flagellate; 10"^ g per
amoeba, and 10"** g per ciliate (Clarholm, 1981).
Nematodes: Nematodes were isolated by extract-
ing 15 to 20 g of soil in Baermann extraction
funnels (Anderson and Coleman 1978), then
counted and identified using a Zeiss dissecting
microscope and Olympus DIC microscope, re-
spectively.
Statistical comparisons
Statistical comparisons (ANOVA, correlation
coefficients) were performed using the Statistical
Package for the Social Sciences (SPSS; Nie et al.
1980). Analysis of variance was performed using
treatment and site as main effects. Only those
main effects or interactions between main effects
indicated as significant by the F test at P < 0.05
were considered. For organism values, the mean
separation tests used were either Least Significant
Page ]
Task 7 Soil Biology
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Experimental Tasks and Facilities
Differences (LSD) calculated for a significance
level of P < 0.01, or standard deviations (SD).
STATUS/RESULTS:
Soil Moisture:
Soil moisture at the Cascade field sites compared
with the Terracosms was slightly, but not signifi-
cantly, greater in the Fall 1993. Soil moisture in
the A horizon in the field was 0.42 grams of water
per gram fresh soil (SD 0.13) while the A horizon
in the Terracosms contained, on average, 0.23
grams of water per gram fresh soil (SD 0.14). For
both the field and Terracosms, soils contained
more water as depth increased, generally increas-
ing to between 0.30 and 0 36 grams water per gram
fresh soil in the C horizon.
Seasonal comparison
Comparison of organism numbers for soil in place
in the forest during mid-Summer 1993 with num-
bers for soil in the Terracosms in Fall 1993 showed
that bacterial activity remained about the same
(Table 1), although an increase between summer
and fall is generally expected based on seasonal
change Variability was decreased in the Terra-
cosms compared with field values.
Total bacterial numbers and their variability de-
creased by about an order of magnitude, indicating
some adverse effect of moving the soil from the
field to the Terracosms (Table 1).
Analysis of protozoan numbers has not been com-
pleted for the Terracosms.
Active fungal biomass increased sjightly in the
Terracosms, as expected, between the summer
and fall (Table 1). Total fungal biomass did not
change significantly when soil was moved from
the field into the Terracosms, although, as with
bacteria, variability decreased in the Terracosms
compared with the field.
Nematode numbers decreased slightly but not
significantly with the disturbance to the soil (Table
1), and no overall change in nematode community
composition was observed.
Field site and Terracosm organism numbers
and activity, fall 1993
Comparison of A horizon values for the fall samples
collected from the Cascade field plots and the
Terracosms (Table 2) shows that increases in
activity occurred between the summer and fall
samples, while total biomass remained about the
same (cf. Tables 1 and 2).
Comparison of samples from the field plots with
Terracosm samples showed that total and active
bacteria were still lower in the Terracosms, while
total and active fungal biomass, and total nema-
tode numbers were not different (Table 2).
Climate treatment effects
Elevated temperature and elevated CO2 treat-
ments showed significant reductions in, active
fungi, total fungi and nematodes compared with
the ambient treatment (Table 2).
DISCUSSION:
Lower active and total bacterial biomass occurred
in the Terracosm soils, most likely the result of
reduced substrate input from trees during the trans-
fer of the soil to the Terracosms and as a result of
the loss of understory plants in the Terracosms.
There was no significant difference in active or
total fungal biomass, or total nematode numbers
between the forest soils and the Terracosms, pos-
sibly because these organisms can utilize the more
recalcitrant carbon compounds in the soil and are
not as dependent on constant plant inputs to sus-
tain their biomass. Certainly the connection be-
tween the ectomycorrhizal fungal component in
the bulk soil and the host trees was lost when the
soil was moved, but it has been suggested that
these fungi can exist without the host for a limited
period of time, which appeared to be the case.
Page 2
Task 7 Soil Biology
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Experimental Tasks and Facilities
In general, the ambient Terracosm treatments
showed reduced bacterial numbers, but similar
fungal and nematode numbers compared with the
field plots. However, some climate effects could
be observed within these first few months. The
question remains whether these are transient ef-
fects with numbers quickly returning to ambient
levels, or whether these differences will be magni-
fied in months to come as the climate treatments
persist.
Reductions in fungal biomass in the elevated tem-
perature treatment suggest it will be important to
continue to monitor the effects of global climate
change on the soil organism component. If fungal
biomass continues to decrease in the Terracosms,
survival of the Douglas-fir seedlings may be in
jeopardy, since shifts in fungal-to-bacterial biom-
ass ratios to favor bacteria have been shown to
result in conifer regeneration difficulties (Rose
1988, Perry et al. 1989, Ingham in Mlot, 1993,
Colinas et al. 1994). If these biomass ratios
continue to shift in the Terracosms, some effort
will be required to encourage the appropriate
fungal biomass to develop. However, the shift
observed so far is typical of what occurs after soil
disturbance of this magnitude, and quite often,
forest ecosystems recover through internal pro-
cesses. Unfortunately, we don't fully understand
those internal processes, so at this time our best
effort is to monitor changes. If the shift continues,
indicating a serious problem is developing, then
an effort might be needed to determine how to alter
the soil community to return the soil a fungal-
dominated system.
In general, there was less variability in microbial
and microfauna data of the Terracosms than of the
field Other work on spatial heterogeneity within
forest soils suggests that the field site sampling
must take into account distance from Douglas fir
trees, from grass patches, from rhododendron
patches, and other types of vegetation, thickness
of bryophyte layers, distance from downed woody
debris and so forth in order to reduce the variabil-
ity observed in these samples. Future soil sam-
pling at the three elevational plots will follow
similar tree-to-tree spatial considerations as is
followed for the Terracosms. In future sampling
in the forest surrounding the elevational plots,
greater efforts will be taken to control for these
factors, and to choose sampling points similar
distances from saplings as is done in the Terra-
cosms.
REFERENCES:
Anderson, R. V. and D.C. Coleman. 1977. The use
of glass microbeads in ecological experiments
withbactenophagic nematodes. J Nematol. 9:319-
322.
Babiuk, L.A. and Paul, E.A. 1970. The use of
fluorescein isothiocyanate in the determination of
the bacterial biomass of a grassland soil. Canadian
Journal of Microbiology 16: 57-62.
Clarholm, M. 1981. Protozoan grazing of bacteria
in soil - impact and importance. Microbial Ecol-
ogy 7: 343-350.
Colinas, C., E. Ingham and R. Molina. 1994.
Population responses of target and non-target for-
est soil organisms to selected biocides. Soil Biol.
Biochem. 26: 41-48.
Darbyshire J.F., Wheatley, R.E., Greaves, M.P.
and Inkson, R.H.E. 1974. A rapid rrucromethod
for estimating bacterial and protozoan popula-
tions in soil. Ecology 61: 764-771.
Ingham, E. R. in Mlot, C. 1993. Clearcutting's soil
effects. 1993. Science 262: 116.
Ingham, E.R. and D.A. Klein. 1984. Soil fungi:
Relationships between hyphal activity and stain-
ing with fluorescein diacetate. Soil Biol. Biochem.
16: 273-278.
Page 3
Task 7 Soil Biology
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Experimental Tasks and Facilities
Nie.N.H ,C.H Hull, J G Jenkins,K.Steinbrenner
and D.H Brent. 1975 SPSS: Statistical Package
for the Social Sciences, 2"d ed. McGraw-Hill,
NY
Perry, D.A., M.P. Amaranthus, J.G. Borchers,
S.L. Borchers and R.E. Brainerd. 1989.
Bootstrapping in ecosystems. Bioscience 39 (4):
230-237.
Rose, S.L. 1988 Above and belowground com-
munity development in a marine sand dune eco-
system Plant and Soil 109: 215-226.
Van Veen, J A., Ladd, J N , Martin, J.K. and
Amato, M 1987 Turnover of carbon, nitrogen
and phosphorus through the microbial biomass in
soils incubated with ^C-, ^N- and ^^P-labelled
bacterial cells. Soil Biology and Biochemistry 19:
559-565.
Van Veen, J.A. and Paul, E.A. 1979. Conversion
of biovolume measurements of soil organisms,
grown under various moisture tensions, to biom-
ass and their nutrient content. Applied and Envi-
ronmental Microbiology 37: 686-692.
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Task 7 Soil Biology
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Experimental Tasks and Facilities
Table 1 T otal biomass (p.g g~ ^), numbers g" * and active biomass (ng g" ^) of dry soil food web organism
groups: forest soil sampled in mid-Summer 1993, and Terracosm soils sampled in October 1993.
Organism Group
Forest Soil
Horizon
A
B
C
Terracosm Ambient Treatment
Horizon
A B C
Active Bacteria
3.55
4.49
3.61
1.38
2.04
1.49
(2.11)
(2.25)
(1.55)
(1.0)
(1.30)
(1-43)
Total Bacteria
61.6
60.3
53.7
5.47
4.96
4.58
(33.4)
(30 3)
(34.6)
(2.25)
(1.59)
(1.06)
Active Fungi
0.86
0.04
0.01
1 31
0.24
0.07
(0.72)
(0.03)
(0.03)
(0.70)
(0.63)
(0.67)
Total Fungi
1903
133
242
946
280
266
(1681)
(128)
(134)
(280)
(60)
(54)
Protozoa
Flagellates
0.04
ND
ND
ND
ND
ND
(0.23)
Amoebae
0.27
ND
ND
ND
ND
ND
(0.25)
Ciliates
0.02
ND
ND
ND
ND
ND
(0.05)
Total Nematodes
5.6
2.2
1.6
1.26
0.53
0.50
(5.0)
(1.3)
(1.0)
(0.59)
(0.50)
(0.83)
Values in parentheses are standard deviations of the mean.
Page 5
Task 7 Soil Biology
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Experimental Tasks and Facilities
Table 2 Numbers and activity of soil foodweb organism groups in A horizon in the Cascade forest
soil, and the same forest soil placed in the Terracosm in June 1993; sampling of both soils took place
in October 1993
Terracosm Treatment
Organism GrouD
Forest Soil
Tt
ct
tcT
Ambient
Active Bacteria
44.5
2.20
2.80
4.03
1.38
(25 2)
(1.03)
(1.80)
(2.35)
(1.00)
Total Bacteria
105
3 67
3.73
5.50
5.47
(74.5)
(1.80)
(2.03)
(3.00)
(2.25)
Active Fungi
25 6
0.42
2.07
0.74
1.31
(35.5)
(0.31)
(1.11)
(0.35)
(0.70)
Total Fungi
4001
340
604
378
946
(2905)
(225)
(267)
(108)
(280)
Total Nematodes
8.80
0.64
081
1 47
1.26
(7.90)
(0 26)
(0 05)
(0 10)
(0.59)
Values in parentheses are standard deviations of the mean.
Page 6
Task 7 Soil Biology
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Experimental Task and Facilities
TASK 7 B: SOIL BIOLOGY- ECTOMYCORRHIZAE COLONIZATION
AND DIVERSITY
PARTICIPANTS: Paul Rygiewicz (EPA), Mark Johnson (MET!) - Principal Investigators
Ken Cullings (OSU) - Molecular Ecology of Mycorrhizae
Elaine Ingham (OSU) - Mycorrhizae Colonization & Morphotyping
Amy Tuininga (OSU) - Mycorrhizae Colonization & Morphotyping
Task7: Soil Biology
-------
Experimental Tasks and Facilities
TASK 7 B: SOIL BIOLOGY- ECTOMYCORRHIZAE COLONIZATION
AND DIVERSITY
PARTICIPANTS: Paul Rygiewicz (EPA), Mark Johnson (METI) - Principal Investigators
Ken Cullings (OSU) - Molecular Ecology of Mycorrhizae
Elaine Ingham (OSU) - Mycorrhizae Colonization & Morphotyping
Amy Tuininga (OSU) - Mycorrhizae Colonization & Morphotyping
OBJECTIVE:
To quantify the effects of elevated CO2 and
climate change on the colonization of roots by
mycorrhizal fungi, and on the diversity of
mycorrhizal fungi colonizing roots
APPROACH:
Colonization
A scheme was designed to subsample the roots of
whole seedlings (e.g , To) for mycorrhizal coloni-
zation, and mycorrhizae morphotyping. The root
system of each seedling was washed, and then five
secondary roots were randomly selected from the
primary root One of the secondary roots was laid
out on a plastic sheet with nested grids, and a grid
was randomly selected. The number of root and
mycorrhizal tips (no distinction about mycorrhizal
status was noted) within a selected grid was
counted. If twenty or fewer tips were found, all
tips in the grid were selected. For grids containing
more than twenty tips, a sub-grid (sub-grid equals
one-fourth of a grid) was randomly selected. If
twenty or less tips were counted (which was al-
ways the case), all the tips in the sub-grid were
collected. The process was repeated for the next
secondary root, and so on for the remaining three
secondary roots The whole process was then
repeated with the first secondary root until 120 tips
were collected.
All tips were taken for analysis from each depth
segment of the Fall 1993 Terracosm cores because
so few tips were encountered. We anticipate that
all tips will be selected from each depth segment
of the Spring 1994 cores. A variant of the
subsampling method will be used when the num-
ber of tips in each depth segment gets beyond a
few hundred. Our goal is to analyze a maximum
of 120 tips from each of the depth segments.
Percent mycorrhizal colonization is assessed on
all the 120 tips selected. First, the root systems
were washed for 10 min. in tap water in a 45 mesh
soil sieve and inspected under the dissecting scope
to be sure that all soil was washed off (excluding
small clay particles imbedded in the mycorrhizal
hyphae). If not, they were washed for an addi-
tional 5 min. (10 minutes was generally suffi-
cient). Mycorrhizal status was determined using
non-destructive methods (e.g., dissecting micro-
scope) to observe gross moiphological character-
istics. Diagnostic features, using traits described
by Agerer (1987), Agerer (1991) and Ingleby et al.
(1990), included (diagnostic # 5 alone would
suffice to indicate a mycorrhizae, diagnostics 1-4
would need to be present in conjunction with one
or more other diagnostics to indicate a mycor-
rhizae):
1)absence of root hairs (but not as only
diagnostic used)
2) swelling (but not as only diagnostic
used)
3) branching pattern (but not as only diag
nostic used),
4) coloration (but not as only diagnostic
used), and
5) presence of mantle.
These gross morphological traits were considered
Page 7
Task 7. Soil Biology
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Experimental Tasks and Facilities
indicative of putative and confirmed mycorrhizal
colonization (depending on morphotype) after
preliminary or concurrent work was done using
squash mounts or cross-sections and light micros-
copy
Biodiversity
Traditionally, specific identification of a mycor-
rhizal fungus on a given host plant has been
accomplished by morphotyping (gross morpho-
logical features), the rationale being that a given
fungus will impose a unique morphology upon a
given plant root. However, this technique has two
major drawbacks (Harley and Smith 1983).
1) most morphotypes cannot be linked to a
single known fungal species, thus identi-
fications are often limited to descriptions
such as "the brown, bifurcate type on
spruce", and, therefore,
2) it is not known how the genetic
and species diversities of fungi
forming a single morphotype are
represented
These problems can be illustrated and overcome
using recently-developed molecular methods in-
volving the Polymerase Chain Reaction (PCR)
(Bruns and Gardes 1993). First, DNA from an
individual specimen of a morphotype is isolated.
Then, PCR is used to amplify a specific portion of
the fungal DNAs that are in the DNA preparation
(Gardes and Bruns 1993). Next, the amplified
DNAs are digested with appropriate restriction
enzymes, and the digested DNAs are subjected to
gel electrophoresis. Finally, the resultant finger-
prints (RFLPs - restriction fragment length poly-
morphisms) are compared to determine if the
fungus of all "replicates" of a morphotype is the
same (Gardes et al 1990; Cullings 1993). Spe-
cific taxonomic identifications are made by di-
rectly sequencing the DNAs and identifying the
fungi in question to family phylogenetically
(Cullings 1993)
A recent study which approached the mycorrhizal
biodiversity issue along molecular lines can serve
to illustrate the usefulness of these approaches.
DNAs from several "replicate" mycorrhizae of a
single morphotype of Iodgepole pine roots col-
lected in Yellowstone National Park were sub-
jected to these analyses Results indicate four
distinct RFLP patterns within the single
morphotype (Y1 through Y4, Figure 1). Subse-
quent sequencing of the DNAs indicates that the
fungi forming the four "replicate" mycorrhizae of
this single morphotype belong to three fungal
families (Figure 2). Thus, morphotyping is not
always a reliable means of identifying fungi in-
volved in specific symbioses. The results indicate
the need to utilize and expand current PCR meth-
ods available for mycorrhizal fungal identifica-
tion
While assessing mycorrhizal colonization rates,
the mycorrhizal morphotypes were categorized
and placed into microtiter plate wells, dried and
passed on for molecular analysis. All "replicates"
(but no more than ten) of each morphotype were
subjected to the molecular analysis
STATUS/RESULTS:
Colonization
A book (guide) illustrating the various mycor-
rhizal morphotypes with photographs and notes
on morphology is being constructed to aid in
categorizing the morphotypes. All confirmed and
putative mycorrhizae morphotypes will be in-
cluded in the guide
We assessed the colonization rates of twelve To
seedlings. There is one confirmed (based on
microscopy) black/white morphotype (approx.
31 % of tips colonized by this morphotype) and we
suspect it to be a Rhizopogon sp. There is one
putative morphotype (approx. 45% colonization
rate), and it is branched but does not exhibit strong
Hartig net and mantle development. We have not
made any attempts to place its taxonomy. There-
Page 8
Task 7 Soil Biology
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Experimental Tasks and Facilities
fore, root colonization rates by mycorrhizal fungi
can range greatly depending on whether the puta-
tive mycorrhizal morphotypes are included, from
31% to 76%, leaving 24% of the tips non-colo-
nized. Work to understand this morphotype is
progressing; see Biodiversity section immediately
below.
Distribution of mycorrhizal root tips and mycor-
rhizal colonization rates from the Fall 1993 coring
event of the Terracosms have been calculated
(Table 1; values include the putative mycorrhizal
morphotype) Variability in the root occupation
and mycorrhizal colonization data are high. There
appears to be a trend of a greater number of
mycorrhizal root tips in the unchambered and
ambient Terracosms compared with the altered
climate treatments. Mycorrhizal colonization rates
among treatments are similar as are the standard
deviations and very high. Little can be surmised
from the colonization data.
More mycorrhizal tips and a higher percent of
mycorrhizal colonization were found in the A and
Bj soil layers compared with deeper soil seg-
ments. Also, root occupation and mycorrhizal
colonization rates appeared to decreased within
the B horizon (cf. B i and B2 data).
Biodiversity
A molecular biology lab has been established in
the project with ail necessary equipment to deter-
mine the molecular identity of the fungal and host
components of mycorrhizae. Training sessions
have begun for personnel.
Molecular analysis has begun on To seedlings
(seedlings coming from the nursery, before plant-
ing them into the Terracosms or field). The
seedlings exhibited a single, confirmed mycor-
rhizal morphotype, and some putative mycor-
rhizal morphotypes. In initial work we isolated
DNAs from four "replicates" of the confirmed
mycorrhizal morphotype, and amplified the fun-
gal DNAs from the Internal Transcribed Spacer
region of the nuclear nbosomal RNA (nrRNA)
repeat unit using PCR. Amplified DNAs were
digested with two restriction enzymes (Figure 3).
Only one genetic type was indicated by the band-
ing pattern of the Hinf I digest. However, the four
"replicates" can be placed into two genetic types
using the banding pattern of the Alu I digest. Thus
using, two digestions we can say that these four
"replicates" of the morphotype are actually formed
by at least two organisms. We can not say for sure
how the organisms differ, but the consensus-at-
large that is forming would place the two organ-
isms in at least two species of the genu s Rhizopogon.
The Rhizopogon designation is based on the
strength of believing the validity of using gross
morphological characteristics to place the tips into
morphotype classifications. Actual taxonomic
designations of the two organisms requires the
DNAs be sequenced, and the sequences compared
with data of fungi available in published data
bases. In the future, morphotypes sharing identi-
cal RFLP patterns for three enzymes will be con-
sidered as being formed by the same fungal spe-
cies.
We are continuing with the initial molecular work
on the confirmed mycorrhizal morphotype from
the To seedlings. DNAs of many more "repli-
cates" of the morphotype will be digested and
analyzed in order to establish a relationship be-
tween the number of "replicates" analyzed and the
amount of genetic diversity encountered in the
morphotype designation (i.e., a diversity versus
number plot). The relationship will help us deter-
mine how many "replicates" of the morphotypes
we will need to analyze for genetic identity to
evaluate how the diversity of the mycorrhizal
fungal community is changing under altered cli-
mates. We wish to:
1) know how the various morphotypes
increase and decrease in relative abun-
dance due to climate change, and
2) make estimates on how the genetic
Page 9
Task 7 Soil Biology
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Experimental Tasks and Facilities
diversity of the morphotypes will change
due to altered chemical and physical
environments in the rhizosphere and bulk
soil.
Work on the putative mycorrhizal morphotypes is
continuing along several lines additional micros-
copy analysis, selective PCR amplification of
DNAs from the fungus and plant components, and
plating of morphotypes in an attempt to isolate the
mycobiont(s) into pure culture. The additional
work is needed in the event that the relative pres-
ence of these putative mycorrhizal morphotypes
increases as the experiment proceeds.
REFERENCES:
Agerer, R. 1987. Colour Atlas of Ectomycor-
rhizae. Einhorn-Verlag Eduard Dietenberger
GmbH. Munich.
Agerer, R. 1991. Characterization of Ectomy-
corrhiza in Methods in Microbiology, Vol. 23, pp.
25-73.
BrunsT.D. and M. Gardes 1993. Molecular tools
for the identification of ectomycorrhizal fungi:
taxon-specific oligonucleotide probes for suilloid
fungi Molecular Ecology (In press).
Cullings, K W. 1993. Doctoral dissertation, Uni-
versity of California, Berkeley.
Gardes, M.J., A. Fortin, T.J. White, T.D. Bruns
and J.W. Taylor 1990. Identification of indig-
enous and introduced mycorrhizal fungi by ampli-
fication of the internal transcribed spacer. Can. J.
Bot. 69:180-190.
Gardes, M.J and T.D. Bruns. 1993. ITS primers
with enhanced specificity for higher fungi and
basidiomycetes: application to identification of
mycorrhizae and rusts. Molecular Ecology
2(2)-115-118.
Harley, J.L. and S E. Smith. 1983. Mycorrhizal
Symbiosis. Academic Press, London.
Ingleby, K., P.A Mason, F.T. Last and L.V.
Fleming. 1990. Identification of ectomycor-
rhizas Institute of Terrestrial Ecology. Natural
Environment Research Council. London: HMSO.
Page io
Task 7 Soil Biology
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Experimental Tasks and Facilities
Table 1 Number of mycorrhizal root tips and percent mycorrhizal colonization of confirmed and putative
morphotypes per soil depth segment for theTerracosms cores, October 1993 Values aremean of three Terracosms
per treatment (one core per Terracosm), except unchambered treatment which is mean of two Terracosms Values
in parentheses are standard deviations of the mean
Horizon
A Si B2 _C Mean
NUMBER OF MYCORRHIZAL ROOT TIPS
Unchambered 157 (206) 17 (5) 36 (6.4) 7.6 (7.8) 54 (69)
Ambient 148 (246) 160 (243) 6.7 (0.18) 3.3 (0.03) 80 (86)
Ct 12 (12) 25 (19) 0.7 (1.2) 0 (0.0) 13 (12)
Tt 7 (8.1) 99 (126) 3 (0.02) 1.7 (2.9) 28 (48)
CTt 43 (61) 57 (31) 0.3 (0.6) 0 (0.0) 25 (29)
Mean 73 (74) 72 (59) 9 (15) 2 (3)
PERCENT MYCORRHIZAL COLONIZATION
Mycorrhizal tips
Unchambered 44 (0 44) 10 (0.07) 27 (0.27) 8 (0.10) 22 (17)
Ambient 27 (0.2) 57 (0.21) 10 (0.18) 2 (0.03) 24 (24)
CT 35 (0.39) 62 (0.39) 2 (0.03) 0 (0 0) 25 (30)
Tt 34 (0.57) 63 (0.55) 1 (0.02) 1 (0.01) 25 (30)
CT? 28 (0.22) 70 (0.23) 1 (0.01) 0 (0.0) 25 (33)
Mean 34 (6.8) 53 (24) 8.2 (11) 2.2 (3.4)
Page 11
Task 7 Soil Biology
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Experimental Tasks and Facilities
SM Y1 Y2 Y3
:Miiu:
wmm
is?
Figure 1. Depiction of Mbol restriction enzyme digest of DNAs amplified from the Internal Transcribed
Spacer (ITS) of four root tips (Y1 through Y4; SM= molecular weight size marker, number of base pairs
indicated to the left of the gel depiction) from a single mycorrhizal morphotype on roots collected from
a mixed lodgepole/white bark pine forest in Yellowstone National Park.
Page 12
Task 7: Soil Biology
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Experimental Tasks and Facilities
' Patillus atroiomentosus
PaxtUus statuuni
Cotnoptiorapuieana
PhAeog\roporus portetuosus
C\ rodon niemhotdes
C\roporus c\anescens
Pisahthus (inctonus
-c
' H\gropftoropsis aurantianca
Merultopsts tncrasMia
¦J Serputo funvwntiotdes
^ Tapttttlla panuoides
Chroogomphus i micolor
4 M hypos 3 12 92
I Rhizopogon subcatndesctns
~V el lows tone 2
Compfiiritus gluttnosus
Melanogastrr tubffomus
Smllus smuipaultanus
Suillus coupes
Yellowstone 1
Boletus ptperaius
suilloid group
Boletus satanus
Boletus \idinfUnus
Xerocontus Chr\stnieron
Ph\lloporus rhodotanthus
Paragsrodon sphaerospons
PaxiUus imwlutus
H\grophorus hspothtjus
Yellowstone
r- C tubae/pr
C cinnatxir
Ccibanus
Hygrophoreceae
Sarcodes
ConthareUaceQe
Sarcodes s4
mPanus Conthatui
Poiidnr^f* i niottlOJirt
ThelepharA terresirts
Bolriopsis subsquamosa
Thelephora
Bcleiopsis
¦ Bonderze * i
r Thelef.
_P- Bolri
"1r'
I Bole
£
r Lactanus \otemus
"!(" M uniflora
Russula rosacea
- Yellowstone 4
Lactanus ptperaius
M uniflora 0023
Russula lauroctrasi
Russulaceae
Albatrellus ellisn
Albrtrellurellus
Macroitptoia rachodes
Agarxus bnutnescens
Amanita francheiil4l7
Pluieus cen hius
Hemitomes spoint
I Con man u»toiaceous
Anrultanaalbonanl404
LeucopaxtUtu gennaneus
' TncolomA pardtnum
Asterophora hcopcrdotdts
Tncoloma fla\t\trl 395
Bolbmus vitelhnus
EniolomA sertceum
Inonbf soronal427
Nematoioma auranitaco
_ Gomphus floccosus
, Gomphus cl
Figure 2. Phylogenetic analysis of partial mitochondrial small nbosomal RNA subunit sequences from
each ITS Mbol pattern produced using fungal-specific PCR primers. Sequences were analyzed along
with a data base containing over 70 mycorrhizal fungal genera using PAUP (vers. 3.0L), and each ITS
pattern was identified to family or near-family level monophyletic group. Yellowstone 1 through 4
corresponds to Y1 through Y4 of Figure 1.
Page 13
Task 7 Soil Biology
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Experimental Tasks and Facilities
Alu I Hinfl
SM R1 R2 R3 R4 SM R1 R2 R3 R4
1114
900
692
501
489
404
320
242
190
124
110
67
53
Figure 3. Depiction of electrophoresis gel of two restriction enzyme digests (enzymes: Alu I and
Hinfl) of DNAs amplified from the Internal Transcribed Spacer (ITS) of four "replicates" (R1 through
R4) of the confirmed mycorrhizal morphotype (suspected to be Rhizopogon sp.) from the To seedlings.
SM = molecular size marker, number of base pairs indicated to the left of the gel depiction.
Page 14
Task 7: Soil Biology
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Experimental Tasks and Facilities
TASK 7 Cs SOIL BIOLOGY - SOIL GASES
PARTICIPANTS: Mark Johnson (METT), Paul Rygiewicz (EPA) - Principal Investigators
Ricky King (MET!)
Markus Naujok (CDS, German Student Exchange Program)
Suean Ott (METI)
Task 7: Soil Biology
-------
Experimental Tasks and Facilities
TASK 7 C: SOIL BIOLOGY - SOIL GASES
PARTICIPANTS: Mark Johnson (METI), Paul Rygiewicz (EPA) - Principal Investigators
Ricky King (METI)
Markus Naujok (CDS, German Student Exchange Program)
Suean Ott (METI)
OBJECTIVES:
To measure trace gas production and loss
within the soil profile and the physical, chemi-
cal and environmental factors affecting their
production and loss.
APPROACH:
The concentration of CO2, CH4, 02, and N2O
within the Terracosm soil profile is quantified and
the flux of C02 out of the soil (soil respiration) is
measured on a monthly basis. The data obtained
from these measurements will be characterized as
a function of treatment, soil water content, soil
temperature, soil chemistry, root dynamics, and
other variables to determine the important con-
trolling factors.
STATUS/RESULTS:
When the Terracosm soil and litter layer were
placed in the Terracosms, two gas wells were
buried at five different depths (location of the gas
wells is shown in Figure 5-8 of the Research
Plan.) In the last part of 1993 the methods for
collecting and analyzing samples from the soil gas
wells were developed. Beginning in January 1994
the first monthly samples were collected and ana-
lyzed. Figure 1 shows the mean concentrations of
CO2, CH4, and O2 by Terracosm soil horizon and
treatment. Nitrous oxide concentrations are be-
low the detection limit of the gas chromatographthat
we are using to analyze the gas samples.
Soil gas data from March 1994 (Figure 1), indicate
that soil air CO2 concentrations appear to be a
function of both soil horizon and treatment. The
CO2 in the soil air in the chamberless (CL) Terra-
cosms had the lowest CO2 concentrations in the
litter layer and the greatest in the C-horizon. The
CO2 in the soil air of the elevated CO2 treatments
(Ct and CT) had similar concentrations, and in the
ambient CO2 plus elevated temperature treatment
(cT) soil CO2 was greater in the B1 and B2
horizons over that observed in the other treat-
ments.
Methane in soil air of the chamberless Terracosms
decreased with depth. Excluding the chamberless
Terracosms, CH4 was the greatest in the litter
layer and decreased to its lowest values in either
the B1 or B2-horizon and increased with depth
from that point. Oxygen concentrations in soil air
were similar in all the chambered Terracosms
(21.2 - 21.8 %) but somewhat greater in the
chamberless ones (22.8 - 23.6 %).
Soil Profile Gas Analysis
It is too early to draw many conclusions from the
soil gas data we collected so far other than to state
that the methodology seems to be working. When
data from several more months are collected it will
be easier to see trends and perhaps treatment
effects. As far as N2O is concerned, it is not
possible to measure N2O at extremely low levels
with the GC we currently use. To do this requires
a GC with an electron capture (EC) detector.
Because the Terracosm soils are low in N it seems
unlikely that N2O concentrations will ever be
Page 15
Task 7 Soil Biology
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Experimental Tasks and Facilities
significant. Therefore, until another GC with the
EC detector becomes available we will only be
quantifying CO2, CH4, and O2 in the Terracosm
soil air on a monthly basis.
Soil Respiration/Headspace Gas Analysis
We purchased a LI-COR soil respiration chamber
that we will use to measure the flux of CO2 (g/m^/
min) in a headspace created by the instrument
above the soil/litter. We will develop the methods
for using this instrument early in 1994 and then
begin applying them in the Terracosms and at the
Cascade field sites beginning in late Spring 1994.
Page 16
Task 7 Soil Biology
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Experimental Tasks and Facilities
| Utter Layer | A gj B1 §| B2 QD C
0 500 1000 1500 2000 2500 3000
Soil C02 (ppm)
CT
cT
CL
02
04
06
08
Soil CH4 (ppm)
0 5 10 15 20 25
Soil 02 (%)
Figure 1. Mean Terracosm soil air concentrations of CC>2, CH4, and 02 as a function of treatment and soil horizon
(Litter Layer, A, Bl, B2, and Q. The treatments are: CL = chamberlessTerracosms; ct = ambient CO2 & ambient
temperature; Ct = ambient CO2 plus 200 ppm CO2 & ambient temperature; cT = ambient CO2 & ambient
temperature plus 4°C; and CT = ambient CO2 plus 200 ppm CO2 & ambient temperature plus 4°C. Each bar in
the ct, Ct, cT, and CT treatments is the mean of 6 soil air samples. Each bar in the CL Terracosms is the mean
of 4 samples. The data were collected the week of March 7,1994.
Page 17
Task 7: Soil Biology
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Experimental Tasks and Facilities
TASK 7 D: SOIL BIOLOGY - SOIL & LITTER FAUNA
PARTICIPANTS: Paul Rygiewicz (EPA), Mark Johnson (METI) - Principal Investigators
Nancy Baumeister (OSU)
Andy Moldenke (OSU)
Jim Wernz (OSU)
Task 7: Soil Biology
-------
Experimental Tasks and Facilities
TASK 7 D: SOIL BIOLOGY - SOIL & LITTER FAUNA
PARTICIPANTS: Paul Rygiewicz (EPA), Mark Johnson (METI) - Principal Investigators
Nancy Baumeister (OSU)
Andy Moldenke (OSU)
Jim Wernz (OSU)
OBJECTIVES:
To monitor changes in the density and species
composition of soil arthropods with respect to
the effects of elevated CO2 and climate change
APPROACH:
Arthropod traps are being used. Traps consist of
20-cm-diameter stainless steel, expanded metal
hardware cloth formed into a cylinder, and nylon
mesh bags filled either with the litter layer or the
A horizon. At trap installation, the litter and soil
was excavated, and the cylinders were placed into
the A horizon. Traps extend upward through the
litter layer. The excavated soil (top 0.5 cm of A
horizon) or the litter layer was placed into the
nylon mesh bags. Filled bags were then put in the
cylinders. Two traps per Terracosm were installed
as described in the Research Plan (Figure 5-6).
Two traps per elevational field site were also
installed. The traps in the field were placed in the
buffer zone between two of the sub-plots to be
used for annual harvests..
The traps are sampled to coincide with taking the
Terracosm soil cores. The two bags (litter and A
horizon) are removed from the cylinders and poly-
foam is used to replace the bags' to maintain
uniform temperature and moisture in the litter and
A horizon. Bags are placed in a Berlese funnel
extractor system and the arthropods are extracted
using heat. Arthropods are collected alive, enu-
merated to species and returned to the Terracosms.
Moisture content of the extracted soil and litter is
adjusted to match the soil moisture of the respec-
tive horizon. Bags are then returned to the cylin-
ders.
STATUS/RESULTS:
Terracosms
Two samples were obtained separately from each
of two horizons from each Terracosm in the Fall
1993. Samples included (1) the litter layer, and (2)
the top 0.5 cm of the A horizon. At this start-up
sampling time the mineral soil supported extremely
low densities of invertebrates; the only species
encountered were Onychiurus (springtail), preda-
ceous gamaselid mite, enchytraeid worm and
pauropod (Table 1; total counts; chamberless con-
trols x 1.5; * = significance p< 0.10). There were
no significant treatment effects of elevated CO2 or
elevated temperature or any significant interac-
tion effects.
Each taxon was examined separately for treatment
effects. Oppiella (oribatid mite), pauropods, and
rhagidiid mites showed a significant effect (p<0.5)
in the chamberless controls. Only one species, the
oribatid Odontodamaeus veriornatus, showed a
significant (p<0.5) treatment effect at To for
both elevated CO2, and elevated CO2 - elevated
temperature; we assume this result is spurious buts
bears watching subsequently.
Cascade Field Plots
In the field sites, however, numerous significant
differences were found in the faunas at the sepa-
rate elevations, even though they had started with
a homogenized resource and faunal inoculum (e.g.,
the litter layer placed at the three elevational plots
came from the high elevation site and was charac-
Page 18
Task 7 Soil Biology
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Experimental Tasks and Facilities
tenstic of that altitude). Sampling was the same as
for the Terracosms As a general rule, most
functional groups decreased in density with in-
creasing altitude (Table 2, total counts in 6 repli-
cates; significance p<0.5)
On an individual species basis, 16 of the most
common 34 taxa displayed significant (p, < 0.05)
altitudinal treatment effects Eight of these taxa
(springtails: Isotoma sp P, Tomocerus: oribatids:
Oppia, Quadroppia, Phthiracarus,
Nanthermannia, Hermanniella, Jacotella) showed
mid-elevation numbers distinct from either ex-
treme. Seven of the taxa (enchytraeids; spring-
tails: Entomobrya, Onychiurus sp L,
Hypogastrura; oribatids: Tectocepheus,
Odontodamaeus, unident. immature category)
show low altitude to be different from both mid-
and high-elevation One species of onbatid,
Galumna, is significantly distinct at each eleva-
tion. As a generality, it appears that many of the
taxa characteristic of the high elevation (drier
sites) are undergoing severe reductions at the
lower sites, combined with unrestricted immigra-
tion and earlier reproduction of local populations.
It is most likely that the onset of the rainy season
is responsible for the abundance of worms at the
lower/warmer site.
Samples of microarthropods (1-500 ^.g wet
weight) will serve as robust estimators of
experimental densities but mesoarthopods and
worms (1000-10,000 ng) are too sparse to yield
robust density estimates. Mesoarthropods and
worms will continue to be monitored for indica-
tions of major populations shifts and potential
changes in Terracosm resource allocation; most
of the larger species of insects will probably
become extinct by the end of year 1 in the
Terracosms.
Table 1. Total counts of soil/litter fauna in Terracosm traps, samples Fall 1993.
Fauna
ct
Tt
ctT
Ambient
Chamberless
Springtails (9 species)
1329
2037
1145
1577
1107
Onbatid mites (28 species)
1416
1039
1031
1311
1074
Predaceous mites (7 species)
222
210
173
170
350
Fungivorous mites (4 species)
297
201
20
17
8
Enchytraeid worms (1 species)
4
0
12
0
3
Predaceous mesoarthopods (7 species)
9
12
6
6
17*
Fungivorous mesoarthropods (11 species)
7
24
17
11
56*
Herbivorous mesoarthropods (2 species)
0
0
2
0
0
Summed fauna (69 species)
3321
3352
2424
3178
2777
* Indicates significantly different from chambered treatments at p < 0.10.
Page 19
Task 7 Soil Biology
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Experimental Tasks and Facilities
Table 2 Total counts of soil/litter fauna in Terracosm traps, samples Fall 1993.
Elevational Plot
Fauna
Low
Mid
High
Spnngtails (13 species)
2386a
1618b
647c
Onbatid mites (32 species)
2572a
2086b
836c
Predaceous rmtes (9 species)
214a
189a
104b
Fungivorous mites (4 species)
9b
5b
42a
Enchytraeid worms (1 species)
106a
3b
3b
Predaceous mesoarthopods (16 species)
29a
35a
14b
Fungivorous mesoarthropods (18 species)
50
37
56
Herbivorous mesoarthropods (4 species)
9a
3b
2b
Summed fauna (97 species)
5375a
3958b
1704c
* Fauna values between elevations with different letters indicate significantly different at p < 0 10
Page 20
Task 7 Soil Biology
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Experimental Tasks and Facilities
TASK 7 E: SOIL BIOLOGY - MICROBIAL TRANSFORMATION
RATES/ENZYMATIC ACTIVITIES
PARTICIPANTS: Paul Rygiewicz (EPA), Mark Johnson (METI) - Principal Investigators
Bruce Caldwell (OSU)
Robert Griffiths (OSU)
Task 7: Soil Biology
-------
Erperimemal Tasks and Facilities
TASK 7 E: SOIL BIOLOGY - MICROBIAL TRANSFORMATION
RATES/ENZYMATIC ACTIVITIES
PARTICIPANTS: Paul Rygiewicz (EPA), Mark Johnson (METI) - Principal Investigators
Bruce Caldwell (OSU)
Robert Griffiths (OSU)
OBJECTIVES:
To Characterize the carbon transformation
rates of the bulk soil microbial population
under elevated CO2 and climate change by
measuring the activities of enzymes process-
ing organic compounds to integrate the contri-
bution of several taxonomically distinct groups
of microorganisms.
APPROACH:
Enzyme analyses were done on soil samples col-
lected during the first Terracosm soil coring event
(October 1993) following planting of seedlings in
the Terracosms.
Five enzymes were initially selected to monitor
the effects of the Teracosm treatments on initial
transformations of carbon (B-glucosidase, peroxi-
dase, phenoloxidase), nitrogen (proteinase) and
phosphorus (phosphatase). Assays were:
B-glucosidase and acid phosphatase as-
sayed by release of p-nitrophenol from the
respective glucoside and phosphate esters.
Proteinase determined as release of acid-
soluble tyrosine-equivalents from casein.
Peroxidase-mediated phenol oxidation
measured as the oxidative polymerization of
o-dianisidine using H2O2 as electron accep-
tor.
Phenoloxidase-mediated oxidation of p-
dihydroxyphenol measured by the coupled
reductive decoloration of 2-nitro-5-thiobenzoic
acid.
STATUS/RESULTS:
Soil enzyme activities (Table 1) were typically
highest in the upper horizons (A or B1) and lowest
in the C-horizon material We found no consistent
differences in enzyme activities for ambient and
elevated CO2 and/or temperature treatment within
a soil horizon for the closed Terracosms. How-
ever, peroxidase activities in the B1 horizon of the
unchambered Terracosms were substantially
higher than those assayed in the closed Terra-
cosms.
Because of the soil storage prior to terracosm
construction, these results can only generally indi-
cate the homogeneity of each horizon material,
rather than actual initial soil activities. Absence of
rhizosphere activity and detrital inputs, as well as
leaching, during the storage period, may have
caused caused transient changes in microbial popu-
lations and activities that reduced the differences
between treatments. Subsequent sampling after
seedling establishment should show greater defi-
nition between horizons, as well as the emergence
of treatment-related trends.
Page 21
Task 7 Soil Biology
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Experimental Tasks and Facilities
Table 1. Mean and standard error of mean (± SEM) of Terracosm soil enzyme activities.
Horizon
A
Bl
B2
C
B-Glucosidase (junol p-nitrophenol released/g/h)
Unchambered
1.22 (1.05)
1.11 (0.20)
1.02 (0.04)
1.03 (0 06)
Ambient
1.16(0.06)
1.15 (0.21)
1.22 (0.13)
0.86 (0.09)
CT
1.28 (0.07)
1.30 (0.13)
1.10(0.11)
0.76 (0.09)
Tt
1.35 (0.18)
1.21 (0.14)
0.91 (0.10)
0.75 (0.05)
CTt
1.20 (0.09)
1.16(0.19)
0.87 (0.08)
0.83 (0.09)
Phosphatase (^mol p-nitrophenol
released/g/h)
Unchambered
6.10(1.21)
4.68 (0.25)
5.20 (0.31)
3.62 (0 45)
Ambient
6 27 (1.03)
5.07 (0.62)
5.17 (0.29)
3.55 (0.32)
ct
5.42 (0.24)
4.73 (0.40)
5.08 (0.10)
2.47 (0.32)
Tt
6.34 (0.39)
4.53 (0.28)
4.14(0.51)
3.41 (0.16)
ctT
>
5.35 (0.80)
5.28 (0.57)
4.40 (0.21)
4 03 (0.39)
Proteinase (nmol tyrosine-eq/g/h)
Unchambered
25.4 (3.40)
15.9 (2.95)
10.7 (3.21)
6.80(1 7)
Ambient
26.3 (3.52)
22.2 (3.10)
17.4 (4.04)
9.10(1.3)
ct
22.9(1 60)
21.7(1.15)
12.8(6 02)
7.91 (0.8)
tT
19.5 (1.68)
174(1.17)
10.9(1.20)
10.2 (2.62)
CTt
27.1 (3 91)
19.0 (2 88)
14.7 (2.01)
5.82(1.33)
Peroxidase (x 100 change in Abs 460 nm/g/min)
Unchambered
9.13 (0.84)
12.1 (1.65)
5.50 (2.35)
4.20(1.80)
Ambient
10.4(1.14)
4.90(1.50)
2.63 (0.38)
3.53 (0.38)
Ct
10.7 (0.90)
7.20 (0.08)
4.57 (0.66)
3.70 (0.45)
Tt
7.20(1.20)
4.50 (0.57)
2.97 (0.39)
2.67 (0.30)
ctT
8.53 (0.71)
6.03(1.09)
3.53 (0.47)
3.17(0.41)
Phenoloxidase ( x 100 change in Abs 400 nm/g/min)
Unchambered
6.92 (0.44)
4.2 (0.01)
4.14(0.26)
3.67(0.19)
Ambient
7.25 (0.67)
4.60 (0.42)
4.87 (0.29)
3.65 (0.19)
Ct
6.74 (0.25)
4.18(0.43)
3.93 (0.18)
2.91 (0.20)
Tt
7.99(1.14)
4.69 (0.60)
4.40 (0.30)
3.39 (0.16)
CTt
7.55 (0.58)
4.07 (0.93)
3.92 (0.12)
3.20(0.15)
Number of replicates = 3, except for Unchambered treatment (n=2) and B^-Ct (n=2).
Page 22
Task 7 Soil Biology
-------
Collaborative Research Efforts
COLLABORATIVE RESEARCH EFFORTS:
PART A - COLLEMBOLA SPECIATION
PART B - LIGHT AND ELECTRON MICROSCOPY OF ECTO-
MYCORRHIZAE
PART C - COMMUNITY STRUCTURE OF PHYLLOSPHERE
MICROORGANISMS
PART D - 13C/lsO AND SOIL RESPIRATION
Collaborative Research Efforts
-------
Collaborative Research Efforts
COLLABORATIVE RESEARCH EFFORTS
It is the intention of the Project Staff to utilize the
TER A facility and the experiments done therein to
the maximum extent possible. Project Staff agreed
that after outlining the core portions of the Project
(originally Tasks 1 through 7, Modeling tasks
using TREGRO; and now including Task 8) that
we would welcome and seek out colleagues from
outside ERL-C to utilize the facility, the samples
we could provide and data we collect to support
their research. We envisioned working arrange-
ments (financial and personnel) for this participa-
tion would range widely, including conducting
research at the TERA facility:
with financial support provided by the off-site
researchers, and
by co-operative participation between off-site
and Project Staff to secure funding other than
Project resources
Several collaborative efforts have been estab-
lished and are presented in greater detail in this
section of the booklet. Briefly, the joint research
efforts underway are:
COLLEMBOLA SPECIATION Dr.
Juliane Filser, Institut fur Bodenokologie,
Neuherberg Germany, is enumerating
springtails obtained from the semi-annual
collections of litter and soil fauna taken by
Oregon State University researchers Ms.
Nancy Baumeister (Ph.D. student funded
by a ProjectTraining Co-Operative Agree-
ment) and Drs. Andrew Moldenke and Jim
Wernz.
LIGHT AND ELECTRON MICROSCOPY
OF ECTOMYCORRHIZAE Dr.
Carolyn McQuattie, US Forest Service,
Northeast Forest Experiment Station, Dela-
ware OH, is examining the ultrastructural
changes in prominent mycorrhizal
morphotypes obtained from the semi-an-
nual Terracosm soil cores.
COMMUNITY STRUCTURE OF
PHYLLOSPHERE MICROORGAN-
ISMS Drs. Ralph Crawford, Iwan Ho
and Chang-Yi Li, US Forest Service, Pa-
cific Northwest Experiment Station,
Corvallis OR, are taking needle samples
twice per year from each seedling in the
Terracosms to plate on selective media to
assess changes in phyllosphere microor-
ganisms.
. 13c/18q AND SOIL RESPIRATION
Drs. Jim R. Ehleringer, Guanghui Lin and
Nina Buchmann, University of Utah, Salt
Lake City, UT, are taking root and litter
samples, and soil gas and solution samples
from the Terracosms and from field sites
in Oregon and Utah. They want to evalu-
ate the potential of using stable isotope
techniques to partition root and microbial
respiration, and deep root and shallow root
respiration. The contained environment
of the Terracosms offers the opportunity
to study these processes. The results will
aid in understanding changes in carbon
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COLLABORATIVE RESEARCH: PART A -COLLEMBOLA SPECIATION
PARTICIPANTS: Paul Rygiewicz (EPA)
Juliane Filser (Institut fur Bodenokologie - Neuherberg Germany) - Speciation of
Collembola
Nancy Baumeister (OSU)
Andrew Moldenke (OSU)
Jim Wernz (OSU)
Collaborative Research Efforts
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Experimental Tasks and Facilities
COLLABORATIVE RESEARCH: PART A -COLLEMBOLA SPECIATION
PARTICIPANTS: Paul Rygiewicz (EPA)
Juliane Filser (Institut fur Bodenokologie - Neuherberg Germany) - Speciation of
Collembola
Nancy Baumeister (OSU)
Andrew Moldenke (OSU)
Jim Wernz (OSU)
INTRODUCTION:
Considering the theorized shift to increased pro-
duction of dry matter belowground by forest trees
grown under elevated atmospheric CO2 concen-
trations and associated climate change, changes in
soil physical, chemical and biological processes
become an important component of understand-
ing the forward (atmosphere to plants/soils) and
feedback (plants/soils to atmosphere) effects of
altered climates.
Virtually all processes in soil ultimately are driven
by carbon allocated belowground through indirect
or secondary effects mediated by plants. The
carbon fixed by plants eventually is passed to soil
through litterfall; roots with subsequent release
via exudates, allocation to symbionts, sloughing
and root turnover; and to numerous soil pools via
decomposition of coarse woody debris This
carbon movement can alter the soil food web (both
flora and faunal components) through the quality
and quantity of carbon allocated belowground.
A significant and little understood component
controlling both the short- and long-term seques-
tration of carbon in forests is the role of inverte-
brate fauna These organisms are some of the first
to process newly-fallen detrital material, and also
are highly inter-connected with organisms at nu-
merous trophic levels of the soil nutrient and
decomposition cycles. In order to assess the
forward and feedback effects indicated above, a
better understanding of the role of invertebrate
fauna is needed.
As part of this project, fauna traps have been
installed in the Terracosms and the Cascade Moun-
tain field sites (Task 7: Soil Biology). The faunal
work is focused on enumerating litter layer and
soil fauna to the lowest taxonomic level possible.
Collembola, small and primitive insects, are one
of the most numerous and widely distributed of the
soil arthropods, numerous both in numbers and
species diversity. Collembola exist only in moist
situations, but some can resist desiccation slightly.
Some collembola species live in the surface layers
while others are commonly found deeper in the
soil. Populations are largest in the surface layers
of the soil, especially where the macropore space
is greatest Their feeding habits are varied. Their
food may consist of bacteria, fungal hyphae and
spores, decomposing organic material, feces, and
living plants or animals. Collembola play a role in
fragmenting plant litter and may not be minimally
involved in the direct turnover of soil nutrients.
Because of their role in the soil food web, and high
population numbers and species diversity, addi-
tional attention is being given to collembola. To
the extent possible, collembola will be enumer-
ated to species. Changes in total numbers of
individuals in the community will not indicate
changes in diversity of the population, and there-
fore, the linkages to changes in ecosystem nutrient
and detritus processing to which this organism
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group contributes will not be fully identified un-
less measures of community structure can be ob-
tained.
OBJECTIVE:
to determine if effects of elevated CO2 and
elevated temperature alter the relative species
mix of the collembola population in the litter
and soil profiles
APPROACH:
After the litter and soil fauna are extracted by Drs.
Moldenke and Wernz, and Ms. Baumeister as part
of Task 7 (Soil Biology) collembola are stored in
ethanol and shipped to Dr. Filser at the Institut fur
Bodenokologie - Neuherberg Germany for spe-
cies analysis. There the ethanol is removed using
a lOO^im sieve The sieve and the retained
collembola are placed into lactic acid. Every
individual is transferred onto a slide by transfer-
ring the liquid using a pair of tweezers or a fine
pencil (approximately 10 specimens per drop).
Samples can be stored for up to two weeks for
species determination.
Individuals are identified to species using the most
important morphological traits:
body shape
morphology of extremities
position and number of the body setae
pigmentation
Collembola will be identified to species using the
keys of Christiansen and Bellinger (1980),
Fjellberg (1980), Gisin (1960), Palissa (1964) and
available revisions.
STATUS/RESULTS:
Drs. Moldenke and Wernz sent collembola to Dr.
Filser from the first set of Terracosm cores (Octo-
ber 1993) in Feb. 1994. The second harvest of soil
and litter fauna traps is scheduled for Spring 1994
to coincide with the Terracosm coring event.
Collembola will be sent to Germany from that
sampling event.
REFERENCES:
Christiansen, K. and P. Bellinger. 1980. The
collembola of North America north of the Rio
Grande. Gnnnell College, Grinnell, IA, 1322 pp.
Fjellberg, A. 1980. Identification keys to Norwe-
gian collembola. Norsk Entomologisk Forening.
152 pp.
Gisin, H. 1960. Collembolenfauna Europas. Mu-
seum d'histoire naturell, Genf. 312 pp.
Palissa, A. 1964. Insekten Teil I/I. In: Brohmer,
P., P. Ehrmann, G. Ulmer (eds.). Die Tierwelt
Mitteleuropas Quelle & Meyer, Leipzip. 1-299.
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COLLABORATIVE RESEARCH: PART B - LIGHT AND ELECTRON
MICROSCOPY OF ECTOMYCORRHIZAE
PARTICIPANTS: Paul Rygiewicz (EPA)
Carolyn McQuattie (USFS- NE For. Exp. Stn )
Collaborative Research Efforts
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Experimental Tasks and Facilities
COLLABORATIVE RESEARCH: PART B - LIGHT AND ELECTRON MI-
CROSCOPY OF ECTOMY CORRHIZ AE
PARTICIPANTS: Paul Rygiewicz (EPA)
Carolyn McQuattie (USFS- NE For. Exp Stn )
INTRODUCTION:
Increased atmospheric C02 concentrations may
alter the amount of carbon allocated to roots and
may in turn affect mycorrhizal root structure.
Increased numbers of ectomycorrhizal roots and
increased mantle thickness were found on pitch
pine (Pinus rigida) seedlings grown under condi-
tions of elevated CO2 (McQuattie and Schier
1993). These seedlings were grown in growth
chambers in sand/nutrient solution culture and
inoculated with a known mycorrhizal fungus
(Pisolithus tinctorius)
Characterization of mycorrhizal morphology in
field soils under conditions of elevated CO2 has
not been studied in most tree species Assessing
the number and percentage of roots that are myc-
orrhizal will aid in understanding carbon alloca-
tion under altered climates. However, using these
data only to interpret carbon allocation may be
problematic partially because fewer and similar
numbers of mycorrhizae may be found under
altered climates while the amount of carbon allo-
cated to produce these mycorrhizae may be differ-
ent (e.g., ectomycorrhizae with thicker or thinner
mantles, and more or less extramatrical hyphae
are formed). Understanding the microscopic
morphology of ectomycorrhizae in conjunction
with the other mycorrhizae measures (percent
colonization and diversity) taken in this project
offers a more complete analysis of responses to
treatments.
OBJECTIVE:
to determine if cellular changes can be found
in Douglas fir mycorrhizae (i.e., mycorrhizal
mantle thickness, Hartig net formation, or root
cell structure) in the native soils of the Terra-
cosms as an indicator of carbon allocation
influenced by climate effects.
APPROACH:
Six replicates of each of the one to three most
prominent mycorrhizal morphotypes identified in
Task 7 (Soil Biology) will be used for light and
electron microscopy to make an initial assessment
during 1994. Mycorrhizal roots from cores in two
replicate Terracosms from each of the treatments
will be studied. Mycorrhizae morphotypes are
placed in cold buffer solution (or in a fixative if
sampling of all Terracosms takes more than three
days) and stored in the refrigerator. Samples are
sent "overnight mail" to Dr. McQuattie in Ohio.
There they will be processed for light and electron
microscopy; microscope work will be done using
USFS equipment.
STATUS/RESULTS:
Microscope work will be done using the mycor-
rhizae collected from the Spring 1994 Terracosm
cores if sufficient root material is removed by the
soil corer to meet all analytical needs of the project.
A priority scheme to analyze the tissue obtained
from the soil cores has been established [as parts
of Task 6 (Root Growth and Phenology) and Task
3 (System Nutrients)]. If there is root material
remaining after partitioning to do the molecular,
dry weight and nutrient analyses, mycorrhizal
morphotypes will be sent to Dr. McQuattie for
microscopy work If insufficient material is avail-
able from the Spring 1994 coring event, mycor-
rhizal morphotypes will be sent from the first
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subsequent coring event where a sufficient amount
of material is recovered.
REFERENCES:
McQuattie, C.J. and G.A. Schier. 1993. Effect of
elevated carbon dioxide on changes in growth,
cellular structure and elemental localization in
aluminum-treated pitch pine mycorrhizae 9^ N.
Amer. Conf. on Mycorrhizae. Guelph, ON.
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COLLABORATIVE RESEARCH: PART C - COMMUNITY STRUCTURE
OF PHYLLOSPHERE MICROORGANISMS
PARTICIPANTS: David Olszyk (EPA)
Paul Rygiewicz (EPA)
Ralph Crawford (USFS - PNW Exp. Stn.)
Iwan Ho (USFS - PNW Exp. Stn.)
Chang-Yi Li (USFS - PNW Exp. Stn.)
Collaborative Research Efforts
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Experimental Tasks and Facilities
COLLABORATIVE RESEARCH: PART C - COMMUNITY STRUCTURE
OF PHYLLOSPHERE MICROORGANISMS
PARTICIPANTS: David Olszyk (EPA)
Paul Rygiewicz (EPA)
Ralph Crawford (USFS - PNW Exp. Stn )
Iwan Ho (USFS - PNW Exp. Stn.)
Chang-Yi Li (USFS - PNW Exp. Stn.)
INTRODUCTION:
The phyllosphere as habitat for microorganisms
has been recognized for over a century. Recent
work has shown a variety of microorganisms live
in the phyllosphere; some microorganisms are
beneficial (nitrogen fixers) while others are patho-
genic (leaf-blight). The physical environment
(e.g., radiant energy, wind, temperature, atmo-
spheric C02 concentration, rainfall) will affect
numerous plants processes including photosyn-
thesis, respiration and production of leachates.
Changes in these physiological processes may
induce changes in the distribution and succession
of microbial populations in the phyllosphere.
Understanding the microbial ecology of the
phyllosphere may be important under changing
climates as these organisms can affect forest health
and perhaps serve as indicators of forest health.
OBJECTIVE:
to determine the succession and distribution of
microorganisms on needles of Douglas fir
seedlings under different physiological condi-
tions imparted by elevated CO2 and elevated
temperature
APPROACH:
Two needles per seedling of the most recent age
class from each seedling in the Terracosms are
sampled by Drs. Ho, Crawford and Li. Needles
are placed in zip-lock plastic bags and transported
to the USFS PNW Station in Corvallis. Needles
were plated with 6 different agar media for bacte-
ria, fungi, and yeast. Detection of diazotrophic
bacteria in needles was conducted with the nitro-
gen free semi-solid medium. Ahquots of the
buffer solution, and segments of the needles are
plated onto selective media. Buffer and needle
samples are incubated and then microorganisms
growing on the various media are identified, and
colonies enumerated where possible.
STATUS/RESULTS:
Needle samples were taken in the summer of
1993. The study was proposed for three years; the
US Forest Service scientists received funding from
US Forest Service sources for FY 93 but not for
FY 94. The continuation of the study is uncertain.
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COLLABORATIVE RESEARCH: PART D - 13c/lsO AND SOIL RESPIRA-
TION
PARTICIPANTS: Paul Rygiewicz (EPA)
Ray Shimabuku (EPA)
Mark Johnson (METI)
Ronald Waschmann (METI)
Jim R. Ehleringer (Univ of Utah)
Guanghui Lin (Univ of Utah)
Nina Buchmann (Univ of Utah)
Collaborative Research Efforts
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Collaborative Research Efforts
COLLABORATIVE RESEARCH: PART D - 13c/180 AND SOIL RESPIRA-
TION
PARTICIPANTS: Paul Rygiewicz (EPA)
Ray Shimabuku (EPA)
Mark Johnson (METI)
Ronald Waschmann (METI)
Jim R. Ehlennger (Univ of Utah)
Guanghui Lin (Univ of Utah)
Nina Buchmann (Univ of Utah)
INTRODUCTION:
Release of CO2 from soil is a critical process for
carbon cycling within terrestrial ecosystems as
well as for the global carbon budget Understand-
ing the dynamics of terrestrial carbon pools is
critical to understanding atmospheric carbon
fluxes. Since two-thirds of total carbon in the
terrestrial biosphere is stored in soils and associ-
ated peat deposits (Dixon et al. 1993), soil CO2
efflux is an important process within terrestrial
ecosystems and for the global carbon budget.
Many recent reports have improved greatly our
understanding of temporal and spatial variations
in soil CO2 efflux (Mathes and Schriefer 1985,
Coxson and Parkinson 1987), environmental fac-
tors controlling soil CO2 release rates (Zak et al
1993), relationships between vegetation charac-
teristics and soil CO2 production (Amundson and
Davidson 1990, Jurik et al. 1991), and refixation
of soil respired CO2 by leaves within a forest
canopy (Broadmeadow andGriffiths 1993). How-
ever, there is very limited information available on
the relative contribution of organisms responsible
for producing the soil respired CO2 in forest
ecosystems.
Many attempts have been made to separate root
and microbial respiration, but very few have been
successful mainly due to the complexity of soil
systems and technical limitations (van Veen et al.
1991). Controversy still exists on the relative
contribution of microbial vs. root respiration to
overall soil CO2 release. Stable isotope tech-
niques may offer a unique opportunity to separate
root and microbial respiration in natural forest
ecosystems without many of the problems and
technical difficulties associated with radioactive
isotopes.
Carbon isotope ratio of soil respired CO2 is deter-
mined by d^C levels of its carbon sources in
soils, mainly litter and live roots. d^C levels of
CO2 escaping from soils to the atmosphere should
have the same d' ^C value as its sources in the soil
(live roots or litter) at steady state, even though
there is a fractionation during CO2 diffusion
through soil horizons. Live roots, litter etc. all
may have different d^C because of different
chemical constituents (cellulose, lignin, starch
lipids, etc.) (O'Leary 1981, Benner et al. 1987,
Gleixner et al. 1993). Also, surface roots may
have a different d' ^C than deep roots because they
are formed at different times Because the carbon
sources for soil respired CO2 have different car-
bon isotope ratios, it may be possible to separate
root and microbial respiration by applying carbon
isotope techniques.
The oxygen ratio of soil respired CO2 depends on
Page 7
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Experimental Tasks and Facilities
the d'^0 value of the soil water, since CO2
derived from soil respiration can reach equilib-
rium with soil water (Hesterberg and Siegenthaler
1991). Surface water of moist forest soils has a
higher d' value soil water in the deeper hori-
zons because H2 evaporates more slowly from
the soil surface than does H2^0 (Craig and
Gordon 1965). Thus, CO2 derived from soil
respiration in the surface and the deep horizons
should have different oxygen isotope ratios, un-
less CO2 produced in the deep soils will reach
equilibrium again with surface water during diffu-
sion through the soil surface layer. This hydroly-
sis reaction is very slow (minute range) and may
not be important in soils due to residence time of
the CO2.
The Terracosms offer a good opportunity to test
these hypotheses because of their closed-environ-
ment control systems; long-term nature of the
experiment; and because the major source of CO2
is from tanks (some open-atmosphere CO2 will
enter the Terracosms for the brief periods when
the doors are opened), the d 1 signature of input
carbon is known.
OBJECTIVES:
to explore the potential of applying stable
isotope techniques to identify the relative roles
of the major contributors of soil respired CO2
(microbial vs. root, and deep roots vs. shallow
roots)
to determine the effects of elevated CO2 and
temperature on soil CO2 efflux in Douglas fir
ecosystems with respect to sources of soil
CO2 efflux.
APPROACH:
Litter, roots and soil water are collected by Drs.
Lin and Buchmann from the Terracosms and from
Douglas fir forests (Oregon), and then samples
undergo cryogenic processing in Corvallis for
d^C and d'^O analyses. Also, efflux rates and
stable isotope ratios of soil respired CO2 are taken
with a LiCor soil respirometer in these natural
forest stands of contrasting climate conditions,
and in the Terracosms. Similar measurements and
samples are taken from Douglas fir forest in Utah.
Samples of soil leachate from the Terracosm soil
profile are being analyzed for d^ ^O to determine
stratification potential of the isotope ratio in the
Terracosms.
STATUS/RESULTS:
Drs. Lin and Buchmann measured the rate of soil
CO2 release at the soil surface, and they collected
this gas from the Terracosms in March 1994. Gas
samples were cryogenically treated in Corvallis
for isotope analyses in Utah. Samples of seedling
tissue (needles, stems and roots), harvested from
seedlings set aside at the time of planting in the
Terracosms, are also in Utah for isotope analyses.
Drs. Lin and Buchmann will return to Corvallis
during the Spring 1994 Terracosm soil coring
event to collect samples of roots for isotope analy-
ses. They will also collect another set of soil gas
samples and samples of water throughout the soil
profile.
Dr. Lin is selecting specific field sites in Oregon
and Utah. It is anticipated that the sites will be: one
located in the Coast Range Mountains of Oregon,
the three elevational Cascade Mountain field plots
coupled with the Terracosm experiment, and one
in the Uinta Mountains of Utah.
REFERENCES:
Amundson, R.G. and E.A. Davidson. 1990. Car-
bon dioxide and nitrogenous gases in the soil
atmosphere. J. Geochem. Explor. 38:13-41.
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Benner,R ,M.L Fogel.E.K.SpragusR.E.Hodson
1987 Depletion of in lignin and its implica-
tions for stable isotope studies. Nature 329 708-
710.
Broadmeadow, M.S. and J.H. Griffiths 1993.
Carbon isotope discrimination and the coupling
of CO2 fluxes within forest canopies, p. 190-129.
In - J R. Ehleringer, A.E. Hall and G D Farquhar
(eds.). Stable isotopes and plant carbon-water
relations Academic Press, San Diego.
Coxson, D.S. and D. Parkinson. 1987. Winter
respitory activity in aspen woodland forest floor
litter and soils. Soil Biol Biochem. 19: 49.
Craig, H. and L. Gordon. 1965 Deuterium and
oxygen-18 variation in the ocean and marine
atmosphere p 9-130. In: Proc. Conf. Stable iso-
topes in oceanographic studies and
paleotemperature Laboratory of Geology and
National Sciences. Pisa
Dixon, R.K, S. Brown, R A. Houghton, A.M.
Solomon, M.C. Trexler and J. Wisniewski. 1993.
Carbon pools and flux of global forest ecosys-
tems. Science 263: 185-190.
Gleixner,G ,H.-J. Danier, R.A.Werner and H.-L.
Schmidt 1993. Correlations between the ^C
content of primary and secondary plant products
in different cell compartments and that in decom-
posing Basidiomycetes. Plant Physiol. 102:1287-
1290.
Hesterberg,R and U. Siegenthaler 1991. Produc-
tion and stable isotopic composition of C02 in a
soil near Bern, Switzerland. Tellus43B: 197-205.
Junk, T.W., G.M. Bnggs and D M Gates. 1991.
Soil respiration of five aspen stands in northern
lower Michigan Am Midi. Nat. 126:68-75.
Mathes, K. andT. Schnefer. 1985. Soil respiration
during secondary succession: influence of tem-
perature and moisture. Soil Biol. Biochem 17:205-
211
O'Leary, M.H. 1981 Carbon isotope fraction-
ation in plants. Phytochem. 20:553-567.
van Veen, J. A., E. Lijeroth and L.J. A. Lekkerkerk.
1991. Carbon fluxes in plant-soil systems at el-
evated atmospheric CO2 levels. Ecol. Appl. 1:175-
181.
Zak, R.D., D.F. Gngal and L.F. Ohmann. 1993
Kinetics of microbial respiration and nitrogen
mineralization in great lake forest. Soil Sci. Soc.
Am. J. 57:1100-1106.
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TERA: A STATE-OF-THE-SCIENCE RESEARCH FACILITY
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Experimental Tasks and Facilities
TERA: A STATE-OF-THE-SCIENCE RESEARCH FACILITY
PARTICIPANTS: Ron Waschmann (METI), Bruce McVeety (Battelle PNW Labs), Glenn Jarrell
(METI), Mark Johnson (METI), Marjorie Storm (METI), Brent Baker (CSC),
Dave Tingey (EPA), Paul Rygiewicz (EPA), and David Olszyk (EPA).
INTRODUCTION:
The Terrestrial Ecophysiological Research Area
(TERA) is a state-of-the-science research facility
designed to investigate the effects of elevated
CO2 and climate change on Douglas fir seedlings.
These seedlings are growing in sunlit, controlled-
environment chambers, or Terracosms, that en-
able us to examine simultaneously a variety of
above- and belowground processes. The Terra-
cosms are extensively instrumented, semi-closed
systems, that will allow mass budgets for carbon
and water to be developed TERA is unique
compared to other facilities because it is designed
to accurately monitor and track local changes in
ambient CO2, air temperature, and dew point
depression while operating continuously for sev-
eral years Designed and constructed with a pro-
jected life-span of 20 years, or more, we are
striving to achieve a high level of accuracy and
precision in our estimates and regulation of above-
and belowground environmental control variables.
This write-up-
1) provides a brief summary of the TERA
facility,
2) identifies those facility components that
have been completed since the 1992 peer re-
view,
3) identifies those tasks yet to be completed at
the facility, and
4) examines the performance of the facility in
applying the experimental treatments.
Additional, more detailed information on the TERA
experiment and the TERA facility can be obtained
from the Effects of CO2 and Climate Change on
Forest Trees Research Plan.
TERA FACILITY - SUMMARY:
The TERA facility is comprised of several interre-
lated systems (Figure 4-8 of Research Plan) that
are used to support and implement the experimen-
tal procedures detailed in the Research Plan. Each
of these major components will be briefly de-
scribed below.
The experimental design is a 2 x 2 factorial of
temperature and CO2 with three replicates per
treatment. The air temperature levels are ambient
and ambient + 4°C. The CO2 levels are ambient
and ambient + 200 ppm. Air temperature, dew
point temperature, and CO2 are measured at a
local site weather station. These values are trans-
mitted to each Terracosm and provide the set point
value that the Terracosm should attempt to track.
The dew point depression (air temperature minus
dew point temperature) will be held constant across
treatments, thereby providing a similar gradient
forevapotranspiration in all Terracosms. Because
the treatments are based on fluctuations in ambi-
ent conditions, and treatments are continuously
implemented by each Terracosm, diurnal, sea-
sonal, and annual variability are preserved.
Terracosms
The central component of TERA is 12 growth
chambers, or Terracosms, that are capable of pro-
viding complete environmental control for an en-
closed plant/soil system (Figure 4-8 and 4-9 of
Research Plan). Two chamberless controls are
also present in order to assess the effect of the
Terracosm enclosures on seedling growth. The 12
canopied Terracosms are covered with Teflon
film and have a total canopy volume of 3.2 m^
(Figure 4-9 of Research Plan). These are mounted
on a water tight soil lysimeter that has a soil
volume of 2 m . Aluminum air handlers, attached
Page 1
TERA A State Of-The-Science Research Facility
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Experimental Tasks and Facilities
to the north side of each Terracosm, contain the
hot and cold water heat exchangers and resistive
heaters required for Terracosm climate control.
The above- and belowground portions of each
Terracosm are extensively instrumented in order
to address a variety of process oriented questions.
The various above- and belowground measures
we expect to collect from each Terracosm, the
specific sensors that are to be used for this pur-
pose, and their current status are outlined in Table
1
Physical Plant
The hot and cold water, and compressed air re-
quired for climate control are produced and deliv-
ered by a large-scale physical plant that is located
in the northwest quadrant of the facility (Figure 4-
8 of Research Plan). Because of the central role
that the physical plant plays in Terracosm climate
control, redundant, backup systems have been
installed to minimize experimental downtime due
to equipment failure.
Data Acquisition
Located adjacent to each Terracosm are the data
acquisition/process control and analytical instru-
mentation required for each Terracosm to operate
independently of the other Terracosms (Figure 4-
9 of Research Plan) This distributed control
strategy ensures that equipment failure at one
Terracosm will not affect the ability of the other
Terracosms to operate. The aboveground data that
are acquired at each Terracosm are averaged every
1 minute and transmitted, via a communications
network, to a database program located in the
TERA Mission Control Building (inside the
Polyhouse). Additional information about the
database program and the method of data retrieval
from the Terracosms can be found in the Database
Section of this Booklet.
Site Weather Station
One of the unique features of this facility is the
ability of each Terracosm to track diurnal and
seasonal fluctuations in air temperature, dew point
temperature, and CO2 concentration. Ambient air
temperature, dew point temperature, and CO2
concentration are monitored continuously at a site
weather station located 30 meters to the west of the
TERA facility. Data collected at the site weather
station are averaged every 1 minute and the" values
are transmitted to each Terracosm every 1 minute
over a communications network. Terracosm pro-
cess control systems use the site ambient condi-
tions and internal computer logic to determine and
control the individual target CO2, air temperature,
and dew point temperature treatments. With this
design, we expect to track dew point depression
and air temperatures to ± 0.5°C, and CO2 concen-
tration to ± 5 ppm
Mission Control
The Mission Control Building, located inside the
polyhouse, is the site of several functions that are
critical to the daily operation and success of the
TERA experiment (Figure 6, Appendix A of Re-
search Plan). First, the equipment needed to
collect and store individual Terracosm data is
located here (see Database section in this Booklet
for a more detailed description of this process)
Second, a PC computer that serves as a network
interface between the Terracosms and site weather
station is housed here. This computer is also used
for on-line programming and provides graphical
status displays of Terracosm climate and experi-
mental control parameters. Third, a host analyti-
cal system located here will house the: 1) instru-
mentation to measure individual Terracosm leak
detection gas (SFg), 2) equipment to deliver
calibration gases automatically to each
Terracosm's CO2 monitor, 3) instrumentation to
perform frequent, automated, data quality control
checks on Terracosm instrumentation, and 4) data
acquisition/process control system to control and
acquire data from the instrumentation located in
the central analytical system.
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TERA A State-Of-The-Science Research Facility
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Experimental Tasks and Facilities
TERA FACILITY ¦ CURRENT STATUS:
Terracosms
Seedlings were planted in all Terracosms on 7
June and 8 June 1993 Over the next six weeks the
seedlings were grown under ambient air tempera-
ture, dew point temperature, and CO2 conditions.
After developing and testing the climate control
software, Teflon was attached to the Terracosms
and air temperature and dew point temperature
control were initiated on 23 July 1993. Prior to
this, the site weather station was collecting mea-
sures of ambient air and dew point temperature
and transmitting these values as treatment set
points to each Terracosm. Shortly after beginning
Terracosm air and dew point temperature control,
data collection from the aboveground environ-
mental variables and the transmission of their 1
minute averages to the database in the Mission
Control Building was completed (See Table 1 for
a list of on-line sensors).
Terracosm CO2 Control
The seedlings were grown under treatment air and
dew point temperature control for one month
before an initial CO2 control system was com-
pleted on 23 August 1993. This initial control
strategy (still in use today) relies on quantifying
the amount of CO2 injected to raise the Terracosm
CO2 to the treatment set point. When Terracosm
CO2 exceeds the set point, two pneumatically-
controlled dump valves located on the air handler
are opened until the desired Terracosm CO2 is
reached through the mixing of ambient and Terra-
cosm air Since we are unable to quantify the
amount of C02 that is lost from the system during
these venting cycles, we can not accurately derive
Terracosm carbon mass balance estimates at this
time. The strategy for closing the carbon balance
will be outlined below.
We do, however, have a method to frequently
estimate photosynthesis and Terracosm dark res-
piration. On every even hour Terracosm pneu-
matic dump valves are closed and the CO2 control
software withholds all CO2 injection for the next
20 minutes. Over the last 15 minutes of this period
the rate of change in CO2 is measured using a CO2
monitor Since this value is collected throughout
the day, it provides us with a measure of either
photosynthesis or respiration, depending on the
time of day when the estimate was derived.
Terracosm Climate Control
The hot and cold water proportional control valves
and electrical resistive heaters required for Terra-
cosm climate control are fully operational. During
the early development of the climate control soft-
ware it became evident that our temperature and
dew point control was affected by the insufficient
availability of water vapor in the recirculating air
column (excess water vapor in this air column is
required for air and dew point temperature con-
trol) Installation of warm-steam humidifiers to
inject water vapor into each Terracosm tempo-
rarily solved this problem. However, the humidi-
fiers are unable to produce sufficient quantities of
water vapor during the cool rainy season to main-
tain dew point depression. Additionally, warm
steam that is injected into the Terracosm con-
denses on the Teflon walls further complicating
dew point control The condensate remains until
there is sufficient solar energy for evaporation to
occur. Our plans to improve control of dew point
depression are presented below. In spite of some
difficulties with fine-grained control of air and
dew point temperature, Terracosm climate control
is quite high. We will present these results below.
Automated Terracosm IRGA Calibration Sys-
tem
Most of the InfraRed Gas Analyzer (IRGA) auto-
calibration system is completed and is currently
being used for bi-weekly instrument adjustments.
The simultaneous delivery of a zero or span gas to
each IRGA allows us to adjust zero and span
potentiometers quickly and frequently.
Physical Plant
All major elements of the physical plant are on-
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TERA A State Of-The-Science Research Facility
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Experimental Tasks and Facilities
line. Hot and cold water generation and delivery
systems are operational. Also, the air compressor
and heatless air dryer, used to actuate pneumatic
dump valves on each Terracosm, are operational.
The proportional valves used to regulate the flow
of hot and cold water into each Terracosm heat
exchanger have been operating since 23 July 1993.
Mission Control
Since July 1993, Terracosm environmental con-
trol conditions have been monitored and displayed,
in real-time, on a PC located in the Mission Con-
trol Building. The operational status of all Terra-
cosms and the site weather station are also moni-
tored continuously by an elaborate alarm-paging
system. This alarm system provides 24-hour
protection to the experiment and is presented in
greater detail in Appendix A of this Chapter.
All the equipment needed to collect and store
individual Terracosm data is on-line. The PC
computers that provide graphical status displays
of Terracosm environmental control performance
and serve as the network interface between the
Terracosms and the data base are operational
Those components of the host analytical system
that will: 1) provide automated, daily quality con-
trol checks on Terracosm instrumentation, 2) au-
tomatically delivercahbration gases toTerracosm
IRGA's, and 3) estimate Terracosm leakage rates,
will be completed over the next several months.
TERA FACILITY - PLANNED ADDITIONS:
CO2 Scrubbing System
Although the majority of the environmental con-
trol components are operational, several control
and calibration elements remain uncompleted.
We have a plan for a CO2 and hydrocarbon scrub-
bing system that will be installed at each Terra-
cosm by July 1994 Currently, we are unable to
calculate a carbon mass balance for each Terra-
cosm due to the non-quantified exchange of CO2
through the dump valves. Completing the scrub-
Page 4
bing system will allow us to estimate accurately
each Terracosm's carbon balance and remove any
hydrocarbon buildup. Until the system is com-
pleted, estimates of Terracosm photosynthesis
and respiration will continue to be measured by
examining the increase or decrease of CO2 when
Terracosm dump valves are closed.
Automated Terracosm IRGA Calibration Sys-
tem
Although InfraRed Gas Analyzers located at each
Terracosm are manually calibrated bi-weekly, we
are installing the remaining mechanical elements
required to calibrate each IRGA automatically on
a daily basis. After completion in July 1994, the
system will automatically deliver zero and span
gases to each IRGA, collect stabilized output
readings, calculate a new linear regression equa-
tion, and apply this equation to the instrument
readings for the next 24 hours. In addition to daily
calibration, we will continue to adjust IRGA elec-
tronic components bi-weekly in order to maxi-
mize instrument output resolution.
Refinement of Dew Point Temperature Con-
trol
Dew point temperature control will be improved
after designing and installing a proportionally-
controlled water vapor injection system. Ex-
pected to be on-line by mid-summer 1994, the
system will improve air and dew point tempera-
ture control, minimize water condensation on the
Terracosm walls, and allow us to estimate Terra-
cosm water mass balances.
Terracosm Leak Rate Determination
Currently we have no direct method of estimating
the rate of gaseous leakage from the aboveground
portion of the Terracosms. We are implementing
a system that will utilize Sulfur Hexaflouride
(SFg) as a leak detection gas. Using a mass flow
controller, SFg will be injected into each Terra-
cosm in order to maintain a steady concentration.
Terracosm SF^ concentrations will be analyzed
on a time-shared basis using a photoacoustic de-
TERA A State-Of-The-Science Research Facility
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Experimental Tasks and Facilities
tector located in the Mission Control Building and
will provide the feedback control for Terracosm
SFg injection (Figure 6, Appendix A of Research
Plan)
Central Analytical System
The central analytical system data acquisition/
process control equipment has been operating
since July 1993. The required CO2 and SFg
injection/sampling tubing has been installed in the
facility raceways and routed to the central analyti-
cal systems heated cabinets. Over the next several
months we will install the SF^ photoacoustic
detector and complete the plumbing necessary to
estimate Terracosm leak rates. During this time
we will also install the valves, plumbing, and
sensors (dew point hygrometer and CO2 monitor)
required to conduct frequent quality control checks
on several of the Terracosm instrument systems.
Irrigation System
Irrigation water is manually added either through
a soaker hose system during the summer months
or through a hand-held wand during the winter
months. Most of the irrigation delivery lines are
installed in the raceways, but are not linked to the
Reverse Osmosis (RO) system. We plan on at-
taching these lines to the RO unit and installing
rate meters and computer-controlled flow valves
at each Terracosm by Fall 1994. Completion of
this task will allow us to automatically deliver and
quantify summer Terracosm irrigation events.
However, we will continue to apply winter irriga-
tion events through a hand-held wand.
Physical Plant
Flow sensors and fluid level indicators for the
physical plant were installed in November 1993,
but are not interfaced with the data acquisition
system. These sensors will be hooked-up to the
physical plant data acquisition system and will
provide real time data display and alarm capabili-
ties in 1995.
TERA FACILITY - QUALITY ASSURANCE/
QUALITY CONTROL:
Quality Assurance (QA)
A laboratory-approved Quality Assurance Project
Plan (QAPP) is in place for the Terracosm experi-
ment. The QAPP describes how the QA program
of the Project is managed, audited, and results of
audits reported to Lab Management. The QAPP
also explains where various QA/QC (Quality As-
surance/Quality Control) information can be found.
All QA/QC aspects concerning environmental
measurements are addressed in detail in the nu-
merous Standard Operating Procedures (SOP's)
that support the QAPP. The QAPP and supporting
documentation are described in the Quality Assur-
ance booklet.
The successful operation of the TERA facility
relies heavily on manual and automated quality
control checks in order to assure data quality and
instrument accuracy. Using a QA check sheet,
Terracosm and physical plant operational status
and performance are examined and logged daily.
The QA check also includes quantifying the amount
of water required to replenish the climate control
humidifiers and calculating daily ET estimates for
each Terracosm.
By August 1994, automated QA checks will be
performed daily on the Terracosm and weather
station CO2 monitors and dew point hygrometers.
This will be accomplished by continuously pull-
ing an air sample from each Terracosm into the
Mission Control Building where multisampler
valves will divert the samples from one ambient
and one elevated CO2 Terracosm through a dual-
channel CO2 monitor and two dew point hygrom-
eters. Comparing the values obtained by the
central analytical system with those obtained from
the sampled Terracosm air will allow us to identify
any instruments that fall outside of preset limit
conditions.
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TERA A Stale Of-The-Science Research Facility
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Experimental Tasks and Facilities
Terracosm and weather station thermocouples will
be checked manually using a hand-held thermo-
couple calibrator. Currently each Terracosm has
two thermocouples mounted in aspirated, ther-
mally-shielded, cylinders. Eventually, the Terra-
cosm control software will compare the 1-minute
average and standard deviation of each thermo-
couple with defined limit values. When the pri-
mary thermocouple falls outside of this range, a
flag will be set indicating a problem and the value
from the secondary thermocouple will be used for
climate control decisions. If the value from the
secondary thermocouple also falls outside of range,
an alarm condition will be sounded indicating that
they need to be promptly repaired.
The remaining sensors and data acquisition hard-
ware will have QA checks performed according to
the manufacturer's suggested procedures and in-
tervals Over the next 9 months we plan to
complete the Standard Operating Procedure for
Terracosm sensors and data acquisition compo-
nents. The document will outline the procedures
and frequency at which calibrations and data qual-
ity control checks will be performed for the entire
field of Terracosms
Preventative Maintenance
The TERA physical plant and experimental sup-
port facilities operate under manufacturer-sug-
gested preventative maintenance schedules and
procedures. Over the next 9 months we will
integrate this information into the TERA Facility
Quality Assurance documents.
Facility Troubleshooting
Terracosm environmental conditions are continu-
ously monitored and evaluated against defined
alarm limits. When environmental conditions in
any Terracosm exceed these limits, an audible
alarm and pager system is triggered indicating the
general nature of the problem(s) to the on-call
personnel. This system allows us to respond
rapidly to hardware or software related problems
that could jeopardize the experiment. Over the
next year we will continue to expand the number
of sensors and environmental conditions that are
covered by the alarm system.
As hardware or software problems are encoun-
tered they are repaired, the short-term and long-
term implications are discussed by Project Staff,
and long-term solutions are identified, examined
and implemented. All problems that are encoun-
tered and the corrective actions applied are logged
in an extensive Quality Assurance notebook sys-
tem located in the Mission Control Building. The
logbooks serve as a permanent record of which
Terracosms or specific pieces of equipment have
operated improperly. The logbooks allow us to
examine the nature and frequency with which
specific components experience failures and will
provide us with historical records needed to iden-
tify recurrent problems and begin implementing
long-term solutions.
TERA FACILITY - SYSTEM PERFOR-
MANCE:
Because of the complex nature of the TERA
facility and experiment it is important to have
numerous measures of how well the system is
operating. Using data collected between 21 Feb-
ruary and 27 February 1994, the performance of
Terracosm environmental control and the overall
operational integrity of the TERA facility was
examined. The data are presented as an example
of the quality of Terracosm environmental control
and of our ability to implement the experimental
treatments delineated in the Research Plan.
Air Temperature Control
Ambient air temperature set points ranged from
1.5°C to 14.1 °C with the weekly average of 7.8°C
(SD = 2 84). Both the ambient and elevated
temperature Terracosms were equally effective at
tracking diurnal fluctuations in ambient tempera-
ture (Figure la and lb). The air temperature
within the ambient and elevated (ambient + 4°C)
Page
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TERA A State-Of-The-Science Research Facility
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Experimental Tasks and Facilities
temperature Terracosms was controlled to within
± 1°C of set point 90% of the time (Figure lb).
Over the course of this week the average deviation
from the target value (actual air temperature -
target air temperature) was 0 2°C for the ambient
temperature treatment and 0.1 °C for the elevated
temperature treatment (Table 2 and Figure lb).
The average difference between the ambient and
elevated temperature Terracosms was 3.8°C (Table
2). Figure lb illustrates this treatment separation
or offset The results indicate that, in addition to
closely tracking changes in ambient temperature,
we are also very nearly implementing the 4°C
treatment separation outlined in the research plan
The average temperature treatment applied to the
ambient and elevated Terracosms during this week
were 8 1°C and 11 9°C, respectively (Table 2)
Dew Point Depression Control
Ambient dew point depression (ambient air tem-
perature - ambient dew point temperature) set
points ranged from 3.6°C to 13.9°C with the weekly
average of 7.9°C (SD = 2 07). The ambient and
elevated (ambient+ 4°C) temperature Terracosms
performed equally well in tracking changes in
diurnal dew point depression (Figure 2a and 2b).
Those times when all Terracosms had difficulty
tracking the set point were primarily due to either
the: 1) inability of the humidifiers to add sufficient
water vapor to reach the target value, or 2) con-
densing of injected water vapor on the Teflon
walls.
During the week of 21 -27 February 1994, the dew
point depression of the ambient temperature Terra-
cosms was controlled to within ± 6°C of the set
point 51% of the time, while in the elevated
temperature Terracosms it was controlled to within
± 6°C of the set point 72% of the time. During this
week the average deviation from the target value
(actual dew point depression - target dew point
depression) was 0.6°C and 0.9°C for the ambient
and elevated temperature treatments, respectively
(Table 2 and Figure 2b). The average treatment
applied to the ambient and elevated treatment was
8.5°C and 8.8°C, respectively (Table 2). The
average difference between the dew point depres-
sion values in the ambient and elevated tempera-
ture Terracosms was only 0.3°C (Table 2 and
Figure la). This indicates that, although we can
still improve the fine-grained control of dew point
depression, we are implementing the experimen-
tal treatment as detailed in the Research Plan (i e ,
same dew point depression in all Terracosms).
CO2 Control
During the week of 21 -27 February 1994, ambient
CC>2 set points fluctuated between 351 ppm and
526 ppm with the weekly average of 365 ppm (SD
= 12.1). The elevated CO2 Terracosms (ambient
+ 200 ppm) can more accurately track diurnal
changes in ambient CO2 than the ambient CO2
Terracosms (Figure 3a & 3b) because we do not
have an efficient mechanism for removing excess
CO2 Ambient CO2 Terracosm control improves
dramatically during the daylight hours when pho-
tosynthesis consumes CO2 that accumulated from
dark respiration buildup during the previous night
(Figure 3b). Most of the time the CO2 levels in the
ambient CO2 Terracosms are approximately 20
ppm higher than the set point value (Figure 3b)
even though the pneumatic dump valves are al-
most constantly open in an effort to lower the
CC>2. This is due primarily to the ambient CO2
concentration of the air surrounding the Terra-
cosms. It is approximately 20 ppm higher than the
ambient CO2 measured at 3 meters on the weather
station tower.
The ambient CO2 treatment was controlled, on
average, to within ± 20 ppm of the set point 63%
of the time, while the elevated CO2 treatment was
controlled to within ± 20 ppm of the set point 99%
of the time. During this week the average devia-
tion from the target value (actual CO2 - target
CO2) was 20 ppm for the ambient CO2 treatment
and 2 ppm for the elevated CO2 treatment (Table
2 and Figure 3b), while the average separation
between the ambient and elevated CO2 treatments
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TERA A State Of-The-Science Research Facility
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Experimental Tasks and Facilities
was 182 ppm (Table 2). The average treatment
applied to the ambient and elevated treatments
was 384 and 566 ppm, respectively (Table 2).
Although the ambient CO2 Terracosms may not
adequately track changes in diurnal CO2, we are
very nearly approximating the 200 ppm treatment
separation as outlined in the research plan. Car-
bon dioxide control in the ambient CO2 treatment
will improve after installing the CO2 scrubbing
system
Soil Temperature
We are not controlling soil temperature as an
experimental treatment. Soil temperatures are
influenced by Terracosm air temperature. Terra-
cosm soil temperature is measured every 15 min-
utes at nine locations within each Terracosm;
three groups of three thermistors (Figure 5-8 of
Research Plan). One group of three is distributed
in the middle of the A-honzon, the second group
of three is in the middle of the B-honzon, and the
third group is in the middle of the C-honzon. The
Terracosms were divided into quadrants (NE, SE,
NW, and SW) and the thermistors were distrib-
uted within one of those quadrants within each
Terracosm (Figure 4) Figure 4 also lists which
quadrant contains the thermistors for each Terra-
cosm Since the north side of the Terracosms are
not surrounded by soil, all thermistors exit through
the north wall and are terminated in the electrical
enclosure.
There are three thermistors at each depth location
(Figure 4). One thermistor is centered in the
Terracosm (i.e., 50 cm from both the front and the
back of the Terracosm). Another is halfway be-
tween the first thermistor and the Terracosm wall
(i.e., 25 cm from one wall and 75 cm from the
other). The third thermistor is 10 cm from one wall
and 90 cm from the other. The thermistors were
placed in this array to provide a measure of soil
temperature with depth and a measure of some of
the gradients of temperature within the Terra-
cosms. Since the four walls of the Terracosm soil
compartments have somewhat different levels of
insulation (e g., soil on the south, east, and west
sides, 6 inches of blue foam on the north), the
thermal gradients will vary slightly. Placing the
thermistors throughout the chamber field in the
different quadrants will provide data to assess
thermal performance of the soil compartments
and treatment effects.
Soil temperature data from the middle thermistor
(centered between the front and back walls of the
soil compartment) in each of three soil horizons is
presented in Figure 5. The data are for an ambient
C02/ambient temperature (Terracosm 2) and
ambient C02/elevated temperature (Terracosm 5)
Terracosm
A-horizon soil temperature data show diurnal fluc-
tuations in temperature with an overall increasing
trend with time Even though the air temperature
difference between the two chambers is approxi-
mately 4°C, the difference in soil temperature is
only about 1°C The A-horizon thermistor is
buried 5 cm below the surface of the mineral soil
which is covered by up to 6 cm of forest floor liter.
The litter layer combined with moist soil condi-
tions may account for the 1°C difference.
Soil temperatures decreased as depth increased.
In the B-horizon the elevated temperature cham-
ber soil (Terracosm 5) is slightly less than 1°C
warmer than the ambient temperature chamber
(Terracosm 2). Differences in the C-horizon were
slightly greater than those seen in the B-honzon.
As for the A-horizon, there was an overall increas-
ing trend in the B and C-horizon soil temperatures
with time.
Although we have been gathering soil tempera-
ture data for some time now, we have not devel-
oped a scheme to analyze the data. There are 126
soil thermistors in the Terracosm field. The num-
ber of comparisons possible is large and the analy-
sis of these data is complex. In the next year we
will be looking at these data and will identify ways
to reduce and report them.
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TERA A State-Of-The-Science Research Facility
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Experimental Tasks and Facilities
Mechanical And Experimental Downtime
The Terracosm environmental control software
uses binary flags in the database to indicate se-
lected Terracosm conditions. One flag identifies
when a Terracosm door is opened due to either
mechanical failure (doors are opened when per-
sonnel responding to environmental control alarms
are unable to repair certain problems) or intrusion
into the Terracosm to conduct measurements. The
environmental control software bypasses the cli-
mate control subroutines while the door remains
open A second flag is set for the duration that all
Terracosm IRGA's are simultaneously being
manually calibrated (once every 2 weeks).
While the environmental control results presented
earlier demonstrate the quality of Terracosm cli-
mate control, examining data collected between
21 February and 28 March 1994 (42 days or
12,096 total hours) provides a preliminary mea-
sure of the facility to implement experimental
treatments over a longer period. Table 3 summa-
rizes the total amount of time that environmental
conditions for each treatment (3 replicate Terra-
cosms) and for all Terracosms were not being
controlled due to mechanical failure, sampling, or
on-line programming.
The total time that seedlings were not subjected to
experimental treatment conditions because of open
doors ranged from 0 2% to 1.9%, or 7 to 58 hours
(Table 3). Using just a binary flag, we are unable
to differentiate between an "open door" for sam-
pling and mechanical reasons. However, examin-
ing the logbooks indicates that the doors were
open approximately 1/2 of the time for sampling in
all but the elevated temperature/elevated CO2
treatment. Mechanical failures at one of the Terra-
cosms of this treatment accounted for 79%, or 45
hours of the total treatment downtime. Total
downtime for all Terracosms due to "open doors"
was only 0.8%, or 94 out of a possible 12,096
hours (Table 3).
The time that CO2 treatments could not be im-
posed because of manual IRGA calibration was
0.7%, or 80 hours (Table 3). Downtime due to
IRGA calibration will probably increase slightly
once the IRGA auto-calibration system is on-line;
mathematical offsets (needed to maximize the
analytical sensitivity of the CO2 monitor) will be
derived daily and manual calibrations will most
likely continue on a bi-weekly schedule.
The time when environmental conditions were not
implemented due to on-line programming was
0.2%, or 28 hours (Table 3). When programming
on-line, the processor is in program mode and is
not acquiring or processing data. After the control
software is fully refined, downtime due to on-line
programming should decrease to almost 0%.
Total downtime for the 42 day period was only
1 1%, or 205 out of a possible 12,096 hours (Table
3). Although this value will most likely increase
at times, due to either mechanical failures affect-
ing more of the Terracosms, or more intrusions
into the Terracosms for experimental activities,
these preliminary estimates indicate that the TERA
facility is capable of providing the physical condi-
tions required to conduct a long-term controlled-
environment experiment.
CONCLUSION:
Since the 1992 peer review considerable progress
has been made on the TERA facility. The physical
plant is completely operational. The climate con-
trol software has been written and debugged, and
the climate control hardware is operational and
provides temperature and dew point control. The
hardware and software to control CO2 has also
been completed. Most of the above- and
belowground instrumentation is installed and ter-
minated to the data acquisition system. Most of
the elements of the irrigation system either have
been completed or designed.
Even though many aspects of the facility are
completed, or are nearing completion, more ele-
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TERA A State Of-The-Science Research Facility
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Experimental Tasks and Facilities
ments need to be finished before all aspects of the
Research Plan can be accomplished. Completing
the CO2 scrubbing and water vapor injection
systems by Fall 1994 will allow us to close the
water and carbon budgets and to improve the
quality of environmental control. Completing the
irrigation system by Fall 1994 will allow us to
automatically deliver and quantify irrigation events
ateachTerracosm. Once completed by Fall 1994,
the central analytical system will allow us to: 1)
estimate Terracosm canopy leakage rates, 2) iden-
tify and alarm faulty Terracosm instrumentation,
and 3) conduct frequent quality assurance checks
on numerous environmental sensors.
The overall ability of the Terracosms to track
diurnal changes in ambient air temperature, dew
point temperature, and CO2 is quite good. While
dew point depression control exhibits the greatest
variability, all three control variables very nearly
maintain the treatment separations outlined in the
Research Plan Completing several planned sys-
tems and refining the control software should
improve control of each environmental parameter
and provide better treatment separation. Since the
facility was brought on-line in Fall 1993, all ele-
ments have performed exceptionally well. Total
downtime over a representative 42 day period
demonstrates that the facility is quite capable of
providing the mechanical and physical elements
required to conduct the TERA climate change
research project
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TERA A State-Of-The-Science Research Facility
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Experimental Tasks and Facilities
APPENDIX A
Below is a draft copy of the TERA 24-hour monitoring and emergency response plan that is currently
under Project review.
24-HOUR TERA MONITORING AND EMERGENCY RESPONSE PLAN:
Introduction
The TERA facility is comprised of a number of
sophisticated instruments and equipment. Ex-
periments in TERA are dependent upon the proper
functioning of these instruments and equipment.
When hardware and software failures occur, the
success of an experiment could be at risk. To
reduce the amount of time that any critical com-
ponent of the TERA facility is not functioning a
systems monitoring and emergency response plan
has been developed The rationale for this action
is to produce reliable and documentable experi-
mental conditions.
Routine Monitoring:
On a daily basis, including weekends, the TERA
facility will be inspected by a member of the
TERA staff. This inspection has three compo-
nents- (1) the Terracosms, (2) the physical plant,
and (3) the experimental conditions. This inspec-
tion will occur between 12:00 noon and 2:00 p.m.
Terracosm Inspection: This inspection includes a
visual inspection of each Terracosm to:
(1) assess the integrity of the Teflon film,
(2) note condensation on the Teflon film (a
diagnostic for poor temperature or dew point
control),
(3) verify the operation of the circulating fan,
(4) note the physical appearance of the trees,
(5) observe leaks of hot or chilled water,
(6) observe the water levels in the pit liners,
(7) fill reservoirs on dew point generators
(humidifiers), and
(8) verify operation of gas sample pumps
(flow indicator on east side of air handler).
Physical Plant Inspection: The TERA physical
plant will be inspected to:
(1) identify leaks,
(2) note and record the temperature of hot and
chilled water,
(3) confirm the operation of pumps,
(4) confirm the operation of chiller,
(5) confirm the operation of the compressor,
(6) confirm the operation of the heaters, and
(7) check the fluid levels in the hot and chilled
water reservoirs.
Experimental Conditions: The experimental con-
ditions are monitored in the Mission Control
Building (room to the west in the TERA
Polyhouse) at the real-time diagnostic computer.
On this computer there are several windows that
show the current control conditions and error
terms (the difference between set-point values
and actual conditions) including: temperature,
CO2 concentration, and dew point for each of the
chambered Terracosms; there are also data on
ambient conditions. Each of these windows shall
be viewed and conditions noted in the daily log
sheet. Terracosms that have conditions out of
control need to be noted and steps taken to rectify
or stabilize the situation immediately. These
steps include:
(1) reporting the problem to the TERA Site
Manager,
(2) repairing, resetting, or replacing the prob-
lem equipment, or
(3) stabilizing the facility to minimize poten-
tial damage to the experiment (this is dis-
cussed in detail below).
Page 11
TERA A Slate Of-The-Science Research Facility
-------
Experimental Tasks and Facilities
Reporting: A daily log reporting the results of the
TERA inspection will be completed by the person
conducting the inspections. The notebook with
log sheets is kept in the Mission Control Building.
Problems and corrective actions are to be re-
corded in the TERA log book. This log book is
also kept in the Mission Control Building and is
reviewed on a regular basis by the TERA Site
Manager.
Emergency Response:
In a complicated experiment like the one being
conducted at TERA, situations will arise that
require immediate response to minimize poten-
tial damage to the physical facilities and the
experiment. Since these situations can arise at
any time a 24-hour emergency response plan has
been developed. The emergency response plan
has two levels of response, primary (1°) and
secondary (2°) A 1° response occurs within 30
minutes of an emergency call. A 2° response is
initiated only when the 1 ° response person en-
counters a problem or situation that they cannot
solve. Both the 1° response person and the 2°
response person can be contacted (via a paging
system) The success of this plan depends on a
computer-driven diagnostic expert system that
monitors Terracosm conditions 24-hours a day.
When any of a number of problems occur the
computer can activate an alarm system that re-
sults in an alarm being sounded during normal
working hours, or a page going out to the desig-
nated 1° response person after working hours.
During normal working hours the 2° response
person will deal with system emergencies and
alarms. After hours, emergency calls will be
answered by the 1° response person.
After Hours Emergency Response: The 1° re-
sponse person is responsible for being on-site
within 30 minutes after receiving an after-hours
emergency call. When the 1° response person
arrives he/she assesses the nature of the problem(s)
and takes primary steps to alleviate the problem(s)
or stabilize (a condition in which the system is not
subject to further deterioration and the experiment
is not being compromised) the situation. This is
similar to the Triage Officer in a war or natural
disaster; the problems are identified and the Tnage
Officer (or 1° response person here) prioritizes
each problem and determines the appropriate level
of action that needs to be taken for each problem.
Some problems can wait while others demand
immediate resolution. Part of the response plan is
a key (flow-chart) to corrective actions that are to
be taken when problems arise. When a call goes
out to the 10 response person they will use the key
and their assessment of the problem to take correc-
tive actions immediately. If the problem can be
solved by the 1° response person then he/she
solves the problem and monitors the experimental
conditions long enough to be certain the problem
is fixed. If the problem cannot be solved by the 1 °
response person, the 2° response person must be
notified and steps must be undertaken to stabilize
the system. Secondary response people have the
training and skills to repair or replace most of
hardware and software at TERA.
The first call to the 2° response person is to their
residence. If the 2° does not answer the phone a
message explaining the nature of the situation is
left on the 2° response person's answering ma-
chine. Next a call to the 2° response person's
pager must be made and a phone number where
the 10 response person can be reached sent to the
pager. The 2° response person must answer a call
from the 1° person as soon as possible. An
immediate response is best, but a delay of up to 15
hours for the 2° response person is acceptable. In
some situations the 2° response person will be
able to tell the 1° response person how to fix a
problem over the phone; in other situations he/
she will need to be on site. In some cases even if
a problem cannot be solved by the 1° response
person it can be stabilized so that it may be fixed
during regular working hours. In this situation
there is no need to call the 2° response person.
Page 12
TERA A State-Of-The-Science Research Facility
-------
Experimental Tasks and Facilities
Reporting. Reporting system failures, problems,
and corrective actions is critical to the long-term
management of TERA. Therefore, any problems
and corrective actions are to be recorded in the
TERA log book. This log book is also kept in the
Mission Control Building. Problems and correc-
tive actions must be reported to the TERA Site
Manager orally on the next regular business day
Training:
Both the 1° and 2° response people must have
training commensurate with their respective re-
sponsibilities. The TERA Site Manager is re-
sponsible for determining the level of training,
establishing a training program, and developing
and maintaining the key to corrective actions
(described above). Periodic training updates may
be required as the system is modified.
Page 13
TERA A Slate Of-The-Saence Research Facility
-------
Experimental Tasks and Facilities
TERA A Siate-Of-The-Science Research Facility
-------
Table 1. List of aboveground and belowground environmental parameters to be collected at TERA Facility and projected completion dates
Environmental Parameter
Sensor
Status
Projected Start-up Date
CO2 Concentration
InfraRed Gas Analyzer
Installed
Air Temperature
Thermistor
Installed
Dew Point Temperature
Hygrometer
Installed
Light Level
PAR Sensor
Installed
Needle Temperature
Thermocouple
Spring 1995
Plant Transpiration
Stem Flow Gauge
Fall 1994
Soil Temperature
Thermistor
Installed
Soil Moisture
Time Domain Reflectomelry
Installed
Neutron Probe
Tubes Installed
Technique Applied Summer 1994
Soil Chemistry
Tension Lysimeters
Installed
Soil Gases
Gas Sample Wells
Installed
Roots
Minirhizotron tubes
Installed
-------
Experimental Tasks and Facilities
Measured Air Temperature
Ambient +4
Ambient
Ambient +4
Ambient
-1 - IIIIIHIIIIIIIHHIIIU|IMIIMHIIIHMHIMII[IIIIIIIMIIIIIMIIIIIII|IIIIIIIIIIMUIIIIIIIII|IIHIHIIIIIIHIIIIIIIHHIII llllll llllllllllll|llllll III HUH nnmi
21-Feb 22-Feb 23-Feb 24-Feb 25-Feb 26-Feb 27-Feb 28-Feb
Date
Figure 1 Data are the average (± 1 SD) of 30,1 -minute averages collected between 2/21/94 - 2/27/94. Figure la presents
air temperatures for each treatment and illustrates the ability of the Terracosms to track changes in ambient temperature
Figure 1 b presents the difference between the actual and target (set point) air temperature for each treatment and demonstrates
the quality of Terracosm air temperature control. Ambient temperature data are plotted along a zero base line while elevated
temperature (ambient + 4) are plotted along a baseline of 4 in order to preserve prescribed treatment offset.
Page 15
TERA A State-Of-The-Science Research Facility
-------
Table 2 Terracosm climate and COj control performance characteristics for the week of 2/21 /94 - 2/27/94 Values are derived from analysis of 10,080 1 -
minute averages for air temperature, dew point depression, and CC^.
Variable
Statistics
Deviation From Set
Point (Actual -
Target)
Average
Treatment
Applied
Separation Between
Elevated and
Ambient Treatments
Ambient Temperature
Average (*C)
0 2
8.1
3.8
Standard Deviation
0.6
2.7
0 2
Coefficient of Variation (%)
34
5
Elevated Temperature
Average (*C)
0.1
11.9
3.8
Standard Deviation
0.6
2.8
0 2
Coefficient of Variation (%)
24
5
Dew Point Depression in
Average ("C)
0.6
8 5
0.3
Ambient Terracosms
Standard Deviation
Coefficient of Variation (%)
3 6
3.2
38
1 0
323
Dew Point Depression In
Average ("C)
0.9
8.8
0 3
Elevated Terracosms
Standard Deviation
Coefficient of Variation (%)
3 4
3.0
34
1.0
323
Ambient C02
Average (ppm)
20
384
182
Standard Deviation
11.1
17.0
6 9
Coefficient of Variation (%)
4
4
Elevated C02
Average (ppm)
2
566
182
Standard Deviation
9.3
14.8
6 9
Coefficient of Variation (%)
3
4
I5
3
a
5
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-------
Experimental Tasks and Facilities
16
Measured Dew Point
Depression
mbient
Ambient + 4
Dew Point
Difference
-10
Deviation from Target Value
B
4_-Ambi mt + 4
Ambient
6 |HIIIUIIIMIIIIIIIHI[|IIIIIIMIHIMIHIHIU|IH I1IIIM iniii|iiiif I1IIIITHI iiiiiiiiihim UHI|| 111111111111 n n 11| i»
21-Feb 22-Feb 23-Feb 24-Feb 25-Feb 26-Feb 27-Feb 28-Feb
Date
Figure 2. Data are the average of 30,1-minute averages collected between 2/21/94 - 2/27/94. The top two lines of Figure 2a present dew
point depression for each treatment and illustrate the ability of the Tenacosms to track changes in ambient dew point depression The bottom
line of Figure 2a shows the average difference between ambient and elevated (ambient + 4) dew point depression Table 2b presents the
difference between the actual and target (set point) dew point depression for each treatment and demonstrates the quality of Terracosm
dew point depression control Both lines are plotted along a zero baseline to show how dew point depression control deviated from the
target value with ume
Page 17 TERA A State Of-The-Science Research Facility
-------
Experimental Tasks and Facilities
Deviation from Target Value
40-
Ambient
E 30-
10-
2
concentrations for each treatment and illustrates the ability of the Terracosms to track changes in ambient CO2 Figure 3b presents the
difference between the actual and target (set point) CO2 for each treatment and demonstrates the quality of Terracosm CO2 control
Page 18
TERA A Stale Of-The-Science Research Facility
-------
Experimental Tasks and Facilities
Location of Terracosm Soil Thermistors: Top-down View
1M
2M
90 cm
i 75 cm
50 cm
T~
50 cm
J_
10 cm
I
25 cm
SE Quadrant
Terracosms
1,4,10,11
NE Quadrant
Terracosms
2, 3, 12
SW Quadrant
Terracosms
7, 13,14
NW Quadrant
Terracosms
5, 6, 8, 9
Figure 4. Positions of the 3 soil thermistors located at all depths in the Terracosm soil horizons. Each Terracosm has a total
of 9 thermistors; 3 in each of the 3 soil horizons Also identified are the quadrants in which the soil thermistors are located
for each Terracosm
Page 19
TERA A Siate-Of-The-Science Research Facility
-------
Experimental Tasks and Facilities
Ambient +4
11.5-
A-Horizon
10.5-
Ambient
B-Horizon
Ambient +4
Ambient
o
o
a>
(0
at
a.
E
a>
H
O
(/)
6.5-
12.5
11.5
10.5
9.5-
8.5-
7.5-
6.5-
C-Horizon
Ambient +4
cSI
o>
c\)
C\J
c\l
o>
?5
c3
§1
LO
CT5
to
OJ
cvJ
O)
CO
CVJ
c\)
~r
o>
CM
cvl
Ambient
CT5
00
Figure 5 Diurnal fluctuations in soil temperature for each of the three soil horizons in Terracosm #2 (ambient air temperature/
ambient CO2) and Terracosm #5 (ambient air temperature + 4'C/ambient CO2) Data are the average temperature for three
thermistors located in the same soil horizon Data were collected every 15 minutes between 2/21/94 - 2/27/94
TERA A State Of-The-Science Research Facility
Page 20
-------
Table 3 Estimates of total cumulative amount of experimental downtime1 observed within treatments and across all Terracosms These data
summarize observations collected every 1 minute between 2/21/94 - 3/28/94 Total number of observations collected for each treatment and lor
all Terracosms are 181,440 and 725,760, respectively
Treatment Code2
Time Doors Open
Time
to Calibrate
IRGA's
Time
Terracosms Off-
Llne^
Total Downtime
%
hrs
%
hrs
%
hrs
%
hrs
tc
0.3
9
0.7
20
0 2
7
1
2
37
Tc
0 2
7
0.7
20
0.2
7
1
1
34
tc
0 7
20
0 7
20
0.2
7
1
6
48
TC
1 9
58
0 7
20
0 2
7
2
8
86
Total
0 8
94
0.7
80
0 2
28
1
7
205
1Time Terracosm environmental conditions can not be controlled for one or more reasons
O
Treatment codes t = ambient temperature. T = ambient temperature + 4*C. c = ambient C02, and C = ambient C02 + 200 ppm
O
"'Time that a Terracosm processor Is not controlling the environment or acquiring data due to on-line programming
-------
DATA ACQUISITION AND DATABASE MANAGEMENT
-------
Experimental Tasks and Facilities
TERA DATA ACQUISITION AND DATABASE MANAGEMENT
PARTICIPANT Brent Baker (CSC)
INTRODUCTION
The goal is to establish and maintain a single
cohesive database of the raw data collected from
the TERA project
OBJECTIVES:
Short-term
Database should include data collected by real-
time sensors and data collected manually, e.g.
results of chemical analysis, physical measure-
ments of the trees, etc.
Database should have a very high reliability.
Limitations due to missing data or hardware
or software faults should be well defined and
understood
Database should be accessible from mul-
tiple computer systems and be fairly easy to
convert from its native format to other rel-
evant formats.
Long-term
At the completion of the project the database
should be available to other researchers on
magnetic or optical media or via the Internet.
Link the non-tabular data from the experiment
into the main database in some meaningful
way. For example, the data collected from video
images of the tree roots and root growth infor-
mation, and the digital images of the trees and
leaf area calculations.
DATABASE DESIGN:
Hardware
An Allen-Bradley PLC 5/20 (microprocessor) at-
tached to every chamber performs environmental
control and data acquisition. A PC running
Microsoft Windows with an Allen-Bradley inter-
face card that communicates with the terracosms
and provides data collection. This PC is connected
via Ethernet to a Sun Sparc IPX workstation that
contains the database.
Software
The PLC 5/20 at each chamber creates a packet of
data values once a minute and sends it to the PC
running Windows. A C program running on the
PC captures this data packet and forwards it to the
Unix workstation.
The link from the PC to the Unix workstation is
based on the TCP/IP communication protocol.
This standard guarantees the delivery of all data
packets in the correct order. In practice, this means,
that the PC sending the data has confirmation that
the data was delivered. The software re-sends all
data packets that were not delivered correctly. If
the connection between the PC and the Unix
workstation breaks, the PC sending the data stores
the data until the connection is restored. The PC
can store several days worth of data.
QUALITY ASSURANCE:
Data Validity Checks
The PC takes a message packet from each cham-
ber once a minute. Embedded in this message at
fixed locations are unique values that cannot be
mistaken for data. These values are passed on,
unchanged, to the Unix workstation. If the Unix
system doesn't find these values where it expects
them, it marks the data as tainted when it places it
in the database. At this writing 0.00608% of the
data points have tainted values. Our current policy
is to leave tainted data as is. We will use valid data
within a few minutes of the bad data as a close
approximation of what the tainted value should
have been.
Page 1
TERA Data Acquisition and Database Management
-------
Experimental Tasks and Facilities
Redundancy
Two identical PC's are running Windows. Ei-
ther one can act as the conduit that collects data
from the terracosms and passes it on to the Unix
workstation.
Backups
The database is backed up every night with
standard Unix utilities according to the follow-
ing protocol:
Every night, all the files that have been changed
since the previous night's backups are backed
up. Because the database changes every
minute, it is included in this backup.
The last two weeks of daily backups are saved
before tapes are reused.
On Friday nights, all the files that have changed
since the previous Friday are backed up. This
also includes the database because in changes
every minute It also includes files that change
relatively infrequently
The last four Friday night backups are saved.
Once a month, every file that has changed in
the past month is backed up. This backs up
files that change even more infrequently.
Monthly backup tapes are never reused.
Finally, a complete backup is performed peri-
odically. This backs up everything, including
all system files. Full backup tapes are kept
permanently and never reused. Standard Unix
backup utilities are used so that any compe-
tent system manager can restore the database
in case of a crash.
Possible Data Loss
Probably the greatest risk of data loss is the
failure of the hard disk where the database
resides. In such a case the largest loss would be
no greater than 24 hours, because our database is
backed up every midnight. There are some soft-
ware utilities that allow part of data from this
period to be reconstructed. Another potential
loss is due to power failure. However, both the
PC's and the Unix workstation are on
uninterruptable power supplies. The TERA fa-
cility also has a backup generator.
Problems
The PC sends a data packet to the alarm system
once a minute. If five consecutive minutes pass
without a message from the PC, the database
manager is paged. If the Unix workstation goes
down intentionally, such as for nightly backups,
but does not come back on-line within 1 1/2 hours,
the database manager is paged. If the Unix work-
station goes down unintentionally and does not
come back on-line within 1/2 hour, the database
manager is paged. As long as the PC is running it
can store data for up to several days, even when the
Unix workstation goes down. To date, the data-
base manager has never been paged due to a failure
in the system that hasn't been caused by testing
new software or other maintenance work occur-
ring.
DATABASE PERFORMANCE:
Database Growth rate
The climate control data grows at about 1.6 mega-
bytes a day. The soil chemistry, and other analysis
data will not contribute significantly to this growth
rate. Soil moisture data (TDR) will increase the
growth rate of the database. Currently soil mois-
ture data are not being kept in the main database,
but will be as soon as software to do the data
transfer can be written.
Efficiency
Data collection and logging data into the database
do not consume a large fraction of the computing
power of either the PC or the Unix workstation
This means that when other tasks need to be done,
such as database access or program maintenance,
our system can handle these tasks, and still per-
form data collection.
Database Accessibility
At this writing the main method of data access is
via the charts created for the weekly TERA staff
meetings, although data files can be extracted for
Page TERA Data Acquisition and Database Management
-------
Experimental Tasks and Facilities
use in various statistical analyses. The data files
can be varied fairly quickly by anyone who knows
Unix, Perl and SQL. That's not everyone, but such
skills aren't overwhelming rare in the computer
science market place. In addition, there is a pro-
gram that prints out the data stream arriving from
the PC once a minute as the data arrives. A window
can be created on the workstation that will allow
the user to scroll through the last few hours of
collected data. This program is available to any-
one with access to a Unix workstation on site
(workstations are readily available).
In the future, we'd like to extend access to the
database to Macintosh and PC users. Our rela-
tional database management software has options
that access, via our local network, from a spread-
sheet running on a PC or a Macintosh.
Database Security
Everyone has permission to examine the data if
they have an account on the Unix workstation that
holds the database. However only an individual
with system manager privileges can alter the con-
tents of the database that is a small number of
people.
Page 3
TERA Data Acquisition and Database Management
-------
United States Environmental Research May 1994
Environmental Protection Laboratory - Corvallis
Agency Corvallis, OR 97333
QUALITY ASSURANCE
PROGRAM
Project: Effects of C02
and Climate Change
on Forest Trees
For additional Information fontact:
Dr.David T. Tingey
Program Leader
ERL-CorvalJis
(503)754-4621
-------
ACKNOWLEDGMENT
The Project Staff greatfully thanks S. Volk for developing the
format for this book and producing the booklet in its final form.
-------
Quality Assurance Program
TABLE OF CONTENTS
TABLE OF CONTENTS
I. QUALITY ASSURANCE PROJECT PLAN
II. LIST OF PROJECT SOP's
III. STANDARDIZED PROJECT SOP FORMAT
IV. EXAMPLE OF PROJECT SOP 2.01
V. EXAMPLE OF EXPERIMENTAL PROTOCAL EP.06
Page i
Effects of CO 1and Climate Change on Forest Trees
-------
QUALITY ASSURANCE PROJECT PLAN
EFFECTS OF C02 AND CLIMATE CHANGE ON FOREST TREES
-------
Version Number 1
Senal Number wac^'c*1^ Co\h^4c^
Pc&r R-e- \j \ Sfa y j2> ^
QUALITY ASSURANCE PROJECT PLAN
EFFECTS OF C02 AND CLIMATE CHANGE ON FOREST TREES
April 1993
Approval:
T. Tingey S f
David
Project Leader, Effects of CO2 and climate change on forest trees
0-
David T. Tingey, Program Leader GlfibaJ^r
rocesses and Effects
Peter A. BeedlowTTerrestrial Branch Chief
>£/TP
Roben T. Lackey, ERL^Oualit/Assurance Officer
Dale
y-"2 2^93
Date
2M;
Date
4%
-------
QAPP: Effects of CO2 and Gimate Change on Forest Trees
Page 2 of 11
2. LIST OF FIGURES
Figure 1. General description of Research Tasks for illustrating the comprehensive approach
taken toward above- and belowground components of the Douglas fir seedling system.
Figure 2. Generalized sampling schedule for Research Tasks illustrating the sequencing of
sampling linked to phenological events in both the above- and belowground
components of the Douglas fir seedling system.
3. LIST OF TABLES
Table 1. Overall management of Project Research Tasks and the distribution of responsibility for
tasks among Principal Investigators.
4. INTRODUCTION
The concentrations of several greenhouse gases are increasing in the Earth's atmosphere
(Houghton et al. 1990) largely due to human activities including fossil fuel combustion,
deforestation, air conditioners and land use changes (Houghton and Woodwell 1989). These
gases [primarily CO2, N2O, CH4 and chloroflurocarbons (CFCs) (Houghton et al. 1990)] absorb
some of the sun's energy reflected from the Earth causing the atmosphere to warm. Some
warming of the atmosphere is needed so that life as we know it can exist (Mitchell 1989),
however, as greenhouse gas concentrations continue to increase excessive atmospheric warming
may occur leading to an altered global climate. The over arching research question is, "What will
be the effects of climatic change?"
Green plants require CO2 for photosynthesis, however, cunent concentrations of atmospheric CO2
may limit plant growth, meaning a growth response will be observed at CO2 concentrations above
cunent levels (Oechel and Strain 1985; Allen 1990). This is an important factor when considering
the response of green plants to elevated atmospheric CO2. Will primary production of green plants
increase in response to elevated CO2 and thereby reduce atmospheric CO2 concentrations, or will
primary production be restricted due to some consequent resource limitations?
Understanding the response of forested systems is particularly important because they contain
approximately 90% of the Earth's terrestrial biomass. Depending on the response of forests under
altered global climates they may become a large sink for, or source of atmospheric CO2 (Waring
and Schlesinger 1985; Post et al. 1990).
5. PROJECT DESCRIPTION
A focus of the U. S. Environmental Protection Agency's Global Climate Change Research
Program is to understand better the relationships between atmospheric CO2 concentrations, global
climate and the terrestrial biosphere. The research Project Effects of CO2 and Climate Change on
Forest Trees (U.S. EPA 1993) was developed to provide a scientific understanding of the
interactions between CO2, climate and biospheric processes necessary to make some of the difficult
policy decisions facing the U. S. Government. The Research Project will be conducted in the
Terrestrial Ecophysiology Research Area (TERA) facility in Corvallis. The Project is focused on
and organized around examining the ecophysiological responses of the above- and belowground
components of forest tree systems to changes in atmospheric CO2 and climate (Figure 1). The
research also is structured according to a regimen of spatially- and temporally-organized
-------
QAPP: Effects of CO2 and Climate Change on Forest Trees
Page 3 of 11
measurements based on above- and belowground phenological events of the experimental plants,
and is depicted in the generalized sampling schedule presented in Figure 2. Refer to the Research
Plan for a detailed discussion of the Project (U.S. EPA 1993).
Experimental Research Tasks
Talk 1
Shoot Carbon sod
Witir Flum
Ta*k 4
SjnUm Water
Plaal Wattf
Task 3
Syitem Nutrients
PUal Nuu-kdu
Taik 5
Litter Layer
Taik 1
SoD Biology
Figure 1. General description of Research Tasks for illustrating the
comprehensive approach taken toward above- and belowground compo-
nents of the Douglas-fir seedling system.
-------
QAPP: Effects of CO2 and Gimate Change on Forest Trees
Page 4 of 11
fall-
FALL
WbcbPlaal Gai
Eiekufi
Nndle A Brascfc
Cm bcbtip
Sub Dlaniter
CERLS D«rkn
Plan Wwr
Statu
c
Continuously
]
1 Every Eifthi Wub | Every Four WttU | Every Ei&hi Weeks j
[
Coniinuouily
:
DUaMer^Mli 1 ual I Every Eight Weeks | Every Four Weeks | Every Eight Weeks )
Nttdke Art*
lni|i Analysts
Bud PhtDOlOQT
Branch & Sun
Architecture
Needle
Sample
r
Every Four Weeks
J
Every Four Weeks
Shoou Dormaoi
Soil Surface
Bud
Brtik
Shooi Growth
Bud
Sa
Shoou Dormant
Rspid Root Growth
Rapid Root Growth
I
s,
I
s
Cona-to-Drptb
Soil & Root Samples
Litter Bap &
Needk Pecks
Root Images
X
X
X
X
Every Four Weeks
Soil Solutions
Soil Profile
Gases
Soil Surface
Case*
Soil Water
Content
Every Four
Every Four Weeks
Every Four Weeks
Continuously
Figure 1 Generalized sampling schedule for Research Tasks illustrating the sequencing of
sampling linked to phenological events in both the above- and belowground components of
the Douglas-fir seedling system.
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QAPP: Effects of CO2 and Gimate Change on Forest Trees
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6. PROJECT QA ORGANIZATION AND RESPONSIBILITIES
Achieving the QA goals of the Project is predicated on establishing and implementing an efficient
and manageable organizational structure for QA/QC within the Project, and between the Project and
Branch Management. In developing the Project's QA organizational structure to meet the QA
goals, five essential QA/QC elements were addressed: (1) QA/QC responsibilities, (2) research
responsibilities, (3) communications, (4) document control, and (5) the importance of Standard
Operating Procedures (SOPs).
Although the five QA/QC elements described above are highly interdependent to successfully
execute the Project's research, SOPs have an especially critical role. Standard Operating
Procedures are the keystone elements upon which Project Quality Assurance Program and
experimental procedures will be based; Management and Project personnel will interact in the other
four elements of the Project QA program. Because the Project's QA organizational structure
depends heavily on SOPs, their format was structured to serve as guidelines for all Project
personnel to accomplish both the scientific and QA/QC procedures of the Project (see "Importance
of Standard Operating Procedures" below in this section).
QA/QC Responsibilities:
The ERL-Corvallis management and research staff share responsibility for implementing the
Laboratory's QA policies, and they are accountable for those aspects of QA/QC associated with
their work areas. The QA Responsibilities in this Quality Assurance Project Plan (QAPP) were
derived from Section 1.0 of the ERL-C Plan (U.S. EPA 1989). The duties of theQA officer and
interaction with the Project are detailed in the ERL-C QA Plan (U.S. EPA 1989). The general
Project QA/QC organizational structure can be described as follows:
Program Leader
Project Leader
Pi's
Branch Chief
^Project ^
QA
L Coordinator
Laboratory
QA Officer
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Branch Chief - The Project is managed within the Terrestrial Branch and comes under the
general responsibilities of the Terrestrial Branch Chief. The Branch Chief is responsible for all
projects within the Branch and for ensuring that all technical outputs meet the quality requirements
of the Laboratory and Agency. The Branch Chief also is in the direct management line to the
Project Leaders, and can apply Branch resources to resolve QA issues. The Branch Chiefs key
QA responsibilities include:
review and evaluate work on QA implementation and progress
review and evaluate the quality of outputs generated by each project
review and evaluate audit and performance evaluation reports.
Program Leader - The Project is managed within the Global Processes and Effects Program
(GPEP) and comes under the general responsibilities of the Program Leader. The Program Leader
is responsible for all projects within the Program and for ensuring that all technical outputs meet
the quality requirements of the Laboratory and Agency. The Program Leader is also the direct line
manager to the Project Leader, and can apply Program resources to resolve QA issues. The
Program Leader's key QA responsibilities include:
review and evaluate work on QA implementation and progress
review and evaluate the quality of outputs generated by each project
review and evaluate audit and performance evaluation reports.
Project Leader (Project Officer) - The Project Leader is management's principle contact with
the Project and is responsible for the performance and coordination of the Project. The Project
Leader determines quality criteria based on the intended use of the results to be generated, and
communicates these criteria to the Project participants. The Project Leader's key QA
responsibilities include:
insure the development of the QA Project Plan (QAPP)
negotiate quality requirements with Project participants
ensure that SOPs are developed, review and approve SOPs
review and approve Project QA outputs
decide allocation of resources to resolve QA issues.
Project QA Coordinator - The Project QA Coordinator assists the Project Leader in fulfilling
QA responsibilities and serves as a resource to assist the Principal Investigators (Pis) and other
Project participants in fulfilling their QC/QA responsibilities, however, he receives direct
supervision and performance evaluation from the Program Leader. The Project QA/QC
Coordinator's key QA responsibilities include:
maintain original-approved copies of the QAPP and all SOPs
distribute approved copies of the QAPP, SOPs and manage the Project's document
control policy
conduct or assist in audits
review laboratory notebooks and other primary data sources
review QA data every six months (Performance and System Audits, see Section 14
below) and archive QA/QC data
make data quality determinations based on the QC data collected and document the
determinations
prepare annual QA reports for submission to Project Leader and Branch Chief (QA
Reports to Management, see Section 18 below)
as required, report problems and corrective actions to the Project Leader, and ensure that
the problems and corrective actions are documented.
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Principle Investigator (Researcher) -The Principal Investigator (PI) is responsible for
carrying out the entire or portions of the Project Research Task(s) and insuring the quality of
the results generated from the task(s). The PI therefore places substantial emphasis on the
QC aspects of the Project. The Pi's key QC/QA responsibilities include:
participate in the preparation of the QA Project Plan (QAPP)
negotiate quality requirements with the Project Leader
wnte or assist in writing the SOPs
train Project participants to perform and evaluate QC measurements
train Project participants to perform and document preventative maintenance
report problems and corrective actions to the Project QA Coordinator
verify that QC activities are performed and data quality is determined.
insure that required corrective actions are implemented.
Project Scientists - Project Scientists work directly on the Project's research and QAJQC
procedures by interacting frequently with other Project participants. The Project Scientist's key
QC/QA responsibilities include:
assist in writing SOPs as necessary
implement the SOPs
perform and evaluate QC measurements
perform and document preventative maintenance
report problems and corrective actions to the PI
implement corrective actions.
Research Responsibilities:
To insure that all facets of the Project progress on schedule and that important parts of the Project
are not omitted, individual Pis are assigned specific subtasks of the Research Project (Table 1).
Communications:
The Project is a sophisticated, very complex, multi-discipline, research endeavor. Completing the
Projeci successfully requires effective communication among Project participants, a: all levels.
The Pis (Table 1) and the will meet at least onoe a month and probably more often during periods
of intense activity to: (1) coordinate sampling and various experimental activities, (2) exchange data
and information about the various tasks, (3) share scientific information, and (4) refine and/or
modify the Research Plan, the QAPP and the SOPs.
The Project Leader, Pis and other Project participants will meet at least once a month to: (1)
coordinate experimental activities (e.g, availability of equipment, harvests, etc), (2) exchange
information and (3) to resolve possible problems.
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QAPP. Effects of CO2 and Climate Change on Forest Trees Page 8 of 11
Tablel. Research Responsibilities
TASKS/Subtaki
Principle Investigator (1)
TASK 1 Shoot Carbon and Water Flux
Gas Exchange Needle D Olszyk
Gas Exchange - Canopy D. Tingey
Needle Area Image Analysis D. Olszyk
Stem FIow/Transptrauon D Olszyk
TASK 2 Shoot Growth and Phenology
Shoot Growth/Phenology D Olszyk
TASK 3 ¦ System Nutrients
Biochemistry
Plant Nutrients
Soil Nutrients
D Tingey
D Tingey
M Johnson (2)
TASK 4 System Water
Plant Water
TDR - Soil Water
D Olszyk
M Johnson {2)
TASK 5 - Litter Layer
Litter Layer Addition
Decomposition
M Johnson (2)
M Johnson (2)
TASK 6 Root Growth and Phenology
Cores-to-depth P. Rygiewicz
Minirhizotron M Johnson (2)
TASK 7 - Soil Biology
Microbiological Indices
Soil Fauna
Soil Enzymes
Myconluzae
Biogenic Gases
P Rygiewicz
P Rygiewicz
P Rygiewicz
P Rygiewicz
M Johnson
TASK 8 Soil Organic Matter
B Gnffis
(1) Alt are EPA unless other wise specified
(2) On-site Contractor
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QAPP: Effects of CO2 and Gimate Change on Forest Trees
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Document Control:
The QAPP and the SOPs are key documents for use in implementing a QA program for the Project;
consequently, it is important that all Project participants have acoess to the current-approved QAPP
and SOPs. To insure this is done, a document control procedure will be implemented.
The Project QA Coordinator will be responsible for maintaining the original signed copies of the
QAPP and approved SOPs, and for distributing copies of these documents to Project participants,
as needed. The Project QA Coordinator will assign a version number to the approved QAPP and
each approved SOP, and serial numbers to each authorized copy. The Project QA Coordinator will
maintain a database that registers all authorized version and serial numbers and transmittal copies to
their respective holders to assure that all registered recipients of the QAPP and SOPs can be kept
current with any changes that may occur throughout the Project. When requested, the Project QA
Coordinator will distribute registered copies of the QAPP and approved SOPs to Project
participants. The registered copies will be acoompanied by a memo that the recipients are to sign
and return to the Project QA Coordinator indicating receipt of the QAPP and or SOPs. The date of
this transaction, the new version number, and the document serial number will be recorded in the
database maintained by the QA Coordinator. If the QAPP or a SOP is revised, the Project QA
Coordinator will distribute copies of the revised QAPP or SOP to all registered copy holders,
along with a memo that they are to sign and return with the out-dated QAPP or SOP to the Project
QA Coordinator. This transaction will document the receipt of the revised QAPP or SOP, and
insure that out-dated copies of these documents are not in use. When any additional copies of the
QAPP or SOPs are required, they must be obtained through the Project QA Coordinator to
maintain strict document control and tracking.
Importance of Standard Operating Procedures:
In addition to the Research Plan (U.S. EPA 1993), SOPs provide the detailed information to
conduct the individual research and QA/QC measurements in the Research Tasks. Because SOPs
hold a keystone position in the Project QA program; the format of our SOPs was designed to
accomplish four specific objectives:
1) provide sufficient information for individuals to conduct the research
2) provide a written record of how the data were collected
3) present the QA/QC needs in a form to be used by the Project participants, and
4) provide a standardized format for reporting data.
Standard Operating Procedures will be developed for all environmental measurements required to
fulfill the Research Tasks/subtasks shown in Table 1. Given the central role of SOPs in the
Project's QA program, much of the detailed QA/QC information will be contained in the approved
SOPs. Additional details of and the theoretical basis for the Research Tasks are contained in
Section 5 of the Research Plan (U.S. EPA 1993). As required by the Laboratory Quality
Assurance Program Plan (U.S. EPA 1989), the Laboratory Quality Assurance Officer will review
the SOPs and the Project Leader will review and approve them.
We believe that if the Project participants understand why the QA/QC data are needed and are
shown how the QA/QC data are linked to specific research procedures, they will take greater
efforts to insure that QA/QC practices are followed and produce high quality data. The following
sections of the QAPP make reference to the specific sections of the SOPs where the detailed
QA/QC information is contained.
7. OBJECTIVES OF MEASUREMENT
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QAPP: Effects of CO2 and Climate Change on Forest Trees
Page 10 of 11
To insure that the appropriate data are collected and that the data are collected with appropriate
accuracy, precision and sampled at the proper frequency, data quality objectives (DQOs) will be
established for the measurement methods use to collect environmental data (see individual SOPs
for specific details). As staled in Section 6 above, the Project Leader will work with the Pis to
develop the DQOs for various measurements to insure that the data meet Project goals and are
consistent with those of other similar projects. The specific DQOs for each measurement are
contained in Section D, "Objectives Statement" of the individual SOPs.
8. SAMPLING PROCEDURES
Samples will be taken according to the generalized schedule presented in Figure 2. Specific details
for sampling are presented in the individual SOPs, See Section F, "Sampling Procedures and
Sample Custody".
9. SAMPLE CUSTODY
See Section F, "Sampling Procedures and Sample Custody", of individual SOPs.
10. CALIBRATION PROCEDURES AND FREQUENCY
See Section H, "Quality Assurance/Quality Control", of individual SOPs.
11. ANALYTICAL PROCEDURES
See Section G, "Analytical Procedures", of individual SOPs.
12. DATA REDUCTION, VALIDATION AND REPORTING
See Section J, "Data Reduction, Validation and Archiving", of individual SOPs.
13. INTERNAL QUALITY CONTROL CHECKS
See Section H, "Quality Assurance/Quality Control", of individual SOPs.
14. PERFORMANCE AND SYSTEM AUDITS
In accordance with the Laboratory QA Plan, the project will participate in an extramural QA audit
every two years (U.S. EPA 1989).
In addition, to insure a high level of QA activity and compliance with the QAPP, the Project will
audited by the Project QA Coordinator twice per year. The Project QA Coordinator will collect and
review the QC/QA data collected by the Principal Investigator/Project Scientists from the
performance audits for each individual measurement system (identified in the individual SOPs).
The Project QA Coordinator will use these data to develop a performance and data quality audit
report (twice a year). See Section H, "Quality Assurance/Quality Control", of the individual SOPs
for the details on obtaining QC/QA data for the individual measurement systems used io the
Project. The twice-a-year internal audit reports will form the basis upon which the Project will
develop the annual "QUALITY ASSURANCE REPORTS TO MANAGEMENT" (Section
18, below). In turn, the annual "QUALITY ASSURANCE REPORTS TO
MANAGEMENT" and the QAPP will form the basis upon which the Project and the Branch will
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QAPP: Effects of CO2 and Climate Change on Forest Trees
Page 11 of 11
participate in the extramural (outside the Project) ERL-C QA Program audits every two years as
staled in approved Laboratory QA Plan (U.S. EPA 1989).
Responsibility for ensuring the realization of the twice-yearly audits and reports, and the ERL-C
QA Program audit lies with the Project Leader (Item 1.7.2, and Item 14 of Appendix B-2, U.S.
EPA 1989) who will be supported by the Project QA Coordinator.
15. PREVENTIVE MAINTENANCE
See Section I, "Preventive Maintenance and Corrective Action", of individual SOPs.
16. SPECIFIC ROUTINE PROCEDURES USED TO ASSESS DATA
PRECISION, ACCURACY AND COMPLETENESS
See Section H, "Quality Assurance/Quality Control", of individual SOPs.
17. CORRECTIVE ACTIONS
See Section 1, "Preventive Maintenance and Corrective Action", of individual SOPs.
18. QUALITY ASSURANCE REPORTS TO MANAGEMENT
Annual summaries of the internal Project audits (see Section 14 above) will be prepared by the
Project Quality Assurance Coordinator and presented to the Project Leader for approval, necessary
corrective action and forwarded to the Program Leader and Terrestrial Branch Chief for review and
evaluation (Iiem 1.7.3, U.S. EPA 1989) and any necessary action. The summaries will include
the items specified in Item 5.16 of the ERL-C Quality Assurance Program document (U.S. EPA
1989) plus any other findings contained in the overall performance audit reports. Also Project,
Program and Branch management will receive copies of the external QA audits (every two years)
for review and appropriate action.
Specific Project deliverables are listed in the Peer Reviewed Research Plan (U.S. EPA 1993).
19. REFERENCES
Houghton, J.T., G.J. Jenkins and J.J. Ephraums (eds.). 1990. Climate Change: The IPCC
(Intergovernmental Panel on Gimate Change) Scientific Assessment. Cambridge University Press,
Cambridge, England.
Houghton, R.A., and G.M. Woodwell. 1989. Global climate change. Scicn. Amer. 260:36-44.
Mitchell, J.F.B. 1989. The "greenhouse" effect and climate change. Rev. Geophys. 27:115-139.
Oechel, W.C. and B.R. Strain. 198S. Native species responses to increased atmospheric carbon
dioxide concentration. In: Direct Effects of Increasing Carbon Dioxide on Vegetation. U.S.
DOE, DOE/ER-0238
U.S. EPA. 1989. Quality assurance program plan for the Environmental Research Laboratory
Corvallis. U.S. Environmental Protection Agency, Office of Research and Development, Corvallis,
OR. Document Control No. EPA Corvallis No. 110.001. Revision 2.
U. S. EPA. 1993. Effects of CO2 and climate change on forest trees: Research plan. Global Processes
and Effects Program. Environmental Research Laboratory Corvallis, U. S. Environmental
Protection Agency, Corvallis, OR.
Waring, R.H., and W.H. Schlesinger. 1985. Forest ecosystems: Concepts and management.
Academic Press Inc., San Diego, California.
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LIST OF PROJECT SOPs
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Effects of CO2 tod Climate Change on Forest Trees Project 4/1/94
LIST OF SOPs
SOP# Version* SOP Title
Task 1 Shoot Carbon and Water Flux
1.01 Canopy Gas Exchange
1.02 1.00 NeedJe/Branch Gas Exchange
1.03 Canopy Transpiration
1.04 Stem Flow
Task 2 Shoot Growth and Phenology
2.01 1.10 Shoot Biological Measurements
2.02 Stem Diameter/CERES
Task 3 System Nutrients
3.01 CNS Elemental Analysis
3.02 Nutrient Analysis
3.03 TNC Analysis
3.04 ICP Analysis
Task 4 System Water
4.01 Soil Water Content TDR
4.02 Soil Water Content - Neutron Probe
4.03 Needle Water Status - (Pressure Bomb)
4.04 Plant Water Stress (CERES)
Task 5 litter Layer
5.01 Litter Decomposition
Task 6 ' Root Growth and Phenology
6.01 Ktioirhizotroos
6.02 "ROOTS" Image Analysis
6.03 Morphological Fractionation of Roots
Task 7 Soli Biology
7.01 Terra cosm Soil Cores
7.02 Microbial Population Dynamics
7.03 Total Bacteria
7.04 Nematode Population and Community Structure
7.05 Protozoan Population and Community Structure
7.06 Ectomyconiuzal Colonization of Roots
7.07 Ectomycorrhizae Morphological Classification
7.08 Ectomyeonfaizae Nucleic Acid Classification
7.09 Biogenic Gases from Soil Processes: Gas Chromatography Analysis
7.10 Biogenic Gases from Soil Head Space: Infrared Analysis
7.11 Soil Fauna Population and Community Structure
7.12 Soil Enzymes
Task 8 Soil Organic Matter
8.01 Different SOP are anticipated in this Task
OTHER PROTOCOLS
OP.Ol TERA Weather Station SOP
OP.02 Measurement System Calibration (Temperature etc.)
OP.03 Database Documentation
-------
SOP* version# SOP Title
EXPERIMENTAL PROCEDURES ADDENDA
EP.01 Soil, Litter and Plant Tissue Preparation
(q.v. Tasks 5,6, & 7 - SOP 5.01, 6.03)
EP.02 Litter Layer Addition and Sampling
(q.v. task 5 - SOP 5.01)
EP.03 Litter Bag and Needle Pack Construction
(q.v. Task 5 - SOP 5.01)
EP.04 1.00 Rounding OftfSignificant Figure Rules
(general)
EP.05 Soil Solution Collection
(q.v. Task 4)
EP.06 1.00 Tree Selection Criteria, Shoot & Root Sampling, and Tree Planting & Culture
(q.v. Task 3 - SOP 3.01, 3.02, 3.03, 3.04;
Task 6 - SOP 6.03;
Task 7 - SOP 7.01 ... 7.08, 7.11)
EP.07 1.00 Collembola Identification
EP.08 TERA Chamber Irrigation System Description
(q.v. Task 4)
EP.09 24-Hour TERA Monitoring and Emergency Response Plan
EP. 10 1.00 Electron Microscopy of Mycorrhizal Roots
EP.l 1 Sample Tracking Procedure
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STANDARDIZED PROJECT SOP FORMAT
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Page 1 of 4
GPEP SOP 0
Version: 1.00
Project Standardized SOP Format
Project SOP Number 0
Version: 1.00 . r
Serial Number ? "toe.
March 1993
Prepared bv:
Date:
Approval.
Principal Investigator
bavid T. Tingey, Project Leader
Date:
Date:
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Page 2 of 4
GPEP SOP 0
Version: 1.00
Introduction
The Project (
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Page 3 of 4
GPEP SOP 0
Version: 1.00
established based on the intended use of the data and not solely upon comparability with other
studies or the highest level of precision/accuracy attainable.
E. List of Equipment and Reagents
A list of equipment and reagents used in the SOP should be made. The source of any special
equipment or reagent not normally found in typical research situations as well as calibration and
reference should be listed, as well as their purity.
F. Sampling Procedures and Sample Custody
The sampling procedures will be described. Including: (1) description of sampling procedures to
insure representative samples; (2) sampling schedule; (3) selection and preparation of sample
containers; (4) sample identification and labeling; (5) sample handling, including preservation and
storage, including recommended storage time; and (6) appropriate method of sample disposal,
including waste management problems, if any (if a Waste Disposal Plan is is use, attach a copy). It
is suggested that these details be shown in a flow chart. Discussion should include security within
sample storage areas and sample archiving (location, labeling, easy retrieval).
G. Analytical Procedures
The procedure will be described in sufficient detail that it can be duplicated by qualified personnel.
Interferences and limitations of the procedure, including preparation of calibration standards and
their concentrations will be discussed. If EPA, ASTM, other standard methods and other official
procedures are selected, a copy should be included in Section L. Addendum when possible. If
other published methods are used, a copy of the reference paper should be included in Section L.
Addendum , State any modifications to these methods in this section. If appropriate, hazardous
waste handling methods will be discussed or references to other documents. Also waste
minimization procedures will be included if appropriate. References for all methods should also be
listed in Section K. References.
H. Quality Assurance/Quality Control
For measurement methods and devices, list appropriate calibration procedures. For analytical
instruments the number of standards used, their composition, and concentration should be
provided. State at what point during measures that the instruments are calibrated and the frequency
of recalibration. It is important to identify quality control check samples (QCCSs) that will be used
to indicate the need for recalibration.
This section should also address as necessary 1) Blanks; 2) Calibration Standards; 3) Precision and
accuracy criteria of calibration standards; 4) Detection limit verification; 5) Sample analysis pattern.
This section should also address 1) Sample Replicates precision criteria; 2) Spike samples -
accuracy criteria; 3) Remeasurements to estimate precision; 4) Measures by all observers on the
same plot to estimate accuracy or detect bias. When observational measures are made by more than
one person, it is important to address comparability between or among observers. Calibration can
include staff training and testing, and requirements for routine practice in making measures.
Explain how (by providing algorithms) precision, accuracy and completeness is calculated and
evaluated. These procedures are expected to be completed as quality control (QC) samples are
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Page 4 of 4
GPEP SOP 0
Version: 1.00
analyzed and summarized. Control charts may be used to routinely monitor precision and accuracy.
A summary table is a good way to present the quality control (QC) activities and their frequency.
This will provide information on the quality of the data (precision and accuracy) and the correct
functioning and performance of the instrument. The appropriate warning and control limits should
be identified as well as the associated corrective actions required when the control limits are
exceeded.
I. Preventive Maintenance and Corrective Action
It is important to specify the frequency of required maintenance activities. A copy of the
instrument manufacturer's preventive maintenance/corrective action schedule might be useful as an
addendum. A table or timeline is useful for summarizing preventive maintenance activities for both
field and laboratory instruments and experimental systems. Each analytical instrument will have a
log book(s) to record notes, quality control any preventive action and corrective action activity.
These entries should be dated and signed.
Corrective action procedures will be described for each protocol and will address the following
elements:
The predetermined limits for data acceptability beyond which corrective action is
required.
Procedures for corrective action.
The individual(s) responsible for initiating the corrective action and also the
mdividual(s) responsible for approving the corrective action.
J. Data Reduction, Validation and Archiving
Trace the data collection from the field or raw data sheets (include reporting units or data format
used) to computer file entry. Samples of data sheets will be included as an Appendix to the SOP.
Discuss verification of data entry, summary statistics, range and reasonableness checks and data
file back-up procedures. Provide information on location and custodian of the data and method of
file back-up and back-up frequency.
K. References
List references that are specific to the SOP.
L. Addendum (optional)
This section can include any data sheets, sample of labels, other standard procedures, published
methods, instrument manuals, and minor modifications of the SOP to suit specific use by Project
researchers.
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EXAMPLE OF PROJECT SOP 2.01
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Page ] of 31
GPEP SOP 2 01
Version 1.10
A. Signature Page
Shoot Biological Measurements
Project SOP Number 2.0]
Version: 1.10 ,
Serial Number UnW-ti cvclA
&er- R^o.euO ^ Jfrjg
August 31,1993
Prepared by:
' >Y'
David M. Oiszyk ^ -
Date:
r/W<-
7
Approver- ,
Date: /^ '/^ k
. Oiszyk, Principal Investigator
Date: SS/'fJ
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Page 2 of 31
GPEP SOP 2 01
Version 1 10
B. Table of Contents
ge
C. Introduction
D. Objectives Statement
E. List of Equipment
F. Sampling Procedures and Sample Custody
G. Analytical Procedures
H. Quality Assurance/Quality Control 15
I. Preventive Maintenance and Corrective Action 16
J. Data Reduction, Validation and Archiving 16
K References 18
L. Addenda 19
L-l Lyophilizer Operation 19
L-2 WiJy Mill Operation 20
L-3 Digital Balance Operation 21
L-4 Digital Caliper Operation 22
L-5 Area Meter Operation 24
L-6 Calibration Forms for the Leaf Area Meter, Digital Caliper,
and Digital Scale 26
L-7 Example Quality Control Chart 30
-------
List of Tables
Table 1. Data Quality Objectives for Douglas fir Shoot Biology Measurements
Table 2. Equipment Used for Douglas fir Shoot Biology Measurements
Table 3. Number of Samples per Measurement Time
List of Figures
Figure 1. Physiological-Based Schedule of Above- and Belowground
Sample Collection Timing and Frequency
Figure 2. Sample flow for chemical analysis.
Figure 3 Sample flow for final measurements.
Figure 4 Data Flow for Shoot Biological Measurements.
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Page 4 of 31
GPEP SOP 2 01
Version 1.10
C. Introduction
Shoot biological measurements are key indicators of the integrated response of tree seedlings to
changes in their environment. Shoot biology is especially affected by changes in atmospheric CO2
concentration and air temperatures (Allen et al., 1990). Thus shoot biological measurements are of
critical interest for the project Effects 0}CO2 and Climate Change on Forest Trees, as described for
Task 2 "Shoot Growth, Morphology, AJlometry, and Carbon Partitioning". The goal of the
project is to collect data for use in answering the science questions described for Task 2 in the
Research Plan:
Will shoot growth change in response to elevated CO2 and climate change?
Will shoot morphology and allometric relationships change in response to
elevated CO2 and climate change?
Will shoot phenology change in response 1o elevated COzand climate change'
Will the biochemical partitioning of C in shoots change in response to elevated
CO2 and climate change?
The research described under Task 2 will also provide inputs for modeling and integration and
inference tasks.
D. Objectives Statement
The purpose of this SOP is to document the proper procedures for making shoot biological
measurements. This SOP is designed to be used in conjunction with the Quality Assurance Project
Plan (QAPP) for the project Effects of CO2 and Climate Change on Forest Trees. The shoot data
quality objectives (DQO) are shown in Table 1. The focus is on acceptable precision, accuracy, and
completeness to insure that the shoot growth measurements are of known quality; and insure that
the data will be comparable to other data collected in this scientific field.
E. List or Equipment
The equipment used in the SOP is listed in Table 2. Calibration and reference materials are
included.
F. Sampling Procedures and Sample Custody
(1) Procedures to insure representative sampling
This study involves careful sampling and measurement of tree seedling growth to insure that
changes in response parameters can be attributed to the applied treatments and not due to bias or
error in sampling and measurement. First, to insure representative and uniform sampling and
measurement, all staff involved with measurements will be required to follow this SOP. Staff will
be trained on proper analysis procedures and use of equipment as described below.
Second, to insure representative sampling over the course of the study the following additional
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Page S of 31
C.PIP SOP 2 01
Vctsmn I 10
Table 1. Analysis of Data Quality Objectives Cor Douglas fir Short Biology Measurements
Parameter Analywr/ Units Precision * Accuracy » Completeness
Equipment
Shoot Height
Ruler
mm
5%
5%
90%
Stem Diameter
Digital Calipers
mm
5%
s%
90%
Bud/Stem Length*
Ruler
mm
.1%
S%
90%
Needle Length
Ruler
mm
5%
s%
90%
Architecture d
Counting
* branches, stems, buds
!S%
Not applicable
90%
Leaf Area
U-COR 3000
cm1
5%
5%
90%
Dry Matter
Digital Balance
m8
2%
1%
90%
¦ Precision is defined as repealed measures on same plant. Repeated measure at same location for shoot height, stem diameter, and architecture, and hud/stem
fcngth;for different needle from same general area a king item for needle length; and for same sample for .itca and dry matter. For numbers see Table 3.
* Accuracy determined using calibration standards defined in Table 2 using methodology described in the tcxl.
c Terminal bud and stem or most nearly terminal hud and stem if .here is no clear terminal.
* By age class.
5
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Page 6 of 11
C.PI P SOP 2 01
Version I 10
Tabic 2. Equipment Used for Douglas fir Shoot Biology Measurement*
Pwwiielcis Equipment
Make**
Calibration Standard c
Frequency of Measurement for Accuracy
For Project Data Quality Objective*
Shoot Height Ruler
Bud/Stem Length tidier
Stem Diameter Calipers
Needle Length Ruler
An
Dry Matter
Dry Matter
Dry Matter
Dry Matter
Dry Matter
Area Meier
Digital Balance
Freeze Drier
Grinder
Ovens
Wooden
Plastic
Browne and 9iarpe
Plastic
U-COR 3000
Mettlcr, Sartoris
Metal Ruler at 500 mm
Metal Ruler at 50 mm
Metal Bar. 10 mm.
Metal Ruler at .SO mm.
Metal Disc. 50 cm2
Whenever new ruler to be used
Whenever new ruler to he used
Ik-fore and after each Ml of samples
Whenever new ruler to he used
Before and alter each set of samples
Metal Weight Class S-1.1 or 10 g lie fore and after each set of samples
For Instrument Data Quality Objects (i e. to Prepare Samples)
To he noted
Unitrap II
Wiley Mill
Blue-M
Thermometer in Freezer d
Not applicable
Not applicable
Thermometer in Oven «
When samples put in freezer
Not applicable
Not applicable
When putting in and removing
samples from oven
* Operating institutions are found in the appendices for (be freeze drier (L-1), grinder (Lr2), digital balance (L-3), digital calipers (L-4), and leaf area meter (L-5).
The freezer, freeze drier, grinder, and ovens ate used to prepare .samples for measurements, data quality objectives are to verify conditions where applicable.
* Specific model and serial numbers should he noted at lime of measurement.
e Recommended standards. Others may be used depending on plant material to he measured.
* Freezer to be kept in range of minus 70-WFC, thermometer has digital reading on front of freerer.
* Thermometer reading obtained either from digital or recorder output from of oven.
6
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Page 7 of 31
CPEP SOP 2.01
Vcmon: 1.10
steps will be taken. At the start of the study the trees will be randomly assigned to the different
lerracosms by assigning numbers to the trees, and randomly selecting 14 of these numbers for each
terracosm. sampling will occur in the same spatial and temporal patterns as defined in this SOP
for each sampling event.
(2) Number of Samples. Numbers of samples measured per parameter are shows in Table 3.
If extra intermediate samples are taken they will be from middle subset of trees, i.e. the middle tree
in each row of five and me middle two trees in the row of four trees. These samples likely would
be only during the most rapid period of growth. Architecture measurements will be oo the middle
subset of 4 trees per terracosm. Needle samples from all trees in each terracosm will be pooled for
the needle area, weight, and tissue elements measurements. Final measurements will be made
separately on all trees except that needle samples from all trees in each terracosm will be pooled for
tissue elements measurements.
(3) Sampling schedule. Sampling will occur intermittently over the course of the experiment lo
evaluate dynamic changes in seedling growth (intermediate sampling), and at the end of the
experiment to evaluate the integrated effects of the experimental treatments over the entire course of
the study (final sampling).
The detailed intermediate sampling schedule for the terracosm study shown in Figure 1, is taken
from the Research Plan and QAPP. The intermediate measurements are physiologically-based,
i.e., the schedule was determined according to normally expected phases in seedling growth and
development over course of the year. Measurements are nondestructive (except for sampling of
needles for chemical analysis) to allow for further tree growth. Stem height and diameter, and
phenology (bud, stem, and/or needle length) will be measured more frequently (every four weeks)
during the wanner period of maximum growth (Spring-Summer), and less frequently (every eight
weeks) during the other, cooler, periods with expected slower growth (Fall-Winter). Depending
on availability of staff and time, these measurements may also & measured more frequently (e.g.
every tw o weeks) for a subsample of the trees during periods of major change (e.g. April to early
June for bud break and later summer or early fall for bud set).
Branch and stem architecture (organ numbers) will be measured onoe per year after the fall bud set
and cessation of that season's growth. Needle samples will be collected for chemical analysis
twice yearly, in the spring prior to bud break and in late summer before bud set. These samples
will be measured for area and dry weight prior to preparation for chemical analyses as described in
the appropriate SOPs (3.01 CNS Elemental Analysis, 3.02 Nutrient Analysis, and 3.03 TNC
Analysis).
The final measurements will be during the summer following bud set. These measurements will be
destructive and include stem diameter, height, branch and stem architecture (numbers), needle area
(of representative sample per tree), and dry matter. The architecture, area, and dry matter (buds,
needles, stems/branches) measurements will be made by organ age class.
(4) Selection and preparation of sample containers
Sample flow for chemical analysis will be as described in Figure 2. Plant samples for chemical
analysis are kept in plastic bags and scintillation vials prelabled with sample identification
information described in (4) below. Sample flow for dry matter analysis will be as described in
Figure 3. Plant dry matter samples will be kept in brown kraft paper bags prelabled with sample
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GPEP SOP 2.01
Version: 1.10
Table 3. Number of Samples per Measurement Time foT Intermediate and Final Measurements in
Tenacosms.
Organ
Parameter
Estimated
Remeasurement
Number
Number
Intermediate
Stem
Height
196
14
Stem
Diameter
196
14
Bud/Terminal Shoot
Length
196
14
Needle
Length
196
14
Architecture
# Stems, branches
56
4
Needle
Area/Weight by age class * 28
4
Tissue Elements
Needles by age class c
28
4
Final
Needle
Area
196
14
Needle
Dry Weight
Main
196
14
Secondary
196
14
Stem
Height
196
14
Stem
Diameter
196
14
Stem
Numbers
Main
196
14
Secondary
196
14
Stem
Dry Weight
Main
196
14
Secondary
196
14
Buds
Numbers
Main
196
14
Secondary
196
14
Buds
Dry Weight
Main
196
14
Secondary
196
14
Tissue Elements
For stems, buds,
needles by age class *
168
SeeSOPs
a For 196 remeasurements, data from 1 tree taken at random per terracosm; for 4 remeasurements,
data takes at random from all trees across terracosms.
b If time permits additional intermediate measurements for stem diameter and height, and bud/shoot
and needle length will occasionally be made on all 4 trees per ten-acosm, 56 trees with 4
remeasurements taken at random.
e From one sample per terracosm per organ and estimated two age classes.
a From one sample per terracosm per organ and estimated four age classes.
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GPEP SOP 2.01
Version: 1.10
FALL'
FALL
Whole Plant Gas
Exchange
Needle & Branch
Gas Exchange
Stem Diameter
CERES Devices
Plant Water
Status
Stem Height &
Diameter Manual
Needle Area
Image Aaalvsb
Bud Phenology
Branch & Stem
Architecture
Needle
Samples
Continuously
Every Eight Weeks
Every Four Weeks
Every Eight Weeks
Continuously
Every Eight Weeks
Every Four Weeks
Every Eight Weeks
Every Four Weeks
Every Eight Weeks
Every Four Weeks
Every Eight Weeks
Shoots Dormant
Soil Surface
B»4
Bnik
Shoot Growth
fed
Sm
Shoots Dormant
Rapid Root Growth
Rapid Root Growth
Corts-to-Depth
Soil & Root Samples
Litter Bags &
Needle Packs
Root Images
Soil Solutions
Soil Profile
Case*
Soil Surface
Case*
Soil Water
Content
X
X
X
X
Every Four Weeks
Every Four Weeks
Every Four Weeks
Every Four Weeks
Continuously
Figure 1. Physiological-based schedule of above* and belowground sample collection tuning
and frequency.
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Page 10 of 31
CPEP SOP 2 01
Versioo: 1.10
Sample Needles (30j)er tree pooled per terracosm)
tic Has
Place in Zip-lock Plastic Bag
J
Determine Leaf Area (Appendix L*5), 14% Remeasures
II
Put Plastic Bags in Freezer at -70'C
I
Lyophilize Samples (Appendix L-l), Put in Zip-lock Plastic Bags
Gnnd Samples, to 40 Mesh (Appendix L-2) Put in Plastic Scintillation Counter Vials
Place Groups of Vials within Plastic Bags
Put Desiccant in Plastic Bags to Maintain Dryness for Storage
l
Weigh Samples, 14% Remeasures (Appendix L-3)
Return to Plastic Bags
it
Store at Room Temperature in Laboratory
Chemical Analysis (See appropriate SOP for repeated measures)
Figure 2. Sample flow for chemical analysis. This sequence is used for all needle samples for
intermediate harvests and all samples for final harvest.
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GPEP SOP 2.01
Version: 1.10
Cut off Shoot, Put in Kraft Paper Bag
II
Put Paper Bags in Cool Growth Chamber at 5*C
II
Architecture Measurements, 1% Remeasures
II
Determine Leaf Area for 50 Needles Per Tree, 1% Remeasures (Appendix L-5)
II
Place Paper Bags with Individual Organ Samples in 65*C Oven
II
Weigh Samples, 1% Remeasures (Appendix L-3)
II
Place Paper Bags inside Large Plastic Bags,
Store at Room Temperature in Laboratory
Figure 3. Sample flow for final measurements.
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CPEP SOP 2 01
Version. 1.10
identification information as described in (4) below. After weighing, the individual paper bags
with the biomass samples will be put together in larger plastic bags to insure that no outside
moisture enters which could deteriorate the samples over lime
(5) Sample identification and labeling
Plastic labels will be attached to individual trees at the start of the study. Each label will contain a
plant number 1-14 per terracosm. As necessary labels will also be attached to terminal shoots and
specific branches of each trees to insure repeated measurement of the same organs over time.
For intermediate nondestructive measurements (shoot height, bud/terminal stem length, needle
length, stem diameter) data will be directly entered into a datalogger spreadsheet. Aiea and
architecture data will be entered by hand into a notebook prior to transfer to a spreadsheet. Weight
data will be entered directly into a spreadsheet. In case of datalogger malfunction, all data will be
entered into a notebook followed by entry into a spreadsheet. The final version of each
spreadsheet will contain the following information for identification purposes:
(1) Experiment (T for tenacosm);
(2) Tenacosm Number (#1 to 14),
(3) Terracosm Treatment (A=Ambient CO2 and Ambient Temperature, T=Ambient CO2 and
elevated Temperature, OElevated CO2 and Ambient Temperature, CTsElevated CO2 and
Elevated Temperarure, CL=Chamberless control);
(4) Treatment Replication i.e. blocks, (l=tcrracosms 2,3,4,5; 2=terracosms 7,6,13,14; and
3=terracosms 9,10,11,12);
(5) Within Terracosm Number (1-14;
(6) Sample Type (SH= shoot, ST= stem, NE=needles, BR= branch, BD=buds);
(7) Age 92=year 1, 93=year 2, etc. for main flush per year, and year.x for additional flushes in
year, i.e. 93.2 for second flush in 1993; and
(8) Date (Julian day and year i.e. 20993 for 28 July 1993.
All of the above information should be added to the spreadsheet or notebook record at the time of
sampling or measurement. However, if parts are omitted for some reason, enough information
must be recorded to insure proper identification of the data and the rest of the information will be
added into the spreadsheet file prior to final transfer into the network database. The information in
(1) through (8) above will also be placed on the bags which will hold the plant samples prior to
area measurements and during drying, weighing and storage. The information also will be placed
on the containers which will hold ground, dried plant sample for chemical analysis.
(6) Sample Handling
Samples for chemical analysis are handled as shown in Figure 2. The samples stored in vials are
used in tissue analysis described in SOPs 3.01-3.03. Samples for dry matter are handled as
shown in Figure 3. The growth chamber to hold the tissue until architecture measurements area
made will be in TERF. The temperature will be maintained at 5*C, with QA checks for temperature
determined according to TERF OA protocols. Ovens include a Blue-M Model POM-326E, Blue-M
Model POM-563, and a Blue-M Model DC-366G-HP. If samples have been removed from the
driers, but not weighed within 12 days, they must be returned to the driers for 24 hours prior to
weighing.
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GPEP SOP 2 01
Version 1.10
(7) Sample Preservation, Slorage, and Disposal
Samples for chemical analysis and dry matter measurements will be stored until the end of the
study as indicated in Figures 2 and 3. The samples will be stored in a dry area at room temperature
(Tenestnal Ecology Research Facility (TERF) room 113). Samples will be archived by experiment
(ten-acosm, pots, large lysimeters, field sites) and date. Samples will be stored for at least two
years or until the data have been accepted for publication. Samples will be discarded in municipal
trash when no longer needed.
G. Analytical Procedures
(1) Pattern of sampling Intermediate, nondestructive sampling for stem diameter and height,
phenology and architecture will be made by opening the front (south) panel of a lerracosm. Trees
will be sampled beginning in the northwest comer of the terracosm, working east along the back
row by the air handling system, than west along the middle row, and finally east along front rou
nearest the front. Intermediate architecture measurements will be the middle subset of four trees per
teiracosm. Intermediate needle tissue sampling for elemental analysis will be made by taking five
needles of each age class (current, one year old, etc.) from each tree in the terracosm, and pooled
for all trees per lerracosm. For the final, destructive harvest, the trees will be cut off at the
cotyledonary node and removed from the lerracosm in the same order as for the nondestructive
growth measurements.(2)
Timing of sampling. See F(2) above for seasonal patterns. Intermediate measurements will be
made as rapidly as possible to hold the amount of time that the tenacosms are open (and not
supplying treatments) to a minimum.
(3) Detailed sampling protocols.
(a) Shoot Height. Stem height will be measured from the cotyledonary scar to tip of central
leader of tree by putting ruler parallel to the main stem.
(b) Stem Diameter. Stem diameter will be measured about 4 mm below the cotyledonary scar.
The measurement can not be made on the scar itself as the area has a slight swelling. To insure that
all subsequent measurements are made at the same location, the area will be marked with black
magic marker just before the first measurement is made (and remarked as necessary to maintain
identification over time). Digital calipers will be used as described in Appendix L-4. The calipers
will be held so that they are parallel to the south wall of the tenacosm, i.e. the jaws open on the
side facing the entrance to the terracosm.
(c) Phenology will be determined by measuring terminal bud and needle growth. The focus of
the measurements will the length of the terminal bud and length of the subsequent terminal shoot
and its needles during the growing season. Primary bud break will be defined as the time period
when there is an initiation of rapid increase in length. Continued measurements will be made as the
bud develops into the terminal shoot. After the terminal shoot ceases to elongate, subsequent
measurements will be made on the new terminal bud so that their development and any secondary
bud break during the summer will be detected. The point at which the terminal buds cease to
elongate at the end of the growing season will be defined as bud set. Any changes in appearance
(e.g. color) of the buds will be noted during the growing season. If there is no well defined
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Page 14 of 31
CPEP SOP 2.01
Version' 1.10
terminal bud or stem on a tree or if for some if the terminal bud/stem dies, the nearest axillary bud
and stem which may become dominant wjJJ be measured.
Terminal bud/siem length. The terminal bud and stem length will be measured with a ruler from
the insertion into the main stem to the tip. After the bud begins to elongate the stem will be
identified with a circular piece of while plastic which is open to not restrict stem growth.
Needle elongation. Needle elongation will be determined by measuring the length of a needle
midway chosen at random along the middle part of the terminal shoot of each tree. Indiviudal
needles will not be followed because of possible damage to the tender tissue and difficulty in
returning to the same needle even if it is marked. For the measurement ruler will be placed from
the insertion of the needle on the branch to the tip of the needle. For precision measurements a
second needle will be chosen at random from the same general location on the stem, but the
original needle will not be remeasured.
(d) Branch and Stem Architecture. Intermediate branch and stem architecture will be
determined by counting the numbers of branches and stems per tree by age class in the fall for a
subsample of the four trees in the middle of each terracosm. Final branch and stem architecture
will be determined in the spring at the time of the final harvest as shown in Figure 3. Numbers of
buds per plant will also be counted at that lime.
(e) Leaf Area. The leaf area meter will be used as described in Appendix L-5.
Intermediate measurements of needle area and weight will be based on a subset of the needle
samples taken for chemical analysis. These samples consist of the pooled needles from all trees in
a terracosm (pooled separately per age class). From each terracosm sample, 50 needles will be
taken at random for each age class and used to determine leaf area. For measurement of area, the
needles will be attached to the area meter bell with clear scotch tape.
Ai the final harvest leaf area will be measured for 50 needles taken at random from each age class
per tree as indicated in Figure 3. These needles will be weighed separately from the other needles
per tree so that specific weights per needle area (g/cm2) can be determined. These specific weights
will be used to calculate total leaf area per age class per tree by the formula: Total Area = Total
grams of needles/specific weight per area.
(f) Dry Matter. Samples will be weighed as described in Appendix L-3.
Intermediate measurements of dry weight will be made for each subsample of 50 needles per age
class per terracosm used for the leaf area measurements. These dry weight measurements will be
used to calculate characteristic specific weights of needles (weight/needle area) for the larger needle
sample used for chemical analysis as shown in Figure 2.
At the final harvest, first 30 needles per age class per tree will be removed for chemical analysis
according to the same procedures used for the intermediate measurements (Figure 2). The
remainder of the tree then will be cut off for biomass measurements at the point where the first root
branches off from the main stem. This may involve burying into the ground to expose the base of
the stem. The sampling and handling of the samples for biomass measurements is shown in
Figure 3. The tree will be divided into needles, stems, and buds by rank (current year, one year
old, etc.)-
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Page 15 of 31
GPEP SOP 2 01
VersioD 1.10
H. Quality Assurance/Quality Control
1) Calibration standards. Calibration standards to determine the accuracy of data obtained
with different pieces of equipment are listed in Table 2. For the rulers calibration will only occur
initially when the rulers are received and the data entered into a notebook. The rulers will be
identified by number. Routine calibrations of the rulers will not occur unless new rulers are
purchased or their is some reason to suspect that the rulers have changed their shape such as by
warping. The calibration standards for the digital balance, digital calipers, and leaf area meter will
be used each time the instrument is used, with the data entered on forms shown in Appendix L-6.
The calibration standard for the freezer and oven is to monitor their performance over time. The
temperatures for the freezer and oven will be noted in the Task notebook.
2) Precision and accuracy criteria of calibration standards. Precision will not be determined
for the standards as they are assumed to hold constant over time because they are made of stable
materials (metal). Accuracy of the standard is as determined by the certifiable source. Quality
control for the standards is determined with a control chart for each instrument which is updated
each time a new calibration is made. The control chart will indicate the percent accuracy (y axis)
vs date (x axis). Accuracy will be calculated as indicated under (5) below.
3) Precision for Data Quality Objectives will be verified through remeasurement of a portion
of the plants. This will one chosen at random from each chamber for each measurement (except
annual needle sampling); resulting in approximately 1% (14 total, taken as 1 of from each of 14
terracosms) repeated measures (Table 3). For annual needle sampling there will be only one
pooled sample per terracosm for leaf area and dry weight. Repeated measures will be made for
14% of the samples per organ (2 total, taken from 2 of 14 total samples).
4) Definitions of precision, accuracy, and completeness.
Percent precision for a single plant is defined as: (|xi-x2[/xj> 100, where Xj and x2 are repeated
measures on the same plant. The mean and standard deviation are calculated for all (n) precision
percentages.
Percent accuracy for a single calibration is defined as: ([yi-y2]/yi) * 100, where yt is instrument
reading and yi is standard value. This retains the sign of the accuracy values for control charts.
Across calibrations, the mean and standard deviation are calculated using the absolute values for all
(n) accuracy percentages.
Percent completeness for a data set is defined as: (|zi-Z2|/zi) * 100, where Z| is the number of
possible measurements and ti is the number of actual measurements per data set.
5) Summary tables and control charts. Summary tables and control charts will be used to
routinely monitor precision and accuracy for response parameters listed Table 1. For precision, the
repeated measures data will be copied iato a file and summarized as described in the QAPP.
Calibration data to determine the accuracy of measurements made with the digital balance, digital
calipers, and leaf area meter will be plotted onto control charts (sample shown in Appendix L-7).
The percentage mean differences are immediately entered onto the control charts and compared
visually to the data quality objective for each parameter. As described in the QAPP the calibration
data for the digital balance, digital caliper, and leaf area meter also will be entered into a database,
tabulated, and reported, lite calibration data for the rulers will be reported in the first QA report.
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Page 16 of 31
GPEP SOP 2 01
Version 1.10
The performance monitoring data for the freezer and oven will be available if requested.
6) Sample analysis pattern. All staff will follow the sampling patterns specified in this SOP.
Whenever new staff are added the individuals taking the growth measurements will meet to
compare and standardize techniques to insure comparability between or among observers.
1. Preventive Maintenance and Corrective Action
Routine maintenance of the digital balance, digital calipers, and leaf area meter will occur whenever
the instrument is in use as described in Appendices L-3 to L-5. If calibration standards fall outside
of the accepted accuracy range the instruments will follow the corrective actions described in the
Appendices. Annual calibrations will be made of the digital scale by an independent agent. Each
analytical instrument will have a log book to record notes, quality control any preventive action
and corrective action activity. These entries will be dated and signed.
Precision vu ill be evaluated according to the QA Project Plan. If the data being collected does not
meet the precision DQO, first alt individuals taking the measurement will be questioned to insure
that they are following the SOP. Second, if precision does not improve with careful observation of
the SOP the measurement protocol will be evaluated and improved as necessary. Routine
corrective actions will be made by the individual making measurements with the instrument in
question. They will be reported as soon as possible to the P.I. for Task 2 (Shoot Biology) of the
project. If a problem persists this individual must contact the P.I. before taking any further
corrective action. The P.I. summarizes any problems and corrective actions in the semiannual Q.A.
reports.
J. Data Reduction, Validation and Archiving
Data flow will be as illustrated in Figure 4. Data will be entered directly into a datalogger for all
plant growth, biomass, and phenology measurements. Any unusual accompanying observations
will be noted in the database as comments and in a laboratory notebook with carbon copy pages.
Area and architecture data will be entered in a laboratory notebook. The original of the notebook
will be kept in TERF (Room 105). The P.I. will check the carbon pages, sign them, remove them,
and store them in his office. Calibration data sheets and draft control charts will be kept in plastic
envelopes (to keep them clean) with the instruments. Whenever duplicate pieces of data are present
for a parameter due to repeated measures for precision analysis, only the first piece of data will be
used for analysis.
Measurement data will be transferred from the datalogger (and/or notebook if necessary) into a
computer spreadsheet at the end of each measurement day or as soon as possible. The P.I. or
designee will visually check the spreadsheets for completeness and unusual values, consulting the
notebook as necessary for explanations. A floppy disc version and hard copy of the file will be
stored in the P.I.'s office. The final versions of all shoot growth spreadsheet files will be named
as DF (for Douglas fir), S (for shoot), Julian Date, and year, e.g. DFS20993.WK1 for data taken
on 28 July 1993. The P.I. will check the spreadsheets and sign the top of each hardcopy
indicating that the data are complete. Calibration data and control charts will be kept with
instruments until full of data, at which time they will be transferred to the P.I.'s office. Computer
files with the calibration data will be kept in the P.I.'s office.
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Page 17 of 31
GPEP SOP 2 01
Version 1.10
Record Data in Datalogger
II
Transfer Data to Computer Spreadsheet
II
Check Data for Accuracy and Completeness
U
Make Floppy Disc Copy of Data
Make Hard copy of Data
4
Transfer Data to Main Network Database
U
Routine Backup of Network Database
II
Carry out Analysis of Data
Figure 4. Data flow for shoot biological measurements. This sequence is used for all data except
needle area in which case data are first recorded in a notebook and then transferred by hand to
computer spreadsheet.
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Page 18 of 31
GPEP SOP 2 01
Version 1.10
The P.I. will oversee all statistical analysis of Task 2 data. This will include statistical analysis of
the instrument calibration and control chart data. Measurement data will also be sent to the main
TERA database on the ERL-C network. TTie main network database will be backed-up weekly.
K. References
Allen, S.G., Idso, S.B., Kimball, B.A., Baker, J.T., Allen, L.H.Jr., Mauney, J.R., Radin,
J.W., and M G. Anderson. 1990. Effects of Air Temperature on Atmospheric CO2- Plant
Growth Relationships. US DOE/ER-0450T.
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GPEP SOP 2 01
Version 1.10
Appendix L-l
Unitrap II Lyophilizer
The Unitrap II Lyophilizer is used to freeze-dry various tissue samples io preparation for
grinding and subsequently lor biochemical analyses.
The Unitrap II Lyophilizer is simple to operate and the following steps outline the
lyophilization procedures:
1. Log-in using the logbook located beside the lyophilizer at the start of use.
2. Check the condition and level of the pump oil. The oil level should be approximately
halfway up the window or halfway up tne screw inside; if the oil chamber is too full, the oil
will splatter when the lyophilizer is turned on. Change the oil if necessary and log the date
it is changed into the logbook.
3. Turn on the refrigeration switch. When the temperature reaches *30eC, turn the vacuum
release knob from open lo close.
4. Place sample(s) in cylinder and replace cylinder cover.
5. Turn on vacuum switch. Vacuum should reach 1000 milliton in about 5 minutes and then
drop quickly. If it doesn't, check for leaks around:
a. Plug from drain line
b. Seal around sample cylinder
c. Seal around ice cylinder
6. Shut down the lyophilizer when samples should be dry (minimum of 24 hours). Shut
down procedures are posted on the front of the lyophilizer (by ihe manufacturer-they
cannot be removed).
7. Confirm sample dryness either objectively by weight difference (periodically done) or
subjectively by evaluating the temperature throughout the vacuum chamber and when
handling the envelopes.
8. Continue lyophilization if necessary and recheck sample dryness at a later time.
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CPEP SOP 2 01
Version1 1 10
Appendix L-2
Wiley Mill (Tissue Grinder)
The Wiley Mill is used to grind various sample tissues in preparation for biochemical
analyses. The Wiley Mill is fitted with a 40-mesh screen to ensure uniform particle size for the
processed tissues. The Wiley Mill is simple to operate and the following steps outline the grinding
procedures:
1. Clean Wiley Mill parts, if needed, before beginning sample grinding. Clean the parts
between samples and at the end of a particular grinding session also. The Wiley Mil! is
cleaned by removing or exposing various components, including the grinding chamber and
the grinding chamber face plate (glass), and directing a filtered stream of compressed ai: at
them individually. The compressed air dislodges and removes tissue panicles from these
Wiley Mill components thus cleaning them. Note: Before entering ibe Wiley Mill, the
compressed air stream must be passed tbiough a trap containing glass wool oi absorbent
cotton to facJitate trapping oil, grease ar.d other contaminants.
2. Reassemble the Wiley Mill after its parts have been cleaned.
3. Turn the Wiley Mill on and begin feeding urground tissue into the sample hopper. Use the
wooden dowel to compress the sample and force it into the grinding chamber. Some
samples may require slow addition to the sample hopper and subsequently ihe grinding
chamber to avoid clogging the 40-mesh screen.
4. Turn the Wiley Mill off after the completion of sample grinding and transfer the sample
from the repository chamber to a capped, polyeibylene scintillation vial.
5. Go lo step 1 and repeal the sequence of steps,
The WiJey Mill motor and bell are periodically oiled and replaced, respectively, the cutting
blades ate replaced when samples begin taking & noticeably longer time to grind.
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GPEP SOP 2.01
Version' 1.10
Appendix L-3
Digital Balance Operation
Mettler or Sariorius Toploading Electronic Balances are used to gravimetrically measure
phytomass. These dependable and high precision balances combine several features (compact and
ruped design, simplicity of operation, fast stabilization time and excellent reproducibility) which
enhance the effectiveness of collecting large amounts of plant biomass data.
1. Balance should be maintained in a stable, draft-free location. A cardboard box will be used
as a windscreen around the balance.
2. If the balance is switched off, turn oo by pressing the control bar. Allow sufficient time
(approximately 5 minutes or until digital reading has stabilized) for the balance to warm up.
If balance is to be used several times over the course of a study, it is advisable to leave the
balance switched on.
3. Check balance level by observing the location of the air bubble in the level indicator. If
leveling is required, slowly turn the footscrews until the air bubble is in the center of the
circular level window.
4. Before measurements calibrate the instrument by placing standard weight (generally 1 or 10
g) on balance. Record standard weight and instrument reading. Put calibration data on
sheet provided and calculate percent difference.
5. Place sample on weighing pan and record weight from digital readout, after allowing for a
2-3 second stabilization period.
6. After the weighing session, recalibrate the instrument by again placing standard weight
(generally 1 or 10 g) on balance. Record standard weight and instrument reading. Put
calibration data on sheet provided and calculate percent difference. Record average of
before and after percent differences on control chart. Clean off the weighing pan and
balance housing. Cover the weighing pan with the protective shield.
Ealances are calibrated annually by the manufacturer's service representative as preventive
maintenance. Additionally, operator calibration checks are conducted immediately before each
weighing session. For the calibrations a time of use, a set of NlSTstandard weights (S class) is
maintained for the purpose of operator calibration checks. These weights should never be handled
using fingers but with tweezers instead; oils from hands may change the weights and conode
them. The standard weight used for calibration should be representative of the mid-range of tissue
samples being weighed. The results from these calibration checks must be within 5% of the
known value of the standard weight before proceeding with tissue sample measurements. If the
results from the start-up calibration exceed 5% of the known value, the operator will delay sample
weighing and notify the project leader for corrective action. If the mid- or end-ot-session
calibrations exceed 5% of the known value, all tissue samples weighed since the last valid
calibration will be void. These suspect samples are weighed again and considered valid only when
the start-up and end-of-session calibration checks are within 5% of the standard weight.
Balance operators are responsible for entering the results of each calibration check onto data
sheets entitled, "Quality Assurance Calibration Checks for Weight Measurements". Each balance
has its own data sheet which corresponds to the balance serial cumber. Completed data sheets are
filed by the GPEP Program in a QA Records Folder located in fee P.l.'s office after the data has
been entered into computer spreadsheets. A quality control chart is maintained along side of the
instrument and updatea at the time of each calibration.
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GPEP SOP 2 01
Version. 1.10
Appendix L-4
Digital Caliper Operation
Stem diameter measurements are obtained by using a Digit-Cal Mark II Universal Digital
Caliper or a Digit-Cal + Universal Digital Caliper (Brown and Sharpe). Both digital calipers are
battery-powered, precision tools employed for measuring outside, inside and depth dimensions
(measuring range 0-150mm). At ERL-C, these calipers are most often used to measure stem
diameter (outside dimension). These calipers combine several features (compact design, simplicity
of operation, high resolution and accuracy and excellent reproducibility) which enhance the
effectiveness of collecting data. The same person takes stem diameter measurements throughout
each particular experiment to ensure consistency. To operate the digital calipers:
1. Clean the measuring faces of the caliper jaws and slide with a cotton swab, slightly
moistened with methanol. After cleaning the jaws, close the sliding jaw io make a
light-pressure contact with the fixed outside measurement jaw.
2. Press the "C" button to turn the tool on and clear the display to "0.00". If the display does
not read "0.00", clean the caliper jaws again.
3. Prior to taking stem diameter measurements, check the calibration of the caliper by using
standard rectangular metric gauge blocks (see below).
4. To measure outside dimension (stem diameter), slide the moveable jaw open by using the
thumb pad on the caliper housing and engage the fixed outside measuring face against 1
surface of the material being measured. Then close the moveable jaw with light pressure
against the other surface of the material being measured, while maintaining the measuring
faces parallel or square with the material surfaces. It is important to ensure that the material
surfaces are within the proper section of the jaws (the area closest to the caliper slide is not
used for measuring and will give a much smaller reading).
5. Record the displayed measurement.
6. Close the caliper jaws. If the display reads "0.00", measurements on another plant may be
made. If the display does not read "0.00", clean the jaws and re-measure.
7. After measurements have been completed, check the calibration of the caliper with the
standard gauge blocks.
8. Turn the tool off, clean the caliper jaws and slide as instructed above. Store the caliper in
the provided case.
Operator calibration checks are conducted at the beginning and end of each measuring
session. Calibrations are conducted in the room 105 of TERF which is kept at a constant room
temperature. NIST traceable Brown and Sharpe gauge blocks are used as a set of standard lengths
to ensure the accuracy of the digital caliper. Each steel gauge block has both a serial number and
its length engraved on one surface. The length represents one dimension and has a known
accuracy of .000001 meters.
Prior to and immediately after each measurement session a gauge block is selected which is
representative of the mid-ranee of samples being measured. The results from these calibration
checks must be within 5% of the standard length. If the results from the start-up calibration exoeed
5%, then the faulty caliper is sent back for repair and another caliper is checked out and used. If
the results from the end-of-session calibration exceed 5%, then corrective action must be taken and
all measurements since the last valid calibration will be void. These suspect samples are measured
again and considered valid only when the start-up and end-of-session calibration checks are within
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Page 23 of 31
CPEP SOP 2 01
Version 1.10
5% of the standard length.
Digital caliper operators are responsible for entering the results of each calibration check on
a data sheet entitled, "Quality Assurance Checks for Digital Calipers". Completed data sheets are
filed by the GPEP Program in a QA Records Folder located (n the P.I.'s office after the data has
been entered into computer spreadsheets.. A quality control chart is maintained along side of the
instrument and updated at the lime of each calibration.
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Page 24 of 31
GPEP SOP 2 01
Version- 1.10
Appendix L-5
Area Meter Operation
A. Start-up
1. Move the "ON-OFF" switch to the "ON" position.
2. Press the "LAMP START" button firmly and release after holding for approximately 2
seconds. If fluorescent tube does not illuminate, repeat this procedure.
3. Check transparent belts for cleanliness (refer to "Transparent Belt Cleaning" section below
for correct procedure). Also check for belt tension and tracking (refer to "Instruction
Manual for LI-3100 Area Meter").
4. Allow approximately 15 minutes for instrument and lamp warm-up.
5. Press the "RESET" button to cleaT the display.
6. Calibrate the instrument.
B. Transparent Belt Cleaning
Clean the belts with waier and a cloth or absorbent paper. A detergent may be used for
persistent contamination, but do not allow detergent to fall on the mirrors. Never use benzene or
acetone for cleaning the belts. Any scrubbing of the mirrors to remove detergent spots may
damage the mirror surface. Access to the lower belt is facilitated by momentarily activating the
"ON-OFF" switch to bring the surface near the sample tray. The inner surfaces are cleaned by
reaching inio the access ports in the from panel. Loosen the bells to facilitate cleaning the pulley
surface.
C. Tissue Area Measurements
1. Area measurements of plant tissue should be done as soon as possible after cutting tissue
from plant.
2. Plant tissue should be free of extraneous matter (e.g., soil particles) before placing on
transparent belt of area meter.
3. Ensure that "RESET" button is pressed before each tissue sample measurement.
4. Ensure that all plant tissue samples have exited area meter before placing new samples on
belt.
5. Area measurements of plant tissue samples are recorded on data sheets which are
maintained in a separate logbook or directly into a computer spreadsheet.
D. Calibration Checks
Operator calibration checks are conducted immediately before each area measuring session.
Additionally, operators are required to conduct a calibration check at the end of an area measuring
session and at hourly intervals during lengthy area measuring sessions. A set of 4 calibration test
plates of known area (50.00, 51.33, 77.20 and 101.44cm2) are maintained for the purpose of
operator calibration checks. During each calibration check, the area of a single calibration test
plate, which best approximates the experimental leaf areas, is measured on the area meter being
used. If the observed area reading of the test plate exceeds 5% of the known area, check for belt
cleanliness, tracking and tension. If the observed area of the test plate still exceeds 5% of the
known area, contact the P.I. Do not attempt to calibrate the area meters by adjusting calibration
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GPEP SOP 2 01
Version 1.10
seuings, If ihe results from the end-of-session calibration exceed 5%, then corrective action musi
be taker and all measurements since the last valid calibration will be void. These suspect samples
are measured again and considered valid only when the start-up and end-of-session calibration
checks are within 5% of the standard area.
Once proper calibration has been achieved, record the observed mean (n=3) area and mean
% difference of ihe lest plate on the data sheet entitled, "Quality Assurance Calibration Checks for
Area Measurements", bach area meter has its own data sheet. Completed data sheets are filed by
the GPEP Program in a QA Records Folder located in the P.I.'s office after the data has been
entered into computer spreadsheets. A quality control chart is maintained along side of the
instrument and updated at the time of each calibration.
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GPEP SOP 2.01
Vernon 1.10
Appendix L-6
Calibration Forms for Shoot Biological Measurements
-------
QUALITY ASSURANCE CAI.IORATION CHECKS FOR DIGITAL CALIPERS
ACTUAL LENGTH - KNOWN LENGTH
DIGITAL CALIPER SERIAL HO: % DIFFERENCE -
KNOWN LENGTH
START-*
JP CIIE(
:k
ENI
OF SEJ
SSION <
pHECK
OPER
INIT
START
TIME
ACTUAL
KNOWN
% DIFF
END
TIME
ACTUAL
KNOWN
% DIFF
DATE
PROJECT
IDl
COMMENTS
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Page 2R of 31
Grnp sop 201
QUALITY ASSURANCE CALIBRATION CHECKS FOR AREA MEASUREMENTS Vcision I 10
MEAN OBS.AREA - KNOWN AREA
AREA METER SERIAL HO: MEAN % DIFF =» * 100
KNOWN AREA
NOTE: USE ONE OF THE FOLLOWING CALIBRATION PLATES OF KNOWN AREA (50.00, 51.13, 77.20, OR
101.44 CM ) AND CALCULATE THE MEAN OF THREE AREA MEASUREMENTS (n=3) ON THE SELECTED PLATE.
STI
\RT-irp
CHECK
END Ol
"* SESSION CHECK
OPER
INIT
START
TIME
MEAN
|cn»2)
KNOWN
(cm2)
MEAN
% DIFF
MEAN
-------
QUALITY ASSURANCE CAI.IBnATION CHECKS FOR WEIGHT MEASUREMENTS
ACTUAL WT - KNOWN WT
BALANCE SERIAL NO:
* DIFFERENCE =
Version I 10
X 100
KNOWN WT
OPER
IN1T
START
TIME
DATE
PROJECT
IDl
START -IU* CHECK
ACTUAL
lg>
KNOWN
(n)
% diff
END Ol
ACTUAL
Jq)_
~ SESSIC
)N CHECK
KNOWN
(q)
% DIFF
END
TIME
COMMENTS
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Page 30 of 31
GPEP SOP 2.01
Version: 1.10
Appendix L-7
Example Quality Control Chart:
Used Tor Digital Balance, Digital Caliper, and Leaf Area Meter
-------
5% Upper Control Limit (OOO)
-
¦
2.5% t
Ippev Warn
tg Limil
a
I
11 ;
1 i
i
2.5%
jower Warn
ng Limit
5% Lower Control Limit (DQO)
EXAMPLE COITraOL CHART
-
i
i
¦ -
(i 1
o
¦
1
i
-
DAY OF MEASUREMENT
-------
EXAMPLE OF EXPERIMENTAL PROTOCOL EP.06
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Page 1 of 8
GPEP EP.06
Version: 1.00
EXPERIMENTAL PROTOCOL EP.06
TREE SELECTION CRITERIA, SHOOT AND ROOT SAMPLING
AND TREE PLANTING AND CULTURE
Version: 1.00 - r
Serial Number Itwo-CCieicJ
March 3,1994
\J
QjlS
Prepared^:
'fry.C'/i Date: \>/^'' 7 ^
David M. Olszyk
ApproyaTf^
David M. Olszyk, Principal Investigator
X Date: 3/V* ''
'pau^R^iew^^'rojecr^
Date: 3/7/?/
der
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GPEP EP.06
Version: 1.00
Introduction
This Experimental Protocol (EP.06) describes the procedures for selecting, characterizing,
planting, and culturing of the Douglas fir seedlings used in the terTacosms and supplementary
studies (Cascade field sites, lysimeters, pots, RS-Biome) as described in the Research Plan for
"Effects of CO2 and Climate Change on Forest Trees". Additional information regarding the
seedlings is found in the Task 1 and 2, Terracosm, and Field Site notebooks for the TERA project.
Plant Selection
Seedlings were provided by the Weyerhaeuser Company in Aurora, Oregon (contact Ms. Nina
Poole). The location and % of population for the seed source is described on pp. 33-34 of the
Research Plan. The seedlings were grown at a nursery in Aurora, Oregon, The seedlings were
l+l's, i.e., sown into the ground in Spring (e.g. May) of 1991 and grown for one year; dug up in
Fall of 1992, pruned, sorted, and transplanted back into soil for a second year in a nursery bed.
The seedlings were dug up in Spring of 1993 as bare-root stock for storage and transport to
Corvallis The seedling lot was SP03 LOW.
(A) Batch One (batch refers to trees received at different times from Weyerhauser) for low and
middle elevation Cascade field plots (samples from trees at the initiation of the study are termed
"T0").
The 1120 seedlings (seven bags of 160 seedlings each) were received on 13 May, 1993, for low
and middle elevation field plots in the Cascade Mountains, selected, and put into different groups
(group refers to trees for different uses). First, any unhealthy trees (dead buds, needles, and/or
obviously non representative) were culled and discarded. Approximately 170 trees were discarded
in this way. Second, from the remaining healthy trees, 300 seedlings were selected at random
(approximately 45 each from the seven bags of seedlings with 15 extra discarded) and tagged for
stem diameter measurements. The 300 trees were each tagged on the stem with a yellow plastic
tag. Tags were nunbered sequentially from 1 to 300. Stem diameter was marked with a black
permanent marker and measured just below the cotyledon scar (the measurement can not be made
on the scar itself due to localized swelling). After measuring, the tagged trees were placed into
groups of 50 and (along with remaining untagged trees) stored in a cold room at approximately
5'C.
Stem diameter was measured using a digital caliper as described in Appendix L-2 of the Shoot
Biology SOP # 2.01. The data for tree number and stem diameter were entered into a spreadsheet
(QuatTo Pro) on a personal computer (PC). The tree numbers were sorted by diameter. The 184
medium diameter trees (approx. #62 to 245 in rank) were assigned (based on selecting tree
numbers at random) to groups to be used in the field (group "F"), for an initial destructive harvest
in order to characterize growth parameters and tissue nutrients (group "D"), and to be potted as
replacement trees for the field if necessary (group "P"). The shoot and root sampling procedures
for groups A and D are described later in this EP.
A summary of trees and fate is as follows by group.
I. Trees not selected and discarded.
II. Trees that were selected, tagged, and chosen for detailed study (total of 164). The trees
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GPEP EP.06
VersioQ. 1.00
were allocated to groups as follows.
a) Croup D: 14 individual trees measured for architecture aod destructively harvested for
biomass measurements over 17-28 May, 1993.
b) Group P: 30 trees for replacement in the field sites during the seasoo were placed in small
pots as described below. Potting was on 14 May, 1993.
c) Group F: 140 trees for planting in field plots at the low and medium elevation sites on 18
May, 1993.
111. Trees that were selected and tagged but not chosen for detailed study (total of 116). The
trees were allocated to groups asTollows:
a) Group A, 42 trees randomly assigned into three sets (sets are subdivisions of group labeled
1,2,3) of 14 trees each. Needles, stems, and roots were put into different sets over 7-19
May, 1993. Sets used for chemical analysis.
b) Group X, 74 extra trees retained in cool room for one week until discarded.
(B) Batch Two trees for terracosms, lysimeters, pots, high elevation Cascade field plot (additional
T0 trees)
Approximately 2,000 seedlings to be selected for terracosm and supporting studies arrived 3 June,
1993 . 950 of these seedlings were selected, tagged, and measured for stem diameter. Tags were
numbered sequentially from 400 to 1350. After measuring the tagged trees were placed in groups
of 100 and (along with remaining untagged trees) stored in a cold room at approximately 5*C.
As in (A) above, the data for tree number and stem diameter were entered directly into a database.
The individual tree data were sorted by diameter and the 516 medium trees (approx. #259 to 775 in
Tank) were identified. The data were put into a file on the P.C. These trees were randomly
assigned to the six groups (D,T,F,L,A,P) shown below by randomly picking plant numbers.
These numbers were entered into the data base. During planting some switching and replacement
of trees were made for the different groups based on needs for more trees than originally
anticipated and damage to a few trees. Any additional trees were taken from the remaining group of
434 tagged but not selected trees.
Some of the unselected trees were used for buffer areas around the upper field site on 10 June,
1993. The remaining trees were kept in a cold room for one month as additional replacements for
the selected trees if necessary. The shoot and root sampliog procedures for groups A and D are
described later in this EP.
A summary of trees and fate is as follows:
I. Trees not selected and discarded. Some of these were used in buffer areas at Cascade field
plots.
II. Trees that were selected, tagged, and chosen for detailed study (total of approximately
516). The trees were allocated to groups as follows:
a) Group D: 14 individual trees measured for architecture and destructively harvested for
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Page 4 of 8
CPEP EP.06
Version: 1.00
biomass measurements.
b) Group T: 196 trees for the terracosms.
c) Group F: 70 trees for planting in field plots at the high elevation site.
d) Group L: 92 trees for planting in the large lysimeters.
e) Group A, 42 trees randomly assigned into three sets (sets are subdivisions of group labeled
1,2,3} of 14 trees each. Needles, stems, branches, and roots were put into different sets.
Roots were further sorted by diameter into subsets as described below. Sets used for
chemical analysis.
f) Group P: approximately 102 trees that were potted. Approximately 23 trees in small pots
for replacement in terracosms, lysimeters, or field sites; and which were combined with the
trees potted from the first group of seedlings. 79 trees were potted in large pots for annual
destructive harvests.
III. Trees that were selected and tagged but not chosen for detailed study (total of
approximately 434). The trees were allocated to groups as follows:
a) Group R: approximately 30 trees used for additional detailed root analysis.
b) RS-Biome: approximately 100 trees.
c) A number of other trees were selected for additional pots and as extra trees for use in buffer
areas in Cascade field plots.
Shoot Sampling for Architecture, Biomass, and Nutrient Analysis
Seedling shoots for destructive sampling for architecture and biomass (Groups D for To) were
stored in a 5*C dark growth chamber (functioning as a refrigerator) until they could be measured.
Each of the fourteen seedling shoots was measured for total height (base to tip of apical branch),
and subdivided into two types of woody stems (main stem and axillary branches), needles
(separately on main and axillary branches), and buds (separately on main stem and axillary
branches). All main and axillary stems and branches were counted and dried for biomass. A
subsample of 100 needles per main stem or axillary branch per tree was measured for planar area
and dried for biomass. The remaining needles per sample were then dried for biomass. The
sampling, handling and measurement procedures were based on protocols which formed the basis
for SOP 2.01. Repeated measurements were made on two trees for each parameter to determine
precision.
Seedling shoots for nutrient analysis (Groups A) were stored in a -70*C freezer until they could be
lyophilized, ground, and weighed prior to analysis. The sampling, handling and measurement
procedures were based oo protocols which formed the basis for SOP 2.01.
Root Sampling for Architecture, Biomass, Nutrient Analysis, and Mycorrhizal
Analysis
Whole seedling root systems from To samples were soaked in water for 2-3 hours, to remove any
soil particles adhering to the root system. The soaking time was shorter for root systems to be
used for chemical analysis. The root systems were then rinsed several times with water until the
water ran clear of any soil. The root system was plaoed on a paper towel and air dried for three
minutes and the fresh weight documented.
For chemical analysis of Batch 1-Group A, the root systems were not subdivided. For chemical
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Page 5 of 8
GPEP EP.06
Version 1.00
analysis of Baich 2- Group A, the roots were further sorted by diameter into subsets as described
below.>2 mm (subset III), 1-2 mm (subset II), 0.5-1.0 mm and 0.00-0.05 mml, subsequently the
0.5-1.0 and 0.00-0.05 mm subsets will be combined prior to analysis (subset I).
For the root systems to be used for detailed morphological and mycorThizal measurements (Group
R), the number of primary roots that were attached to the Douglas fir seedling's tap root were
counted and five are randomly removed using a scalpel and forceps, (Statistical Methods George
W. Snedecor and William G. Cochran, Table A pp 543-547). These primary roots were numbered
1 lo 5 and the number of secondary roots counted. If these roots had 1 to 20 root tips then these
root tips were sampled. If more than 20 root tips were present per secondary root, then one
tertiary root was randomly removed from each secondary root. The number of tertiary roots was
counted. If these tertiary roots had 1-20 root tips, then these root tips were sampled. If more than
20 root tips were present, this cycle was continued until such time as the root segment contained
between ] and 20 root tips. The sections were removed randomly, counted, ana placed in a petri
dish This cycle was continued through the remaining root systems until 120 root tips were
selected from the seedlings. The seedlings were returned to the ziplock baggies and stored at 4C.
The root tips were stored in a labeled petri dish at 4C until myconrhizal analysis could be made.
These procedures will also be used for subsequent detailed root system measurements.
Planting
(A)Tenacosms
The trees were planted on 6 and 7 June, 1993, in approximately 15 cm deep holes in the B horizon
dug with a trowel. The trees were planted so the roots were straight down as much as possible to
avoid "J-rooting" (i.e. bending upward) the root systems. See Figure 5-5 of the Research Plan
(US EPA, 1993) for the dimensions of the soil horizons and litter layer. Trees were positioned to
insure that the final tree position was with the stem diameter mark above the A Horizon. Thus,
trees were held in the hole so that the top of the root system would be approximately 2 cm below
the top of the A Horizon (approx. 4 cm. below the top edge of the lysimeter). However, with
subsequent litter addition the original diameter mark usually was no longer visible so most trees
subsequently had to be remarked during the first summer of growth.
Each hole immediately was filled in with B Horizon, which usually came halfway up the root
system. The A Horizon was then added around each tree. The trees were immediately watered
with 16 liters of R/O water added uniformly across the surface of each terracosm. The trees were
planted in three rows of five, four and five trees each according to the spacing specified in the
Research Plan. Approximate tree spacing was as follows. North and south row trees were planted
18.5 cm from the north and south edges of the chamber, with end trees 19.5 cm from the east and
west ends of the chambers. Middle row trees were planted alone the center line of the chamber,
with end trees 395 cm from the west and east ends of the dumber. There was 36 J an between
rows and 39.5 cm between trees within rows. Individual tree placement had to be adjusted by
several cm in a number of cases to avoid hitting the lysimeters, thermistors, and horizontal
minirhizotron tube in the A Horizon. Approximately 6-7 an of litter (seined forest floor) was
added on top of the A Horizon between 10-15 June, 1993.
The lops of the terracosms were covered with black shade doth for 30 davs to simulate cloudy
weather and allow the trees to further acclimate before being exposed to full sunlight. Beginning
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GPEP EP.06
Version. 1.00
on 8 July 1993 the shade cloth was removed in mornings to acclimate the trees gradually to full
sunlight. Over succeeding days the shade cloth was incremental replaced later in the afternoon
(except for weekend of 10-11 July when shade cloth remained on all weekend). However, due to
cloudy weather, from 16 July onward the chambers received full sunlight.
(B) Lysimeters
The trees were planted on 6 June, 1993, in the soil using the same protocol as for the terracosms
and in the same pattern for the Terracosms except that there were rive rows of five, four, five,
four, and five trees at approximately the same spacing from edges and within and among rows as
in the Terracosms. A 6-7 cm deep litter layer was added in June. On 7 June, 1993, shade cloth
was suspended over the lysimeters to simulate cloudiness and allow the trees to acclimate gradually
to full sunlighl.The cloth remained over the lysimeters until 8 July as described for the terracosms.
On the day after planting 24 liters of reverse osmosis (R/O) water were added uniformly across
each lysimeter.
(C) Field sites
There were three field sites at low, middle, and high elevations; with five plots per site. The trees
were planted in each plot using the same protocol and spacing pattern as the terracosms.
Additional trees were planted at the same spacing in border areas. The low and middle elevation
sites were planted on 18 May, 1993; the high elevation site on 10 June, 1993. Additional border
Uees were planted between and around the plots using the same spacing as within the plots. A 6-7
cm deep litter layer was added to the low and middle elevation sites on 10 June, 1993, and to the
high elevation site approximate during the week of 14 June, 1993. Hemlock bark was placed in
lieu of a litter layer on the soil beyond the border tree areas on 14 July, 1993, for the low and
middle sites, and 21 July, 1993, at the middle and high sites as logged in the field notebook.
As necessary, additional trees from the extra trees at Corvallis were planted at the sites to replace
those that died since the original plantings. Notes regarding replacement trees were put in the field
Notebook. On 10 June 1993 trees were replaced at the low and middle elevation sites, and on 8
July trees were replaced at the high elevation site. Measurements were made of initial height,
diameteT, and length of topmost bud as described in the field notebook. At the time of the initial
measurements in the field, if necessary, the diameter was re-marked (just above soil line,
preferably below lowest branch).
(D) Pots
The trees were planted in black plastic pots in late June, 1993. Two pot sizes were used, small for
limited growth of the trees (22.86 cm high x 21.59 an inner diameter at top and 17.78 outer inner
diameter at bottom), and large for long-term growth of the trees (30.48 cm high x 25.4 cm inner
diameter at top and 22.23 cm outer diameter at bottom). For the small pots, trees were planted in
new A horizon with approximately 4.45 cm of space left at the top of the pots. Small pots did not
have a litter layer oo top of the soil. For the large pots, B horizon was placed at the bottom of the
pot. Each tree was then held in the pot and the bottom of the pot filled with B horizon soil to a
height of approximately 10-12 cm. Next A horizon was added to the pot to a height of
approximately 20-24 an. Approximately 4.24 cm deep litter layer was added to the top of the soil
and a few cm were left clear at the top of the pot. Acclimation of the potted trees to full sunlight
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CPEP EP.06
Version 1.00
was gradual. The pots were in the lathe house for approximately three weeks. The trees were
moved to a shaded area to the north of the polyhouse the week of 19 July, 1993. Finally the trees
were moved to full sunlight in the terracosm area. Bark was placed around the pots as a mulch.
(E) RS-Biome
Approximately 100 seedlings from batch 2 were "healed in" in bark in a lathehouse. Twenty of
these seedlings were planted in the RS-Biome area in December, 1993. For more details see the
QA Plan and SOPs to be developed for the supplemental studies.
Tree Cultural Practices
(A) Watering
(1) Terracosms
Trees at ERL-C are watered with R/O water. Immediately after planting was complete on 7 June
1993, each terracosm received 16 L of R/O water. Subsequent waterings and amounts have been
recorded in the TERA site notebook. Watering is usually on Thursdays, with the amount
depended on the evapotranspriation water collected from selected terracosms for the preceding
week. Initial waterings for the tenacosms were with a measured watering can, with watering
through the irrigation system after it was complete in late June, 1993. After full implementation of
the study tenacosm trees are being watered as necessary using the below ground irrigation system
to maintain soil moisture levels within the realm specified in the Research Plan.
(2) Lysimeters
All lysimeteT trees automatically receive liX the amount of R/O water given to tenacosm trees due
to the relative sizes of the soil containers. Lysimeters are watered on the same days as terracosms.
Watering is with a measured watering can. The date and amount are noted in the watering event
notebook kept in the mission control center.
(3) Pots
The trees in pots were watered with R/O water as needed.
(4) Field Sites
Trees at the field sites currently rely on natural rainfall for all water. However, if absolutely
necessary, the trees may be watered from a tanker truck. The amount of water per field site will be
noted in the field notebook.
(B) Pest Control
(1) Weeds
Weed shoots are pinched or cut off and left on the soil surface in order to minimize disruption of
the tree roots and soil. The weed root systems are not to be pulled out but remain in the soil as
-------
Page 8 of 8
GPEP EP.06
Version: 1.00
organic matter. As feasible, the common name for ihe weed will be noted in the tenacosm.
(2) Insects
Each terracosra contains both herbivorous and carnivorous arthropods which were present on the
trees or in the soil at the time of planting. The chambers also contain spiders which were
introduced as controlled numbers as part of the experiment. Herbivorous insects are removed
manually from the trees, identified for at least common name as feasible, and discarded. The
naturally occurring and introduced predators of insects should keep herbivorous insect populations
under control. Application of chemical control will only be used if necessary, as determined on a
case-by-case basis using the least intrusive pesticide.
(3) Diseases
No specific protocols have been identified at this time. However, as for insects, if unusual pest
outbreaks occur al) P.l.s will be consulted to determine the least intrusive control mechanism.
(C) Fertilizer
As of this time, no fertilizers are being used in any study except for specific pot studies. Notes in
this regard will be kept in an experimental notebook as necessary.
Records
Details regarding tree selection, characterization, planting, and culture are recorded in laboratory
notebooks, primarily the TERA and Field notebooks kept in the control room of the polyhouse.
Data files for the To shoot and root measurements are kept with the P.l.s for Task 1 regarding
shoot biology and Task 6 regarding root biology, respectively.
Reference
US EPA. 1993. Effects of CO2 and Climate Change on Forest Trees. Research Plan. ERL-
Corvallis.
-------
United States Environmental Research May 1994
PQA United States Environmental Kesearc
iffSf ¦ "Hf'V Environmental Protection Laboratory - Corvallis
9 m Agency Corvallis, OR 97333
For additional information contact:
Dr. David T. Tingey
Program Leader
ERL-Corvallis
(503)754-4621
-------
ACKNOWLEDGMENT
The Project Staff gratefully thanks S. Volk for developing the
format for this book and producing the booklet in its final form.
-------
Field Studies.
TABLE OF CONTENTS
TABLE OF CONTENTS
I. CASCADE ELEVATIONAL PLOTS
II. APPENDEX A
III. APPENDEX B
IV. APPENDEX C
Page i
Effects of COjOnd Climate Change on Forest Trees
-------
FIELD STUDIES
-------
Field Studies
CASCADE ELEVATIONAL PLOTS
INTRODUCTION:
In conjunction with the experiment at the TERA
facility we decided to compare growth of Douglas
fir seedlings under natural conditions with seed-
ling growth in the Terracosms to provide:
another form of control treatment, and
allometric data from annual seedling har-
vests to parameterize and validate TREGRO
for use with Douglas fir.
We established three small, temporary Douglas-
fir plantations on the west side of the Cascade
Mountains in the Sweet Home Ranger District of
the Willamette National Forest, OR. The three
plantations were established in June 1993, and lie
along an elevational gradient from the foothills to
the upper reaches of the growth of Douglas fir
(Low-site. 537 m; Mid-site: 951; High-site: 1220).
Each plantation has five 1 m by 2 m plots. Trees
were planted at the same density as were seedlings
planted in the Terracosms. Litter bags and needle
packs were buried at 3 cm depth in the 6-cm deep
litter layer placed on the plots. Seedlings will be
excavated in the fall from one plot per year (1994
through 1998) for whole plant allometric data
collection; mycorrhizal colonization and fungal
diversity estimates; and soil, litter and plant chemi-
cal analysis. The harvests will occur shortly
before or after seedling harvest from the Large
Lysimeters and extracting soil cores from the
Terracosms. Additionally, the plots are used to
gather data on seedling growth and phenology,
and decomposition of plant litter. We established
a weather station at the low elevation site in 1993
and will establish similar weather stations at the
mid- and high elevations sites in the summer 1994.
The weather stations will provide year round en-
vironmental data. The field sites are also being
used by some of our Cooperators (litter/soil fauna,
microbial biomass, bulk soil enzymes, stable iso-
tope analysis).
The purpose of the Cascade plots is to monitor the
growth and development of seedlings grown un-
der natural conditions. These studies have been
designed so that once a year a portion of the
seedlings can be excavated and whole seedlings
can be analyzed. Using the data from this study
should provide useful insights into the effects of a
natural range of environmental growing condi-
tions on Douglas-fir seedlings and will be useful
for linking the results obtained with the Terracosm
seedlings to seedlings grown under natural condi-
tions. At the mountain field sites we are also
conducting a forest floor litter decomposition study
that parallels the one being conducted in the
Terracosms to compare decomposition between
Corvallis and the mountain field sites.
SITE SELECTION AND PREPARATION:
We worked with the U.S. Forest Service staff at
the Sweet Home Ranger District to survey poten-
tial sites and to establish an acceptable plan for site
preparation and for building a wildlife exclusion
fence around each site. Our strategy for selecting
sites was to look for sites that been harvested in
within the last five years, that had similar slope
and aspect, and covered the elevational range of
Douglas-fir in the Cascade Mountains. We did not
want sites with atypical climatic conditions or
orographic effects, and from a practical point of
view we wanted all three sites to be within two
hours drive of the Corvallis laboratory and to be
easily accessible from Oregon Highway 20. A
further restriction was that we wanted to establish
one site on the same kind of soil (Forest Service
Mapping Unit 66) that had been used in the
Terracosms.
The Forest Service staff was a tremendous help in
surveying potential sites and for helping us to
complete our plantation establishment plans. We
settled on three sites that follow an elevation-
gradient along the West slope of the Cascade
i 1 Cascade Elevational Plots
-------
Newpo#|';*^
Corvallis ,';i
3T i'--*. . ~iT'-
Sr. V:#?
r ?ifc
1600
1400
1200
f 1000
1 800
C9
| 600
400
200
0
High Site
Elevational Transect
West to East
Bern
Mid Site
Low Site
Corvi
200
250
150
100
Distance - Km
Figure 1. Locatoin of the field sites on the west slope of the Cascades and their elevation.
-------
mountains (Figure 1). The low-elevation site is
approximately 90 kilometers from Corvallis at an
elevation 537 meters. The mid-elevation site is
approximately 107 kilometers from Corvallis at
951 meters Ideally, we would have preferred to
have a mid-elevation site at about 880 meters,
none were available that met our other criteria.
We settled for the mid-elevation site because even
though it is at 951 meters it is situated such that it
functionally behaves as a site that is between 850
and 890 meters This lower elevation characteris-
tic is indicated primarily by the presence of plant
communities found at the lower elevations. The
high-elevation site is at 1220 meters and is 120
kilometers from Corvallis. The high-elevation
site is located on the same kind of soil that was
used in the Terracosms Additional site maps are
found in the Appendices
The specific site location details.
Low-elevation site. [122°22'30" Long &
44°23'30" N. Latt.T13S, R4E, Sec 31,
SE ofNW]
This site, at elevation 1760 feet, is located
in Unit 4 of Topo Falls TBV Timber Sale
(Reforestation No. F16, Compartment
3307, Cell 004B2) Access is from FS RD
203405 which is gated for a winter range
closure from January through April.
Directions to site. At milepost 46 1 on
Oregon Sate Highway 20, turn right (south)
onto FS RD 2032000 (Gordon Road) to
milepost 2.3. Turn right (west) onto FS
RD 2032405. Field site is within a re-
cently-planted plantation on the left (south)
side of the 405 spur at the top of the slope
break, approximately 0.35 miles beyond
the gate closure.
Mid-elevation site: [122° 18' Long. &
44°22' N. Latt.T14S, R5E, Sec 2, SE of
NE]
This site, at elevation 3120 feet, is located
within Unit 4 of the Sheep Snow Timber
Sale (Reforestation No. SI00, Compart-
ment 3313, Cell 155B4). Access is from
FS RD 2000247. This site is also located
behind a winter range road closure from
January through April.
Directions to site: At milepost 61.0 on
Oregon Sate Highway 20, turn right (south)
onto FS RD 2000245 (Burnside Road) to
milepost 1.0. Turn right (south) onto FS
RD 2000247. Field site is within a re-
cently-planted plantation on the left (north)
side of the 247 spur, approximately 1.0
mile beyond the gate closure.
High-elevation site: [ 122° 10' Long. &
44°23' N. Latt.T13S, R6E, Sec 24, SE of
NW]
This site, at elevation 4000 feet, is located
within Unit 2 of the Maude Toad Timber
Sale (Reforestation No. L50, Compart-
ment 3506, Cell 132). Access is from FS
RD 2065174.
Directions to site: At milepost 67.9 on
Oregon Sate Highway 20, turn left (north)
onto FS RD 2065172 to milepost 0.1. Turn
left (northwest) onto FS RD 2065174.
Field site is within a recently-cut planta-
tion on the left (south) side of the 174 spur,
approximately 1.3 miles from the junction
with the 172 road..
FIELD SITE PLAN:
Each field site is approximately 6 m by 13 m and
contain five, 1 m by 2 m, research plots (see Figure
2). Upon the recommendation of Forest Service
revegetation specialists, we had 9 ft. high game
exclusion fence built around each plantation. Some
level of site preparation was required at each site.
This included clearing brush, removal of large
woody debris and hand tilling the soil. Within a
field site, the research plots are separated by a 1 m
-------
Field Studies
6 meters
2 meters
Fence
Planted
Buffer
Area
Soil Faunal
Traps
Plots
Unplanted
Butter
Area
Figure 2: Field site layout.
buffer area that was planted and an additional 1 m
bark-mulched buffer between the planted buffer
and the fence.
At each elevation Douglas-fir seedlings (from the
same lots used in planting the Terracosms) were
planted. Approximately 350 seedlings were
planted in each plantation in May and June of
1993. The planting density (0.13 m^ per seedling)
is much greater than that normally used in reveg-
etation of forest lands, but it parallels that being
used in the TERA facility.
A forest litter layer was estab-
lished in each of the Terracosms
in the TERA facility using for-
est floor litter collected from
established forests near the high-
elevation field site. This proce-
dure was followed at the three
field sites to mimic the natural
forest system and the research
being conducted in TERA. For-
est floor litter from the same
collection area was added to each
of the field sites. The litter serves
as a mulch to help control com-
peting vegetation, and will also
serve as a source of nutrients
through decomposition. Twice
a year an assessment of forest
floor condition will be made in
each of the field sites (using the
same criteria as in the
terracosms), if needed more lit-
ter will be added.
EXPERIMENTAL MEA-
SUREMENTS:
Annually, one research plot will
be excavated and whole seed-
lings with intact root systems
will be collected. By establish-
ing five plots within each plan-
tation, plant materials may be
sampled for up to five years. At the end of the
experiment the EPA will remove all fencing and
either thin the remaining vegetation to Forest
Service specifications or remove it entirely.
Plant Measurements
As part of our comparative study morphological
and physiological data are being collected on the
seedlings at each of the field sites. These data
include: gas exchange, shoot growth, shoot archi-
tecture, and collecting plant samples for chemical
analysis. Soil respiration will be also be a routine
Page 4
Cascade Elevahonal Plots
-------
Field Studies
measurement beginning in the Summer of 1994.
These seedlings will provide root and shoot biom-
ass data and samples of soil will be used for
chemical and biological analyses.
Decomposition study
At each of the field sites a litter decomposition
study, using liner bags and needle packs (see Task
5 of the Research Plan) has been established.
Eighteen fine mesh nylon bags, containing a
pre weighed amount of forest floor litter, and 18
needle packs (needles strung on a string) were
placed in one research plot at each of the field sites.
Twice a year, at the same time they are removed
from the Terracosms, three litter bags and three
needle packs will be removed from each site The
removed bags and packs will be replaced by new
litter bags and needle packs.
Soil Fauna
Soil fauna are being monitored in the field sites to
compare with the patterns observed in the
Terracosms and provide a measure of typical
population changes in natural communities under
ambient environmental conditions. The same meth-
ods of trapping and faunal extraction are used as in
the TERA study, traps are being used (see Task 7).
Six arthropod traps per elevational field site were
installed (Figure 2). The traps are sampled coinci-
dent with the faunal sampling in the Terracosms.
The two bags (litter and A horizon) are removed
from the cylinders and poly-foam is used to re-
place the bags to maintain uniform temperature
and moisture in the litter and A horizon. Bags are
placed in a Berlese funnel extractor system and
the arthropods are extracted using heat. Arthropods
are collected alive, enumerated to species and
returned to the Terracosms. Moisture content of
the extracted soil and litter is adjusted to match the
soil moisture of the respective horizon. Bags are
then returned to the cylinders.
Environmental data
We have established a goal of collecting environ-
mental data [air and soil temperature, solar radia-
tion (PAR), soil moisture, precipitation and rela-
tive humidity] at each of the field sites. In early
1994 a meteorological station was established at
the low-elevation field site Hourly data collected
at the site is down-loaded on a monthly basis An
example of the data are shown in Figure 3.
Meteorological stations will be established at the
nud- and high-elevation sites in the Spring of 1994
following snow melt. Because both the mid- and
high-elevation sites are at elevations that receive
large amounts of snow, environmental data col-
lection will likely be suspended following the first
significant snowfall in the Fall and remain closed
until snow melt in the Spring because it is likely
that the access roads into these sites will be closed
due to snow. We are considering the feasibility of
remotely accessing the data during this period.
To provide an initial assessment of precipitation
patterns with elevation at the field sites the PRISM
Model (Daly, C., R.P. Nelson, D.L Phillips. 1994.
A statistical-topographic model for mapping cli-
matological precipitation over mountainous ter-
rain. Journal Applied Meteorology 33:140-158)
was used PRISM uses a digital elevation model
(DEM), to estimate the "orographic" elevations of
precipitation stations. It partitions a region into
topographic facets (e.g., west slope of the Cas-
cades). For each facet, precipitation is regressed
against DEM elevation (typically 10 km DEM
cells) using observed data from all weather sta-
tions within a certain radius. Precipitation esti-
mates are made for each DEM cell in the topo-
graphic facet by using this regression equation and
the DEM elevation. The estimated precipitation
amounts and monthly patterns for Corvallis and
the 3 field sites are shown in Figure 4. The 30-year
precipitation record (1961 to 1990) was used. The
measured precipitation at Corvallis is included to
provide an indication of model performance.
Page 5
Cascade Elevational Plots
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Field Studies
18-
16-
14-
12 ¦
§ 10
t H
(D
§* 6
Q)
H 4.
2-
0'
-2-
18-
100-
Air Temp
Soil Temp 15 cm
Soil Temp 30 cm
1200
1000
800
¦600
0>
¦400 I
OS
<
¦200
-0
cu
ii'ii1i1i1i 1 i r~
Feb 20-Feb 22-Feb 24-Feb 26-Feb 28-Feb 1-Mar 3-Mar 5-Mar
Date
-200
£
90-
80-
;o
J 70-
X
u
>
I 60
u
OC
50 H
40
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a
a.
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18-Feb 20-Feb 22-Feb 24-Feb 26-Feb 28-Feb 1-Mar 3-Mar 5-Mar
Date
Figure 3 Example of environmental data from the low elevation site on the west slope of the Cascades
Page 6 Cascade Elevational Plots
-------
Field Studies
400
Measured Corvallis
Mid Site
350
Estimated Corvallis
High Site
Low Site
300-
g 250-
; 200-
R" 150-
100-
50-
Oct Nov Dec
Jan Feb Mar
Jun Jul
Month
Figure 4 Precipnaiion comparison among Corvallis and eievational plots on (he west slope of the Cascades The measured
Conallis daia are the 30-year mean (1961-1990) while the estimated precipitation data are from the PRISM model
Page 7
Cascade Elevational Plots
-------
Field Studies
Appendix A
Directions and Maps for the Low Site
Page A-1
Appendix A
-------
SALE ARICA MAI* AND SI.ASII DISPOSAL MAI*
SALE HAMK; TOW) FAI.I S TUV
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-------
Field Studies-
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Page A-3
Appendix A
-------
Field Studies
USD A
Page A-4 Appendix A
-------
Field Studies
Appendix B
Directions and Maps for the Mid Site
Page B-1
Appendix B
-------
Field Studies
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-------
Field Studies -
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Page 6-3
Appendix B
-------
Field Studies
Appendix B
-------
Field Studies
Appendix C
Directions and Maps for the High Site
Page C'l
Appendix C
-------
I
o
o
High Elevation
Sight
.E AREA MAP AND SLASH DISPOSAL MAI-
SALE NANF.: MAUDE TOAD
NATIONAL FOREST: WILLAMETTE
RANGER DISTRICT: SWEET IIOHE
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-------
Page C-3
Appendu C
-------
Pagt C-4
Appendix C
-------
United States Environmental Research May 1994
Arm Environmental Protection Laboratory - Corvallis
Agency Corvallis, OR 97333
TASK 8:
Soil Organic Matter
Project: Effects of C02
and Climate Change
on Forest Trees
For additional information contact:
Dr. David T. Tingey
Program Leader
ERL-Corvallis
(503)754-4621
-------
ACKNOWLEDGMENT
The Project Staff gratefully thanks S. Volk for developing the
format for this book and producing the booklet in its final form.
-------
TaskS. Soil Organic Matter
TABLE OF CONTENTS
Table of contents
TASK 8: SOIL ORGANIC MATTER
Introduction
Policy Issues
Background and Rationale
Science Questions
Objectives
Approach
Modeling
Task Outputs
TREGRO Model Inputs
References
Page i
Effects of CO} and Clunate Charge on Forest Trees
-------
TASK 8: SOIL ORGANIC MATTER
-------
TASK 8: SOIL ORGANIC MATTER
INTRODUCTION:
The Project Research Plan Effects of CO2 and
Climate Change on Forest Trees (U.S EPA, 1993)
describes a detailed study of the effects of elevated
CO2 and climate change on Douglas-fir seedlings
growing in natural soil. In this project one of the
important research foci is on the processing of
carbon in the seedlings and in the soil. When the
original research plan was developed, a decision
was made to investigate as many scientific areas as
possible given the budget and staffing constraints
One area that was not included was a detailed
study of soil organic matter. The situation has
changed since the original plan was approved and
now a new Task, Task 8, is being added to specifi-
call> investigate the effects of elevated COj and
climate change on soil organic matter. Task 8
consolidates the research on soil organic matter
including the measurement of soil C and N in
Terracosm soil organic matter that was originally
described in Task 3 In the sections that follow, the
rationale for adding a soil organic matter task and
the specific research questions, scientific objec-
tives, approach, and research activities are de-
scribed. The last section has a listing of outputs
from this task
POLICY ISSUES:
As listed on page 19 of the Research Plan Effects
ofC02 and Climate Change on Forest Trees (U.S.
EPA, 1993), the project was designed to address a
number of critical policy issues. These are:
1. What are the effects of elevated CO2 and
climate change on the growth and produc-
tivity of forest trees?
2. Will elevated CO2 and climate change
alter the sequestration potential of forest
trees''
3. What is the magnitude of elevated CO2
and climate change impacts on forest trees
and will the impacts be widely distrib-
uted?
A fourth policy issue will be added that provides
the basis for the research proposed in Task 8.
4. Will elevated CO2 and climate change
alter the sequestration and processing of C
in forest soils?
This question is critical because it considers the
storage of C in forest soil organic matter. Under
elevated CO2 and climate change this reservoir
may increase, decrease or remain unchanged. If
sequestration increases, this means that the net
CO2 balance is a withdrawal from the atmosphere.
If soil C decreases, C from the soil has been lost to
the atmosphere, increasing the net balance of C in
the atmosphere which could increase atmospheric
forcing. If the amount of C in soils stays un-
changed, then forest soils are neither a sink or a
source of atmospheric C02- Existing data are not
adequate to provide a defensible scientific answer
to this new question. The results of the research
proposed below will provide the data to answer
this question.
BACKGROUND AND RATIONALE:
An extensive review of the literature and the
rationale for conducting this experiment are given
in the Research Plan (see pages 1-17). Additional
information and literature review are presented
here to provide the necessary background for the
research being proposed in Task 8.
Page I
Effects of CO, and Climate Change on Forest Trees
-------
Global Carbon Cycle
On a global-scale, C circulates between three very
large reservoirs [oceans, atmosphere, and terres-
trial systems (e.g, vegetation, soil)] and can be
found in a variety of compounds in each reservoir
(Houghton and Woodwell, 1989, Post etal., 1990).
These reservoirs,orpools,exchange large amounts
of C annually. A fourth reservoir, the geological
reservoir, contains fossil (i.e., oil, coal, and natural
gas) and mineral C, including carbonates, and
consists primarily of inactive or non-circulating
C. Perturbations, disturbances, or additions of C
(e.g., fossil fuel combustion) to any of the reser-
voirs will have a concomitant effect on the others
because of the dynamic linkage of the reservoirs.
The loss of C as CO2 from soils and wetlands is an
example of loss from the terrestrial pool to the
atmosphere Similarly, erosion moves terrestrial
C from the terrestrial pool to the oceans Global
warming is also expected to have an effect on the
global C cycle. One projected effect is the shifting
of global vegetation zones and the amount of C
stored therein (Emanuel et al., 1985a; Emanuel et
al., 1985b; Leemans, 1990; Prentice and Fung,
1990).
Globally, there are approximately 41,000 Pg (Pg =
petagrams =10^ grams) of active, or circulating,
C (Bolin, 1983; Houghton and Woodwell, 1989;
Post et al., 1990). Of this, the oceanic reservoir
contains 38,000 Pg; the atmosphere 750 Pg; and
terrestrial ecosystems about 2100 Pg of C (Post et
al., 1990). Of the terrestrial C, living plants
account for about 550 Pg of C and soils approxi-
mately 1500 Pg (Houghton and Skole, 1990).
Over two thirds of the C in forest ecosystems,
which are the subject of this research, is contained
in soils and peat deposits (Dixon et al., 1994).
Soils are therefore the largest, non-fossil fuel,
terrestrial reservoir of C.
At steady state, the net transfer of C between the
global C pools is in equilibrium (Bolin, 1983; Post
et al., 1990; IPCC, 1990). The amount of C fixed
annually by terrestrial plants through photosyn-
Page 2
thesis ranges from 100 to 120 Pg (Post et al.,
1990). Plant respiration releases approximately
40 to 60 Pg of C annually, and decomposition of
organic residues, including soil C, releases ap-
proximately 50 to 60 Pg. At steady state, the
amount of C oxidized by plant respiration and
decomposition balance that fixed by photosynthe-
sis. Through agricultural practices and various
land uses, including deforestation, the oxidation
of plant and soil C is exceeding the amount of C
being fixed by photosynthesis and thereby con-
tributing to the net 3 Pg annual increase in atmo-
spheric CO2 (Houghton and Woodwell, 1989).
Climate Change and Soils
One of the concerns and unknowns associated
with global warming and the C cycle is the effect
of warming on the distribution of C in the various
reservoirs. Will increased temperatures cause a
massive shift in C now sequestered in soils to the
atmosphere thereby increasing global warming?
Buol et al. (1990) projected that a 3 °C increase in
mean annual surface temperatures over the next
50 years would result in an 11% soil organic C
decrease in pools of the top 30 cm of temperate
zone soils. This would result in the emission of
approximately 58 Pg of C to the atmosphere.
Additional soil C would also be lost from the
tropical and boreal zones because of global warm-
ing. Jenkinson et al. (1991) projected that if global
warming caused mean air temperatures to in-
crease uniformly by 0.5 °C per decade over the
next 60 years (3 °C total increase), that approxi-
mately 100 Pg of C would be released from soils
due to the temperature increase alone. If this
projection included the effects of deforestation
and land use changes over the same period (the
next 60 years), the projected losses of soil C could
easily reach 10% of the global soil C pool, or about
150 Pg. This is roughly equivalent to C emissions
from fossil fuels since 1850 (Houghton and Skole,
1990). The loss from soils could occur, however,
in a much shorter period of time due to any of the
scenarios above. Occurrence of one of these soil
Effects of CO, and Climate Change on Forest Trees
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Task 8 Soil Organic Matter
C loss scenarios would exacerbate the amplitude
and extent of global warming due to increased
greenhouse gases Increased warming could in
turn increase C emissions from soil Recent re-
search indicates that exposure of tenestnal plants
to elevated levels of CO2 can increase the loss of
C from soils (Komer and Amone, 1992).
Soil Organic Matter and Terracosm Soils
Using Figure 1 as a model for the C cycle in the
Terracosms, the Project Plan as currently struc-
tured includes Tasks that consider the Shoot, Lit-
ter, and Root compartments and the CO2 uptake
(photosynthesis) and release (respiration) fluxes
Task 8 is designed to collect data on the Soil
Organic Matter compartment and on the leaching
of C These data, when combined with data from
other Tasks, will be used to calculate a C mass
balance for the Terracosms.
SCIENCE QUESTIONS:
The purpose of this task is to evaluate the effects
of elevated CO2 and climate change on soil
organic matter. Several scientific questions are
germane to this task. The first question explores
the overall change in the total amount of soil
organic matter as a result of elevated carbon diox-
ide and climate change:
Will a net loss or gain of soil or-
ganic matter result from elevated
CO2 and elevated temperature?
Several authors (Buol et ah, 1990; Jenkjnson et ah,
1991) have predicted that soil organic matter will
be lost as a result of global wanning. Others have
reported that increasing CO2 increases fine root
production and turnover (Nortiyetah 1992;Rogers
et ah 1992) thereby increasing the amount of C
allocated belowground, but not necessarily in-
creasing the net amount of soil organic matter.
Still others have reported that exposure of plants to
elevated levels of CO2 caused large increases in
CO2 evolution from soil and loss of soil organic
matter (Komer and Amone, 1992). Forest soils,
which are the focus of the current research, tend to
assimilate organic matter slowly due to rapid
surface decomposition of large amounts of litter
(Oades 1988). The combination of global warm-
ing with slow accumulation of organic matter in
forest soils could have implications on a global
scale. The predictions of loss of soil organic
matter as a result of global warming have not been
verified, however, under any type of rigorous,
controlled research. In the Terracosm study, the
net gain or loss of soil organic matter will be
evaluated under treatments of elevated CO2, el-
evated temperature, and both treatments together.
The second question concerns the use of' and
1 as naturally-occurring stable isotope labels to
monitor the processing of native soil organic mat-
ter and the new plant-derived organic matter sub-
strates that will be added to the Terracosm soils:
Can measurements of and ^C
be used to assess the processing
(decomposition or sequestration)
of soil organic matter and the ef-
fects of elevated CO2 and climate
change on this processing?
It has been established (Natelhoffer et ah, 1988)
for some soils, especially well-drained forest soils,
that and are preferentially enriched dur-
ing the process of soil organic matter decomposi-
tion. Increasing levels of and are also
detected with increasing soil depth as compared to
levels of soil organic matter at the soil surface. If
these enrichment phenomena are duplicated in a
forest tree seedling-based Terracosm, they may
provide a useful tool for understanding how soil
organic matter is being processed under condi-
tions of elevated CO2 and climate change. This
information may lead to the development of an
index of soil organic matter quality and relative
stability.
Page 3
Effects of CO, and Climate Change on Forest Trees
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TaskS Soil Organic Matter
The third question relates to the interaction of the
C (water insoluble, water soluble and inorganic)
and nutrient cycles and the effects of elevated CO 2
and climate change:
Does elevated CO2 ond climate
change effect soil nutrient levels
and will an increase in the levels of
soluble organic and inorganic car-
bon in soil solution result?
Plants generally respond to elevated CO2 by in-
creased photosynthesis and increased growth if
other factors such as water and nutrients are not
limiting. There is some evidence that plants use
water and nutrients more efficiently under el-
evated CO2 which may account for plant response
even under water and/or nutrient limiting condi-
tions. It is likely that plants under elevated CO2
will respond by allocating a larger portion of
photosynthate belowground for nutrient and wa-
ter uptake. The portion remaining belowground
for a longer period may be: 1) used to build roots,
2) sent to mycorrhizae in exchange for nutrients or
water, or 3) excreted into the rhizosphere to fuel
microbial mineralization of soil organic matter or
to promote mineral weathering. Eventually, some
portion of the C allocated belowground will return
to the atmosphere as CO2 through respiration.
Each of these probable outcomes may affect the
character of the soil organic matter. Since the
Terracosm soil is nutrient limited, monitoring the
quantity and quality of C and the content of vari-
ous plant nutrients in soil organic matter will help
explain the observed plant responses to elevated
CO2 and climate change.
A number of factors contribute to the presence of
soluble C in soil water. One is whether or not the
system is dominated by bacteria or fungi. The
relative presence and activity of these organisms
affects the processing of C in soils. Another is the
amount and form of plant exudates in the rhizo-
sphere. Often these exudates are the result of plant
related modification of the chemistry of the rhizo-
sphere to promote the weathering of primary min-
erals or to alleviate the toxicity of certain metal
cations. Since many of these exudates are soluble
along with some of the by-products of decompo-
sition and soil organic matter mineralization,
monitoring the composition of soluble organics in
soil solutions will help depict the processing of
soil organic matter in the Terracosms.
OBJECTIVES:
The following objectives address the science ques-
tions relating to soil organic matter:
Monitor and quantify changes in overall soil
organic matter content in Terracosm soils that
may result from elevated CO2 levels and cli-
mate change.
Characterize the soluble forms of soil organic
matter from the Terracosm soils and soil
leachates to evaluate the effects of elevated
CO2 and climate change on soil organic mat-
ter.
Collect soil data that, when combined with
data from other Tasks, completes a carbon
mass balance for the Terracosms.
Monitor the relationships between soil nutri-
ents and soil organic matter in the Terracosm
soils to assess effects of elevated CO2 and
climate changeonnutrientsupply and the role
of soil organic matter in nutrient supply.
Monitor the processing of soil organic matter
and plant-derived organic substrates to assess
the effects of elevated CO2 and climate
change on the soil carbon cycle.
Page
4
Effects of COt and Climate Change on Forest Trees
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Task 8 Soil Organic Matter
APPROACH:
This task focuses on the long-term effects of
elevated CO2 and climate change on processing of
organic matter in soil The senes of science
questions and objectives stated above is designed
to guide data collection of this task. It may not be
possible, however, to answer all of the questions
we've asked The ability to answer the science
questions depends upon the successful outcome of
exploratory research and the development of ap-
propriate methods. In Task 8 when we refer to soil
organic matter we are referring to the C content of
the fraction or sample being described. In Task 8
soil organic matter does not include carbonate,
bicarbonate or other inorganic forms of C.
Change in Soil Organic Matter Content
Prior to placing the soil in the Terracosms by
horizon it was homogenized and subsampled for
chemical characterization The change in soil
organic matter content will be assessed twice a
year by analyzing the total C content of soil
samples collected by horizon. Sampling by hori-
zon will allow us to determine where changes in
soil organic matter are occurring Also, by sam-
pling two times a year over the life of the experi-
ment we can plot the rate of organic matter addi-
tion or loss as a function of treatment. Results
from other tasks will be useful in explaining the
observed results.
The change in soil organic matter is described by
Equation 8.1:
ASOM ¦ SOMjnitial SOMrinai
Equation 8.1
Where ASOM is the change in soil organic matter,
SOMjnjtia] is the initial soil organic matter C
content, and SOMfina] is the final soil organic
matter C content. The units on SOM are g C/cm^.
Changes in soil organic matter content will be
based primarily on a whole Terracosm basis This
is possible because a uniform volume of soil was
added to each Terracosm. This means that the by
horizon soil organic matter data will have to be
aggregated up to the Terracosm level. To make
these calculations we need to know the bulk den-
sity of the soil and the volume of the soil. Bulk
density of the specific soil horizon is measured
every time soil samples are collected and the
volume of each horizon is known.
Soil Organic Matter Processing
Monitoring naturally-occurring stable isotopes of
C and N is useful because some natural processes
discriminate between the heavy and light isotopes
leading to heavy isotope enrichment or depletion
For example, during photosynthesis C3 plants
(Calvin cycle) discriminate more against 1 ^C than
do C4 plants (Hatch-Slack cycle) leading to C3
plant tissues lower in than C4 plants (Balesdent
et al, 1988). Consequently plant detritus from C3
will have a different l^C signature than C4 plant
detritus The processing of organic matter in soils
also leads to further fractionation of ^C
(Veldkamp, 1994). Nitrogen in soil organic mat-
ter tends to be enriched in suggesting a
microbially-mediated discrimination during de-
composition (Ehleringer and Rundel, 1989).
Monitoring the stable isotopes of C and N in
Terracosm soils and their changes over time is
likely to be a powerful tool for assessing whether
or not new organic matter is being sequestered in
soil and whether or not old soil organic matter is
being preferentially utilized as a source of N.
These measurements, when coupled with other
laboratory soil incubation and fractionation stud-
ies (described below) will aid in quantifying labile
and passive organic matter pools.
The general methodology used is to measure the
ratio of two stable isotopes (e.g., ^c/ 12q on a
mass spectrometer relative to a standard. The
results are expressed as 8%o^C (per milparts
per thousand) values. This is shown for l^C/^C
in Equation 8.2 and in Equation 8.3.
Page 5
Effects of COt and Climate Change on Forest Trees
-------
Task 8 Soil Organic Matter
6^C%t = ¦ 1] x 1000
Equation 8.2
6JS\Sc = [({ls.V14N)sampie/(15N/14N)reference) - H * 1000
Equation 8J
Enrichment in 13C or1 results in greater 81
and 5^N values, while depletion results in lower
8'^C and 8^N values. Atmospheric C02 in
Covallis has a 8^C ratio of -8%o (Guanghui Lin,
1994, unpublished data) while the tank CO2 that is
used to add CO2 to the Terracosms has a S13C
ratio of -36%c. Therefore, new carbon fixed dur-
ing photosynthesis will have a unique 5^C ratio.
We propose measuring 8'^C and 8^N on Terra-
cosm soil samples collected throughout the el-
evated CO2 and climate change experiment fol-
low ing the methods described in Veldkamp (1994),
Natelhoffer and Fry (1988), and Balesdent et al.
(1988)
Soil Organic Matter and Nutrients
Soil organic matter is a major reservoir of plant
nutrients in forest soils. Under conditions of
elevated temperature, organic matter decomposi-
tion rates may accelerate, leading to increased
availability of plant nutrients in the short-term
This increased nutrient availability may lead to
increased plant uptake. Concomitantly, increased
allocation of C belowground (to roots, exudates,
or symbionts) due to elevated CO2. may also
accelerate decomposition by priming decomposer
populations with a labile C source. In the long-
term, these processes may result in a depletion of
nutrient reserves which in turn could ultimately
limit nutrient availability and plant growth. Moni-
toring changes in soil C concentrations and nutri-
ents will provide information on whether or not
nutrient limitations are being caused by elevated
CO2 and/or elevated temperature.
Total and extractable nutrients will be measured
on Terracosm soil samples collected twice yearly
over the course of the study. We will use these data
to calculate C to nutrient ratios (e.g., C/N). Infor-
mation being collected in Task 3 (System Nutri-
ents) on plant nutrient uptake and nutrients in
drainage waters will be used to help explain why
changes in ratios occurred.
Soil Organic Matter in Soil Solutions
Soil solutions are collected fromsix tension ly sim-
eters in each Terracosm on a monthly basis. Nu-
trients and pH are routinely measured on these
solutions (See Task 3 in this Booklet). Our
approach to characterizing soluble C in the solu-
tions is two-fold. First will be a measurement of
total dissolved organic C (DOC) in these solu-
tions. Second will be the identification and quan-
tification of specific water-soluble organic acids
and compounds Initially, we will look for a broad
range of compounds to qualitatively identify spe-
cific ones in the soil solutions. When we know
which compounds are present, we will then de-
velop procedures to quantify them in the rou-
tinely-collected soil solutions using either ion
chromatography (IC) or high pressure liquid chro-
matography (HPLC).
We will also develop a method for extracting
soluble organics in the Terracosm soil samples
collected two times per year. Our plan is to take
small soil subsamples and extract them with water
and then separate the water and soil by centrifuga-
tion These samples will be analyzed for total
DOC and specific organic compounds. The ratio-
nale for adding this water extraction is to deter-
mine the composition of soil waters held under
greater tensions than those obtained from the
tension lysimeters. Because the Terracosm soils
have an abundance of short-range order Fe, Al,
and Si oxides that have a high affinity for soluble
organics we may find that the soil solutions are
low in soluble organics. By extracting soil samples
with water we reduce the contact of the soluble
organics with the adsorbing mineral phases and
increase the likelihood of collecting soluble or-
ganics.
Page 6
Effects of CO, and Climate Change on Forest Trees
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Task 8 Soil Organic Matter
Exploratory and Developmental Studies
Most computer simulation models that character-
ize soil organic matter processing include two or
three pools of kinetically-defined soil organic
matter that have different turnover rates
(Cambardella and Elliott, 1992). Classical soil
organic matter physical or chemical separation
techniques (e.g, base extractable humic acids,
Stevenson et al, 1989) do not identify pools useful
for describing turnover rates or useful for model-
ing (Parton et al., 1989) In Task 8 our will focus
on biologically-meaningful pools (i.e., kinetically-
defined) of soil organic matter; pools that are
related to the processing of soil organic matter.
This focus is of utmost importance when trying to
interpret how organic matter is processed in soils.
We are not excluding the use classical physical
and chemjcal extraction and fractionation tech-
niques, but use them primarily to provide linkages
between our biologically-meaningful pools and
previously published data
Quantifying the "biologically-meaningful pools"
of soil organic matter is not easy since numerous
attempts have been made to do so and no standard
methods or techniques are have been identified. A
recent paper by Cambardella and Elliott (1992)
reports on the development of a separation method
that links measurable fractions of soil organic
matter to kinetically defined pools. This method
includes dispersion, size fractionation, density
separation and chemical analysis. We propose
using this method to characterize the changes in
Terracosm soil organic matter. By using this
method, the fraction data can be used as inputs to
most soil organic matter models (see following
section on Modeling).
In addition to using the Cambardella and Elliott
(1992) method we propose running soil incuba-
tion studies to characterize the CO2 production
and decomposition of TeiTacosm soil organic
matter. Soil samples will be incubated under a
range of moisture and temperature conditions.
During these studies, the production of CO2 and
Page 7
its 6^C value will be determined and the changes
in soil organic matter will be measured. Rates of
CO2 production will provide insight into the ki-
netics of decomposition. The measurement of
6^C at all stages of the incubation study may lead
to the development of a method for discriminating
between labile and passive soil organic matter.
Integrating incubation studies with the separation
methods of Cambardella and Elliott (1992 and
1994) and Baldock et al. (1992) will provide
additional ways to analyze soil organic matter.
Measurement of 8^N in addition to 8^C may be
useful in developing an isotopic index of soil
organic matter quality.
MODELING:
Section 6 in the Research Plan describes the simu-
lation modeling that is currently planned for the
Project. The modeling primarily focuses on the
use of the TREGRO model (Weistein et al., 1992).
TREGRO is an individual tree growth simulation
model that includes a soil organic matter subrou-
tine based upon the MBL-GEM model of Rastetter
et al. (1991). Inputs to TREGRO for the decom-
position subroutine include root chemistry and the
C and N content of soil humus (i.e., non-plant soil
organic matter) and various transformation pa-
rameters. The root chemistry inputs for TREGRO
come from Task 3 and Task 6 (U.S. EPA, 1993).
The C and N content of soil humus inputs will
come from Task 8. The organic matter transfor-
mation parameters for TREGRO are reported in
published literature such as Ryan et al. (1990).
One of the long-term objectives of Task 8 will be
to project the observations and results of our
research on soil organic matter in the Terracosms
to different scales and to different soils and re-
gions. A variety of models for making these
projections exist that deal specifically with soil
organic matter including CENTURY (Parton et
al., 1988), the Rothamsted model (Jenkinson, et
al., 1990), and MBL-GEM (Rastetter et al., 1991).
Effects of CO, and Climate Change on Forest Trees
-------
Each of these models has terms for biologically-
relevant pools (i.e., kinetic pools) of soil organic
matter. Results from Task 8 will provide the input
needed to run these models. We have not selected
a model to use and will wait until later in the
project.
TASK OUTPUTS:
Quantification of net gain or loss of soil or-
ganic matter in response to elevated CO2 and
climate change.
Characterization of the processing of soil or-
ganic matter in Terracosm soils and how el-
evated CO2 and climate change affect this
processing.
Characterization of the interaction between
soil nutrients and soil organic matter and how
these interactions affect nutrient supply and
cycling under elevated CO2 and climate
change.
Qualitative and quantitative characterization
of the effects and elevated CO2 and climate
change cf soluble organic in Terracosm soils
and soil solutions.
TREGRO MODEL INPUTS:
Task 8 will provide the following inputs for the
TREGRO model:
C content of soil organic matter
N content of soil organic matter
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1989. Methodologies for assessing the
quantity and quality of soil organic matter,
pp 173-199 IqD.C.Coleman,J.M.Oades,
and G. Uehara (eds.) Dynamics of Soil
Organic Matter in Tropical Ecosystems.
NifTAL Project, University of Hawaii
Press, Honolulu, Hawaii.
U.S EPA. 1993. Research Plan: Effects of CO2
and Climate Change on Forest Trees.
Environmental Research Laboratory,
Corvallis, OR, 97333.
Weistein.D A.,R D. Yani,R.M. Beloin, andC.G
Zollweg. 1992. The response of plants to
interacting stresses: TREGRO version
174. Description and parameter require-
ments. Electric Power Research Institute,
Pleasant Hill, CA EPRITR-101061.
Veldkamp, E. 1994. Organic carbon turnover in
three tropica] soils under pasture after de-
forestation. SoilSci. Soc. Amer. J. 58.175-
180.
Page 10
Effects of CO} and Climate Change on Forest Trees
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Task 8 Soil Organic Matter
CO
SP!L 5UFIEACP
Soluble Inorganic C
Soluble Organic C
LITTER
LITTER
CARBONATES
SHOOTS
ROOTS
SOIL ORGANIC MATTER
Figure 1 Generalized Terracosm carbon cycle
Page 11 effects of CO, and Climate Change on Forest Trees
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Environmental Research
Laboratory - Corvallis
Corvallis, OR 97333
May 1994
EPA ERL-Corvallis Library
00005045
f/EPA
United States
Environmental Protection
Agency
MANUSCRIPTS AND
ABSTRACTS
Project: Effects of C02
and Climate Change
on Forest Trees
For additional information contact:
Dr. David T. Tingey
Program Leader
ERL-Corvallis
(503)754-4621
-------
ACKNOWLEDGMENT
The Project Staff gratefully thanks S. Volk for developing the
format for this book and producing the booklet in its final form.
-------
Abstracts and Manuscripts
TABLE OF CONTENTS
I. ABSTRACTS
Effects of C02and Climate Change
Effects of C02and Climate Change
Science Research Facility
Effects of C02and Climate Change
Flux Responses
Effects of C02and Climate Change
Effects of C02and Climate Change
mology
Soil Lysimeters for Evaluating the Effects of Climate Change on Forested Soils
Effects of Soil Collection and Storage on Reconstructed Forest Soil Profiles
II. MANUSCRIPTS
Sunlit Controlled-Environment Chambers for Studying Elevated C02 and Climate Change
on Tree/Soil Processes (submitted to Plant and Soil)
on Forest Trees: The TERA Project
on Forest Trees: TERA, A State-Of-The-
on Forest Trees: Shoot Growth and Gas
on Forest Trees: Roots and Mycorrhizae
on Forest Trees: Soil Biology and Enzy-
Page
Manuscript
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ABSTRACTS
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ABSTRACT: ECOLOGICAL SOCIETY OF AMERICA, 1994 ANNUAL
MEETING
Knoxville, TN
EFFECTS OF CO2 AND CLIMATE CHANGE ON FOREST TREES: THE TERA PROJECT
RYGIEW1CZ1, PAUL T., DAVID T. TINGEY1, MARK G. JOHNSON2, BRUCE D. McVEETY3,
DAVID M. OLSZYK^ and RAYMOND SHIMABUKU'. 'U.S. Environmental Protection Agency,
Corvalhs, OR 97333 USA, ^ManTech Environmental Technology Inc., U.S. EPA, ^Batelle-Pacific
Northwest Laboratories, Richland, WA 99352 USA.
Rising atmospheric CO2 and other greenhouse gas concentrations may lead to altered climates, which
may dramatically affect forests. In 1993, Douglas fir seedlings were planted in sunlight, climate-
controlled environmental chambers (Terracosms) at a new research facility (TERA). The Terracosm
experiment imposes altered climate scenarios possible within approximately 50 years: two CO2
(ambient, and ambient + 200 ppm) and two temperature (ambient, and ambient + 4°) treatments. The
elevated CO2 and temperature treatments are added continuously to current ambient levels to preserve
natural climatic variability. Supporting experiments are underway in the Cascade Mountains, and in
Observation and Manipulation Rhizotrons. Seven tasks describe the ecosystem responses being
measured: 1) Shoot carbon and water fluxes, 2) Shoot growth and phenology, 3) System nutrients, 4)
System water, 5) Litter layer, 6) Root growth and phenology, and 7) Soil biology. Data will be used in
a physiological process-based tree growth model (TREGRO) to assess effects of climate change on trees.
Hypotheses, objectives and an overall description of the project are presented to introduce the results
shown in the following four posters.
Paget
Abstracts
-------
ABSTRACT: ECOLOGICAL SOCIETY OF AMERICA, 1994 ANNUAL
MEETING
Knoxville, TN
EFFECTS OF C02 AND CLIMATE CHANGE ON FOREST TREES: TERA, A STATE-OF-
THE-SCIENCE RESEARCH FACILITY
WASCHMANN1, RONALD S., BRUCE D. MCVEETY2, GLENN D. JARRELL1, MARK G.
JOHNSON1, PAUL T. RYGIEWICZ3, DAVID M. OLSZYK3, and DAVID T. TINGEY3.
1 ManTech Environmental Technology, Inc., U.S. EPA, 2Battelle-Pacific Northwest Laboratories, Richland,
WA 99352, and U.S. Environmental Protection Agency, Corvallis, OR, 97333.
The Terrestrial Ecophysiological Research Area (TERA) is a facility being used to examine the effects
of elevated C02 and global climate change on Douglas fir (Pseudotsuga menziesii (Mirb) Franco)
seedlings. TERA consists of 12 climate-controlled growth chambers (Terracosms) that are illuminated
with natural sunlight. Terracosm environmental conditions continuously mimic diurnal and seasonal
changes in ambient air temperature, dew point temperature, and CO2 concentration to ± 1.5°C, ± 5°C,
and ± 20 ppm, respectively. The terracosms are extensively instrumented in order to examine above-
and belowground processes. All sensors, analytical instruments, and data acquisition equipment
required for independent operation are located at each Terracosm. Data quality control is insured with
centralized, redundant instrumentation. The TERA research facility will be described, including overall
system performance and Terracosm climate control.
Page 2
Abstracts
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Abstracts and Manuscripts
ABSTRACT: ECOLOGICAL SOCIETY OF AMERICA, 1994 ANNUAL
MEETING
Knoxville, TN
EFFECTS OF C02 AND CLIMATE CHANGE ON FOREST TREES: SHOOT GROWTH
AND GAS FLUX RESPONSES
OLSZYK1, DAVID M., CLAUDIA M. WISE1, DAVID T. TINGEYl, PAULT. RYGIEWICZ1, and
RONALD S. WASCHMANN2. Ju.s Environmental Protection Agency, Corvallis, OR, 97333 and ^ManTech
Environmental Technology, Inc, U S EPA
Critical questions regarding effects of rising atmospheric CO2 and climate change on forested systems
include: Will shoot growth change in response to elevated CO2 and temperature?. Will net carbon and
water fluxes change in response to elevated CO2 and temperature? Initial shoot growth and gas flux data
are presented for Pseudotsuga menziesii seedlings continuously exposed for one year to target
environments of ambient or ambient + 200 ppm CO2, and ambient or ambient + 4°C air temperature in
closed chambers. Changes in stem diameter, height, terminal shoot and bud length, are reported. Whole
canopy and single branch level gas flux data used to calculate photosynthetic, respiration, and
transpiration rates also are reported. The experiment is continuing so that longer-term impacts of CO2
and temperature on the seedlings can be determined and data obtained for process-based modeling of
tree growth. The aboveground effects will be related to belowground processes to evaluate whole system
responses to atmospheric CO2 and climate change.
Page 3
Abstracts
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Abstracts and Manuscripts
ABSTRACT: ECOLOGICAL SOCIETY OF AMERICA, 1994 ANNUAL
MEETING
Knoxville, TN
EFFECTS OF CO2 AND CLIMATE CHANGE ON FOREST TREES: ROOTS AND
MYCORRHIZAE
TINGEY1, DAVID T., MARK G. JOHNSON2, PAUL T. RYGIEWICZl, and MARJORIE J.
STORM2. ^U.S. Environmental Protection Agency, CorvaJlis, OR and ^ManTech Environmental Technology,
Inc., U.S. EPA.
Rising atmospheric CO2 and climate change may have dramatic effects on forested systems. Under
elevated CO2 most forest species increase their allocation of C belowground to acquire additional
resources for growth. As a part of a long-term study of elevated CO2 and climate change on forest trees
(the EPA's TERA project) minirhizotrons and root coring are being used to investigate effects on root
dynamics and mycorrhizal colonization. Video images of roots are collected every four weeks and root
cores are collected every six months. Data are extracted from the video tapes using the ROOTS software
and used to characterize root dynamics and life history. Data on the extent of mycorrhizal colonization
and morphology of mycorrhizae are collected from roots sampled in the coring. Most roots observed are
fine roots (diameters < 2 mm). Mycorrhizal colonization exceeds 90% of the fine roots. Methods used
and results will be presented including a video presentation of minirhizotron images and data processing.
Page 4
Abstracts
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Abstracts and Manuscripts
ABSTRACT: ECOLOGICAL SOCIETY OF AMERICA, 1994 ANNUAL
MEETING
Knoxville, TN
EFFECTS OF CO2 AND CLIMATE CHANGE ON FOREST TREES: SOIL BIOLOGY AND
ENZYMOLOGY
MOLDENKEl, ANDREW R., NANCY BAUMEISTERl, BRUCE A. CALDWELL1, ROBERT
GRIFFITH1, ELAINE R. INGHAMJ, MARK G. JOHNSON2, PAUL T. RYGIEWICZ3. DAVID
T. TINGEY^, and JAMES WERNZ1. 'Oregon State University, Corvallis, OR, 97331 USA,
^ManTech Environmental Technologies, Inc., US EPA, and ^U.S. Environmental Protection Agency,
Corvallis, OR. 97333.
Samples of Terracosm soils were analyzed shortly after initial setup to determine whether initial
conditions were equivalent and matched expected values for local soils. Total and active fungal biomass,
active bacteria] biomass and protozoan numbers were reduced, with greatest decreases occurring in the
A horizon. No effect was observed on total bacteria] biomass, nematode or arthropod densities, but
changes in nematode and arthropod species composition occurred. Significant differences in total
density and species composition occurred between the enclosed Terracosms and the open controls.
Arthropod and nematode community structure in the three altitudinal field sites had significantly
diverged. No significant differences in activities of key soil enzymes in C- and N-cycling (acid
phosphatase, protease, B-glucosidase, phenol oxidase and peroxidase) were found between initial
samples relative to treatment, but all levels were significantly different relative to depth in soil profile.
Activities were within ranges previously observed in forests of the Pacific Northwest.
PageS
Abstracts
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Abstracts and Manuscripts
ABSTRACT: SOIL SCIENCE SOCIETY OF AMERICA MEETING
Minneapolis, MN
SOIL LYSIMETERS FOR EVALUATING THE EFFECTS OF CLIMATE CHANGE ON
FORESTED SOILS
M.G JOHNSON', B.D. MCVEETY2, P.T. RYGIEWICZ3 and D.T. TINGEY3. 'ManTech Environ-
mental Technology, Inc., U.S. EPA, :Battelle-Pacific Northwest Laboratories, Richland, WA
99352 USA, andJU.S. Environmental Protection Agency, Corvallis, OR 97333.
Climate change is postulated to effect the productivity of forested ecosystems. Soil and plant processes
and feedbacks between these processes will largely determine the overall response. To evaluate the
effects of climate change (elevated CO2 and temperature, altered moisture regimes) conditions on forest
trees, soils and soil biota we have built a new facility that consists of 12 outdoor climate-controlled plant
growth chambers, called "terracosms." The terracosms couple a climate-controlled aboveground
compartment (2.7 nr3) with a large soil iysimeter (2 m^). Both compartments are instrumented to
monitor soil/plant responses and conditions in sillJ. The soil instrumentation includes: TDR, thermistors,
tension lysimeters, gas samplers, and minirhizotron access tubes. The design, instrumentation, and
utility of these lysimeters will be covered. Soil selection criteria, soil collection methods, and Iysimeter
filling techniques will also be presented.
Page 6
Abstracts
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Abstracts and Manuscripts
ABSTRACT: 8TH NORTH AMERICAN FOREST SOILS CONFERENCE, 1993
Gainesville, FL
EFFECTS OF SOIL COLLECTION AND STORAGE ON RECONSTRUCTED FOREST
SOIL PROFILES
M.G. JOHNSON1,P.T. RGIEWICZ2,D.T.TINGEY2,andB.D. MCVEETY3. 'ManTech Environmen-
tal Technology, Inc., ^ US EPA, U.S. Environmental Protection Agency, Corvallis, OR 97333 USA, and
^Battelle-Pacific Northwest Laboratories, Richland, WA 99352 USA.
Soil and plant processes and feedbacks between these processes will largely determine the overall Soil
and plant processes and feedbacks between these processes will largely determine the overall Soil
response of forest trees to elevated CO2 and climate change. As part of an experiment to evaluate these
conditions on forest trees, soils and soil biota we have built a new facility that consists of 12 outdoor
climate-controlled plant growth chambers, called "terracosms." The terracosms couple a climate-
controlled aboveground compartment with a large soil compartment (2 m3). Both compartments are
instrumented to monitor soil/plant responses in situ. To approximate natural conditions we collected
approximately 100 m3 of forest soil by master horizon. The soil was collected in the fall and transported
to the chamber facility where it was stored over winter in covered piles. In following spring soil profiles
were reconstructed and instrumented in the terracosms. Douglas-fir (Tseudotsuea mgnsiesii (Mirb.)
Franco) seedlings are being grown in the terracosms to assess the long-term soil and plant responses to
elevated C02, elevated temperature, and drought treatments. The objective of this presentation is to
evaluate the effects of collection, storage, and reconstruction on our soil and the potential effects on our
experiment. We have characterized the initial and temporal properties of the virgin forest soil, stored
soil, and the reconstructed soil. We will present results on soil carbon, nutrients, water holding
characteristics, bulk density, and microbial biomass. Soil selection criteria and methods for large-scale
soil collection and profile reconstruction will be discussed.
Page 7
Abstracts
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MANUSCRIPTS
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Number of pages of text - 10
Number of Tables -2
Number of Figures - 5
Running Title: Sunlit Controlled-Environment Chambers for Studying Elevated
C02 and Climate Change on Tree/Soil Processes
Corresponding Author
Bruce D. McVeety
Battelle Pacific Northwest Laboratory
U.S. EPA Environmental Research Laboratory
200 SW 35th Street
Corvallis, OR 97333
Telephone (503) 754-4758
Fax (509) 754-4799
E-mail bruce@heart.cor.epa.gov
Key words: Climate Change, Below-ground Processes, C02, Controlled-
Environment Chambers, Pseudotsuga menziesii [mirb.] Franco
The information in this document has been funded wholly by the U.S.
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. Mention of trade names or commercial products does not constitute
endorsement or recommendation for ust.
1
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Sunlit Controlled-Environment Chambers for Studying Elevated C02 and
Climate Change Effects on Above- and Belowground Tree/Soil Processes
by
Bruce D. McVeetyl, David T. Tingey^, David M. Olszyk^,
Paul T. Rygiewicz^, Mark G. Johnson^
U.S. EPA Environmental Research Laboratory
200 S.W. 35th Street
Corvallis, Oregon 97333
iBattelle Pacific Northwest Laboratory
2U.S. Environmental Protection Agency
^ManTech Environmental Technology, Inc
Key words Climate Change, Below-ground Processes, C02, Controlled-
Environment Chambers, Pseudoisuga menziesii [mirb.] Franco
ABSTRACT
The Global Change Research Program at the US EPA's Environmental Research
Laboratory-Corvallis (ERL-Corvallis) recently initiated a four-year study of the
effects of elevated C02 and climate change on Douglas fir seedlings. This
research project integrates experimental and modeling studies to yield data
necessary to make regional-scale assessments of climate change impacts on forest
productivity and to provide data to support other ongoing climate
change/vegetation studies. The construction of the Terrestrial Ecophysiological
Research Area (TERA) occurred simultaneously with the development of the
experimental research approach, assuring integration of experimental
requirements into system design. Located at ERL-Corvallis, the TERA facility
provides a state-of-the-science research capability to investigate the effects of
elevated C02 and climate change on plants. The facility is unique as it accurately
measures and track changes in ambient CO2, temperature and dew point in sunlit
controlled-environment terracosms. The terracosms are well-monitored semi-
closed systems that allow the development of mass budgets for carbon, water and
2
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plant nutrients while applying specific experimental treatments of elevated C02
and/or increased temperature. Designed to facilitate the simultaneous studies of
above- and belowground plant and soil processes, each terracosm presents the
unique opportunity to investigate relationships such as root activity (e.g., root
initiation, root turn-over) and soil C02 flux as a function of soil water content
and/or shoot activity (e.g., bud break, shoot elongation). This paper describes the
research approach, its emphasis on the integration of above- and belowground
research components, and the TERA experimental facility.
Introduction
The U.S. Environmental Protection Agency (EPA) established its Global Climate
Research Program (GCRP) to conduct integrated research on aspects of the trace
gases fluxes and climate change impacts. An important focus of the Agency's
GCRP at its Environmental Research Laboratory in Corvallis, Oregon (ERL-C),
is to understand if and how C02 and climate change will affect vegetation in
North America. Existing data regarding the response of terrestrial vegetation to
C02 and climate change are insufficient to provide these answers. Thus, ERL-C
initiated the study Effects of CO2 and Climate Change on Forest Trees, to help
determine how trees are influenced by elevated CO2 and climate change. The
goals are to determine the effects of elevated CO2 and climate change on (1) the
growth and productivity of forest trees and associated soil processes and (2) the
carbon sequestration potential of forests.
Past and present trends of C02, temperature, and moisture were examined to
establish relevant experimental conditions. In 1990 the atmospheric concentration
of CO2 was 353 ppm, or 25% higher than in pre-industrial times (Houghton et
al., 1990). Assuming an increase of 1%/year, the C02 concentration in the
atmosphere may double to about 700 ppm by the year 2059 (Houghton et al.,
1990). However, other trace gases are also increasing with C02, and will
contribute to global climate forcing over this same period of time. Thus,
realistically concentrations of CO2 will be in the range of only 450-500 ppm
when global climate forcing increases to double the current level.
To predict the impacts of increased "greenhouse gas" levels on future
temperatures in the Pacific Northwest, the output of four atmosphere/climate
models [Oregon State University (OSU; Schlesinger and Zhao 1989), Goddard
Institute for Space Studies (G1SS; Hansen et al. 1983), Geophysical Fluid
Dynamics Laboratory (GFDL; Manabe and Wetherald 1987), and United
Kingdom Meteorological Office (UKMO; Wilson and Mitchell 1987)] were
reviewed . Although the specific details of the models may vary, they all project a
significant warming and an increase in potential evapotranspiration in the Pacific
3
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Northwest using a scenario of a doubling of atmospheric greenhouse gas
concentrations. For example, in the Willamette Valley temperatures could
increase for all months resulting in a mean increase of 2.3 to 5.1°C, depending on
the model.
The model-based projections for precipitation did not show this same consistency
as temperature. Projected changes in annual precipitation ranged from essentially
no change to 27% increase. However, all models forecast that the current seasonal
pattern of relatively dry summers and wet winters will persist, but the proportion
of rain vs. snow from current conditions may change because of the increase in
temperature. Also as a consequence of the increased temperature, potential
evapotranspiration will increase to a greater extent than the potential increase in
precipitation.
Overall, the future climates projected from the climate models represent a
significant change from present conditions. When viewed in a south-to-north
transect, the projected temperature changes were equivalent to shifting current
climates from 200 to 500 km north, i.e., moving the climate of northern
California into northern Oregon. However, strict geographical analogs of future
climate were difficult to define since projected precipitation may remain
unchanged. (Franklin et al., 1991) Similarly, from an elevational perspective, the
climate projections suggested a 500 to 1000 m upward movement of temperature
regimes.
The objectives of this manuscript are to describe (1) an experiment that EPA is
initiating to determine the impacts of elevated C02 and temperature on forest
trees, (2) the unique experimental facility that was constructed for the study and
(3) the initial performance characteristics of the facility.
Experimental Approach
Experimental Design The experimental design is a 2 x 2 factorial with two C02
and two temperature levels, and three replicate per treatment. The CO2 levels are
ambient and ambient plus 200 ppm (an increase associated with double climatic
forcing). The temperature levels are ambient and ambient plus 4°C (a predicted
temperature increase under double climatic forcing). Ambient conditions are
based on continuous measurements from a meteorological tower at the research
site. The increased CO2 and increased temperature treatments are added
continuously to the current ambient levels to preserve natural diurnal, seasonal
and yearly variability. The dew point depression will be held constant in both the
ambient and elevated temperature chambers, tracking the changes measured at the
site weather station providing a constant gradient for evapotranspiration in the
4
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ambient elevated temperature treatments.
Experimental Tasks To develop an integrated understanding of the effects of
elevated C02 and climate change on forest trees and soils, 7 integrated research
tasks are being conducted (Figure 1). The data will be used to understand the
effects of elevated C02 and climate change on tree growth and associated impacts
on soil processes and as specific inputs for a process-based physiological tree
growth model. Because of the highly integrated experimental design and the
relatively long experimental duration (4 years), it is essential that sampling
activities be coordinated to maximize inter-comparability of data.
Soil Selection: In the Cascade Mountains of Oregon, Douglas fir grows primarily
high-elevation sandy loam derived from volcanic ejecta and glacial till (30%) and
the remainder (70%) in a heavy-textured soil derived from colluvium and
residuum. The sandy loam soil was chosen because of the ease with which it could
be reconstituted and its resiliency to disturbance. The soil was collected by
horizon from the perimeter of a 500-600 year-old Douglas fir stand in the
Oregon Cascade Mountains. Large woody debris and rocks were removed as the
soil was homogenized. The soil strata was reconstructed by horizon in the
terracosms (Figure 2) as temperature sensors, lysimeters, gas samplers and
minirhizotron tubes were installed.
Species Selection: Douglas fir (Pseudotsuga menziesii [mirb.] Franco), currently
the most important timber species in the Pacific Northwest, was selected as the
plant material. Douglas fir is widely distributed, growing under a variety of
climatic conditions. Seedlings (1+1) were from "woods run" seed lots, rather than
half-sib or full-sib seed lots, to ensure that the seedling's genetic variability
reflects that of the natural forest. Seed lots were selected from five low-elevation
seed zones (<600 m) on the western side of the Oregon Cascade Mountains in the
Willamette Valley.
TERA Research Facility
To assess C02 and climate change effects on vegetation (Drake et al., 1985:
Allen, 1990), three major types of outdoor facilities including: open-top
chambers (Rogers et al., .1983); free-air C02 enrichment systems (FACE) (Allen
et al., 1985; US DOE, 1987; Men et al., 1992); and closed-circulation, sunlit,
computer-managed, controlled environment plant growth chambers (Soil-Plant-
Atmosphere Research (SPAR) unit). Each system has its advantages and
disadvantages. The SPAR units are unique because they have not only precisely
control gas"concentrations, but also dry bulb temperature, dew point temperature,
and rooting zone conditions (Jones et al., 1984a, b, 1985; Baker et al., 1990).
5
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This gives SPAR units an advantage for measuring the interacting effects of
rising C02 and climatic change factors on vegetation. Furthermore, because these
chambers use solar irradiance rather than artificial light they provide a more
natural irradiance regime than that used by indoor growth chambers.
The Terrestrial Ecophysiological Research Area (TERA) was designed to provide
a state-of-the-science research facility for investigating the effects of elevated
C02 and climate change on plants. This facility is unique because it is designed to
accurately track ambient C02, temperature and dew point depression while
operating continuously for several years. The central component of TERA are 12
SPAR units, called terracosms, that are capable of providing complete climate
control of an enclosed plant/soil system. TTiere are two additional chamberless
soil lysimeters (terracosms) that are similar to the enclosed terracosms for a
measure of possible enclosure effects on the soil/plan system.
The C02 concentration temperature and dew point depression within each
terracosm are controlled independently using a distributed network of Allen-
Bradley 5/20 programmable logic controllers (PLC). The PLCs serves as
intelligent interfaces to the array of sensors, valves, and flow controllers located
on each terracosm. Ambient weather conditions and C02 levels are monitored
continuously at the site weather station and transmitted to the terracosms. The
terracosm PLCs use the ambient conditions and internal computer logic to
determine and control their individual target CO2, temperature, and dew point
depression levels. With this design, dew point depressions and dry bulb
temperatures can be tracked to ±0.7°C, and C02 concentration tracked to ±15
ppm. Two 486/DX?. PC's serve as the network interfaces. One of the computer
receives data from each terracosm PLC at 1 minute intervals. These data,
(temperature, dew point, light level, C02 concentration, soil moisture, and soil
temperature) are relayed to a Sparc IPX workstation over a TCP/IP ethernet
where they are logged into the network data base. The second network interface
is used for direct processor communication, allowing a real time interface to the
performance status of the individual terracosms.
Terracosm Design: The aboveground chambers (1.5 m high at the back, sloping
to 1.2 m at the front) is 2 m wide and 1 m front-to-back, as seen from a front
view, the south exposure with a total canopy volume of 3.2 m^ (Figure 2), The
chamber is covered by a 3 mil clear Teflon film which was selected because of its
optical clarity, resistance to photodegradation, broad spectral transmission which
extends into both the IR and UV bands, and its chemical inertness.
Each chamber is mounted on a water-tight soil lysimeter that is 2 m wide, 1 m
front-to-back, and 1 m deep (total soil volume of 2 m^). An array of sensors and
6
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sampling ports in the belowground portion of each terracosm provide
nondestructive observation of the soil environment. A description of the sensors,
there intended function, and their physical placement are listed in Table 1.
System Performance: Terracosm Climate and C02 Control
To assess the terracosm system performance, the climate and C02 control for a
representative 16 day period (September 18 to October 3) was selected. From this
period 20,646 one minute averages of temperature, dew point depression, and
C02 concentrations were acquired from each of the twelve enclosed terracosms,
comparing the set points, or target conditions, with the actual terracosm
conditions. The data files were not edited to eliminate periods when the chambers
were entered for sample collection which disrupted climate control.
The performance of the terracosms to continuously reproduce a target condition
(temperature, dew point depression, or C02 concentration) was described using
two approaches. One, the average error term which is difference between the
actual condition measured in the terracosm and the set point for that same period
of time was calculated. Second, the difference between experimental treatments
were also calculated for each minute of acquired data collected over the 16-day
period. The terracosms were blocked into three groups, each group having all
four treatment conditions. Then the treatment differential was calculated between
a pair of terracosms holding either the temperature or C02 treatment constant.
For example, one set of temperature differentials were calculated between the
ambient temperature, ambient C02 terracosm and elevated temperature, elevated
C02 terracosm in each block. Table 2 summarizes these data.
The terracosms have been operating continuously for 90 days; during this initial
period, there have been several minor equipment failures but no problems
causing a complete system shut down. The greatest loss of experimental control in
the individual terracosms is attributed to necessary intrusions to make routine
growth and branch gas exchange measurements. These intrusions account for
control interruptions that amount to less than 1% of total daylight hours.
Temperature Control In the ambient temperature terracosms, mean temperature
was 0.9 °C above set point while in the elevated temperature terracosms it held
much closer to set point, having an average error term of 0.1 °C (Table 2). The
standard deviation of the error term in the ambient temperature terracosms is
also larger (1.5°C) than in the elevated temperature chamber (0.9°C), indicating a
slightly greater degree of trouble meeting ambient temperature treatment targets.
The resulting average temperature treatment applied to the chambers during this
16-day period was 17°C and 20°C for the ambient and elevated temperature
7
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treatments, respectively.
Results from the paired treatment comparison showed that the temperature
difference between the ambient and elevated treatment was 3.2 ±1.6°C which
compares well with the target temperature differential of 4°C The standard
deviation of the treatment differentials were on the order of that calculated on the
unpaired treatments, indicating that overall the temperature control errors tended
to be in phase. This is expected, as the diurnal shift in set points are also in phase,
and therefore, both the ambient and elevated temperature chambers are
attempting to either increase or decrease temperature at the same time.
An illustration of typical temperature control in an ambient and elevated
terracosms are shown for a 24-hour period (Figure 3). This data show that both
terracosms have good temperature tracking for the 15-hour period extending
from about 9:00 to 24:00. TTie periods which show temperature control problems
are cause by the competing requirements of temperature and dew point
depression control. The process control code was originally written to use the
cold water coil exclusively for controlling dew point depression. This strategy
sacrifices the systems ability to track downward moving temperature set points to
provide tighter dew point depression control. The process control code is
currently being modified to provide a better tradeoff between temperature and
dew point depression control.
Dew Point Depression The ambient temperature terracosms had a mean dew
point depression of 0.85 ± 2°C above set point, while the mean dew point
depression in elevated temperature terracoms was 0.56 ±1.7°C. These values
compare well. The resulting average dew point depression applied to both the
ambient and elevated temperature treatments was 9.8°C and 9.6°C respectively.
The difference between the dew point depression delivered to the ambient
temperature chambers and that delivered to the elevated temperature chamber
was 0.4° ±1.7°C, which is very close to the target differential of 0°C.
A 24 hour cycle of target and actual dew point depressions measured in two
terracoms is presented in Figure 4. These data show that while the dew point
depression was as much as two degrees greater in the early morning hours, the
diurnal treatment differential was very close to zero. During the-15 hour period
(9:00 to 24:00) when transpiration is highest, the control was excellent, with both
the ambient and elevated temperature terracosms tracking ambient dew point
depressions accurately.
CC>2 Control The effectiveness of C02 concentration tracking strategy is
demonstrated by the average error terms (Table 2). The ambient terracosms had
an average C02 error of 27 ±40 ppm. This corresponds to an average treatment
8
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concentration of 400 ppm, which is 27 ppm above the average ambient C02
concentration measured during the 16-day period. The elevated C02 terracosms
had an average error of -4.7 ppm ±50 ppm, which translated to an average
concentration of 570 ppm. The treatment differential was 170 ppm ±30 ppm and
is in reasonable agreement with the target treatment of 200 ppm.
A typical diurnal C02 cycle demonstrates the ability of the terracosms to track
the ambient C02 concentration (Figure 5). Both the ambient and elevated
terracosms showed good tracking, with much of the night time fine structure
being reproduced.
The accuracy of the CO2 control should improve as the experiment proceeds. As
currently designed, the terracosm CO2 concentration is increased either through
soil and plant respiration, or by metering in pure C02 using a mass flow
controller. The terracosm CO2 concentration can be decreased either through
plant uptake or venting with ambient air. As the experiment proceeds the
increased plant biomass will increase the rate of CO2 uptake. This should result
in more accurate concentration regulation during photosynthetic periods. At night
time, and until the biomass increase is sufficient, venting is used to adjust the C02
concentration downward.
Venting is most effective in the elevated CO2 chambers because of the -200 ppm
concentration difference between chamber and ambient air. It has not been
effective in reducing the ambient terracosms to their set point CO2 concentration
because the CO2 concentration, at ground level, surrounding the chambers can be
as much as 20 ppm greater than measured at the site weather station. We believe
this is caused by the terracosm air inlets entraining C02 evolved from soil
respiration while the CO2 concentration at the site weather station is measured at
3 meters, and is a location relatively free from obstructions which might limit air
movement. The data presented in Table 2 showed that the ambient C02 chambers
were on average 27 ppm above ambient, whereas the elevated C02 chambers
were 195 ppm above ambient, just 5 ppm away from the target elevated
concentration. We intend to improve C02 control using compressed C02 free air
to dilute the terracosm air as necessary.
Conclusion
We have presented an overview of an experimental research plan of an integrated
study of the effects of elevated C02 and climate change on above- and
belowground processes in a Douglas fir seedling system. The research described
here has just been initiated in an experimental facility designed and constructed
9
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simultaneously with the planning of the research. This paper has provided details
of specific elements comprising a terracosm system, and an initial evaluation of
the climate and C02 control capabilities.
Acknowledgments
The authors would like to thank the ERLC staff members who have be
instrumental to the construction of the TERA facility. The information in this
document has been funded by the U.S. Environmental Protection Agency.
Portions of this work have been prepared through Interagency Agreement DW
89934110 and Contract Number 68-C0-0021. It has be subjected to the Agency's
peer and administrative review, and it has been approved for publication as an
EPA document. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
References
Allen L H, Jr., Beladi S E and Shinn S H 1985 Modeling the feasibility of free-air
carbon dioxide release for vegetation response research. 17th Agricultural and
Forest Meteorology Conference, Amer. Meterorol. Soc., Scottsdale, AZ, May 21-
24, pp 161-164.
Allen L H, Jr. 1990. Plant response to rising carbon dioxide and potential
interactions with air pollutants. J. Environ. Qual. 19, 15-34.
Allen, L H, Jr., Drake B R, Rogers H H and Shinn J H 1992 Field techniques for
exposure of plants and ecosystems to elevated C02 and other trace gases. Crit.
Reviews Plant Sci. 11, 85-119.
Baker J T, Allen L H, Jr., Boote K J, Jones J W and Jones P 1990 Developmental
responses of rice to photoperiod and carbon dioxide concentrations. Agricul. For.
Meteorol. 50, 201-210.
Drake B G , Rogers H H and Allen L H, Jr. 1985. Methods of exposing plants to
elevated C02. pp 11-31. In: Strain B R and Cure J D (eds.), Direct Effects of
Carbon Dioxide on Vegetation. DOE/ER-0238, U.S. Dept. of Energy, Carbon
Dioxide Res. Dev., Washington, D.C. .
Franklin J F, Swanson F J, Harmon M E, Perry D A, Spies T A, Dale V H,
McKee A, Ferrell W K, Means J E, Gregory S V, Lattin J D, Showalter T D and
Larsen D 1991 Effects of global climatic change on forests in Northwestern
America. Northwest Environ. J. 7:233-254.
10
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Hansen J, Russell G, Rind D, Stone P, Lacis A, Lebedeff S, Ruedy R and Travis L
1983 Efficient three-dimensional global models for climate studies: Models I and
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IPCC (Intergovernmental Panel on Climate Change) Scientific Assessment.
Cambridge University Press, Cambridge, England.
Jones P, Allen L H, Jr., Jones J W, Boote K T and Campbell W J 1984a Soybean
canopy growth, photosynthesis, and transpiration responses to whole-season
carbon dioxide enrichment. Agron. J. 76, 633-637
Jones P, Jones J W, Allen L H, Jr. and Mishoe J W 1984b Dynamic computer
control of closed environment plant growth chambers. Design and verification.
Trans. ASAE. 27, 879-888.
Jones P, Allen L H, Jr., Jones J W and Valle R 1985 Photosynthesis and
transpiration responses of soybean canopies to short- and long-term C02
treatments. Agron. J. 77, 119-126.
Manabe S, and Wetherald R T 1987 Large-scale changes in soil wetness induced
by an increase in carbon dioxide. J. Atmos. Sci. 44, 1211-1235.
Rogers H H, Heck W W and Heagle AS 1983 A field technique for the study of
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Assoc. 33, 42-44.
Schlesinger M E, and Zhao Z C 1989 Seasonal climatic change introduced by
doubling C02 as simulated by the OSU atmospheric GCM/mixed-layer ocean
model. J. Climate. 2,429-495.
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11
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Table 1: Terracosm Sensors for Monitoring Above- and Belowground
Processes.
Measurement
Sensor Type
Manufacture/Model
Above-Ground
CO2/H2O
Dry-Bulb Temperature
Dew Point
PAR
Stem Diameter
Stem Sap Flow
Below-Ground
Temperature
Soil Moisture
Soil Gas
Soil Water
Root
Growth/Dynamics
IRGA
Shielded Aspirated Thermistors
Chilled Mirror Hygrometer
Silicon Photometer
Strain Gauge CERES Device
Heat Balance
Thermistors
TDR & Neutron Probes
Gas Wells
Tension Lysimeters
Minirhizotron Camera System
LI-COR LI6262
Campbell Scientific 107
General Eastern Dew-10
LI-COR 109SA
Pacific Northwest Laboratory
ERL-Corvallis
Campbell Scientific 107B
Campbell Scientific/Troxler
Site Built
Corning Glass Works
Bartz Technology Company
IRGA = infrared gas analyzer
PAR = photosynthetically active radiation
CERES = sensor for measuring changes in stem diameter
TDR = time domain reflectometry
12
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Table 2 Terracosm Climate and C02 Control Characteristics9
Error Terms
Resulting
Difference
(Deviation
Average
Between
from Set
Treatment
Ambient and
Point)
Applied
Elevated
Treatment
Ambient Temperature
Terracosms
Average (°C)
0.92
17
3.2
Standard
1.5
C.V.=8%b
1.6
Deviation
Elevated Temperature
Terracosms
Average (°C)
0.09
20
3.2
Standard
0.87
R.E = 4%
1.6
Deviation
Dew Point Depression m
Ambient Temperature
Terracosms
Average (°C)
0.85
9.8
0.37
Standard
2
R.E.=20%
1.7
Deviation
Dew Point Depression in
Elevated Temperature
Terracosms
Average (°C)
0.56
9.5
0.37
Standard
1.7
R.E.=20%
1.7
Deviauon
Ambient CO2 Terracosms
Average (ppm)
27
400
170
Standard
40
R.E.=10%
30
Deviauon
Elevated CO2 Terracosms
Average (ppm)
-4.7
570
170
Standard
50
R.E.=9%
30
Deviation
aData summarized 20,446 1 minute averages of temperature, dew point depression, and C02
concentration measured between 9/18/93 and 10/3/93 in each terracosm. During this period
ambient conditions measured at site weather station were as follows: temperature 16.3 ±7.8 C,
dew point depression 9.0 ±6.9°C, CO2 concentration 375 ±28 ppm.
bR.E. = relative error defined as the standard deviation of error term divided by the treatment
average.
13
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Figure Legends
Figure 1 - The seven experimental research tasks designed to provide and
integrated understanding of the effects of elevated C02 and climate change on
plants and soils.
Figure 2 - Side view of terracosm showing details of reconstructed soil horizons,
minirhizotrons root observation tubes, data acquisition/control, and aboveground
climate/gas sensor locations.
Figure 3 - Ambient and elevated temperature treatments for two terracosms.
Dark solid lines show the set point or target treatment, as measured at the site
weather station. The gray dashes are the actual 1-minute average temperatures
measured in the terracosms.
Figure 4 - Ambient and elevated dew point depression as measured in two
terracosms. The thick black line indicates the target dew point depression
measured at the site weather station. The light and dark gray dashes indicate the
actual 1 minute average dew point depression measured in the ambient and
elevated temperature terracosms, respectively. The thin black line indicates the
treatment differential between the ambient and elevated temperature terracosms.
Figure 5 - Ambient and elevated C02 concentrations measured in two
terracosms. The solid dark lines indicate the target C02 concentrations measured
at the site weather station. The gray dashes indicated the actual C02
concentrations measured.
14
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I
Side View of Terracosm
Dew Point Hygrometer
Hot and Cold Water
Heat Exchangers
Data Acquisition/Control,
C02/H20IRGA,
TDR Probe Multiplexer
Host/Chamber CO2
Sampling Ports, Quantum
Sensor, Dry Bulb Temperature
1.5 Meters
Litter Layer
^ =₯ A Horizon
B Horizon
zmt
R<«
s*s»
V'S'S"
s'vs'
WsW
C Horizon
¦vfl
s
Meter
Root Observation
Tubes
-------
F«,
Experimental Research
Tasks
Task 2
Shoot Growth and Phenology
Task 3
System Nutrients
Plant Nutrients
-w {WiW -WW-,
.* >
"V* - a .
. -v. .% /. /. /.
wmmmmm
..* :*.V' V *V'>' v.- v>*V
Task 1
Shoot Carbon and
Water Flux
Task 4
System Water
Plant Water
Task 5
Litter Layer
sz
Sou Water
Task 6
Root Growth and
Phenology
. ,*' .>/. .**
Task 7
Soil Biology
.*, ..* .*. /;
\V v?», .*
.*; / A f.i /, .> .*; o
iwiiiiaiiiM
.¦> ^v>v>v>v v>v.^:
-------
30
25
0 20
5
15
6
1 10
Elevated Temperature
Set Point
Actual
ras*
0:00
2:00
4:00
6:00
J-*
Ambient Temperature
Set Point . . ,
Actual
t_ _i _ - _( -i --
10:00 12:00 14:00 16:00 18:00 20:00 22:00 24:00
Time (hours)
a
-------
c
o
0
a
e
a.
£
o
a.
*
a
Ambient Temperature
Dew Point Depression
Elevated Temperature
Dew Point Depression
0:00
2:00
4:00
6:00
Set Point
Difference Between Ambient and
Elevated Temperature Dew Point Depressions
10:00 12:00 14:00 16:00 18:00 20:00 22:00 24:00
Time (hours)
-------
650
600
& 550
1 500
S
c
8 450
c
o
O
N 400
o
O
350
300
mcmbwbA
\ Elevated C02
Set Point
Actual
Ambient C02
Set Point
Actual
0:00 2:00
i 1- - i t
4:00 6:00 8:00 10:00
12:00 14:00 16:00 18:00 20:00 22:00 24:00
Time (hours)
------- |